i 
 
The Role of FCGBP in Mucus 
Structure, Processing and Function 
 
 
 
Erik Ehrencrona 
 
 
 
Department of Medical Biochemistry and Cell Biology 
Institute of Biomedicine 
 
Sahlgrenska Academy, University of Gothenburg 
 
 
 
 
 
 
 
 
Gothenburg 2021 
ii 
Cover illustration: FCGBP in human ileum by Erik Ehrencrona 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The Role of FCGBP in Mucus: Structure, Processing and Function 
© Erik Ehrencrona 2021 
erik.ehrencrona@medkem.gu.se 
 
ISBN 978-91-8009-468-9 (PRINT)  
ISBN 978-91-8009-469-6 (PDF) 
http://hdl.handle.net/2077/69314 
 
Printed in Borås, Sweden 2021 
Printed by Stema Specialtryck AB  
iii 
ABSTRACT 
In this thesis, a bottom-up approach was used to study the IgG Fc-binding 
protein (FCGBP), which next to the MUC2 mucin is the second main 
component of secreted mucus in small intestine and colon. FCGBP is also 
found in airways during inflammatory conditions with static mucus. Although 
discovered as an IgG sequester, this function was not reproducible here using 
purified proteins. FCGBP includes many von Willebrand D domains (vWDs) 
with most having a GDPH (Gly-Asp-Pro-His) motif. Theoretically, hydrolysis 
of the DP peptide bonds should result in reactive Asp-anhydrides driving 
covalent crosslinking between FCGBP and MUC2. Using mass spectrometry 
(MS), recombinant proteins, electrophoresis and Western blot, we found that 
all motifs were cleaved but FCGBP remained intact as consecutive fragments 
were tethered by single disulphide bonds. Label-free MS quantification of 
proteins in murine mucus showed that Muc2 and Fcgbp are mostly not 
covalently bound, and in silico structural predictions further argued against 
such interactions, with these Asp-anhydrides being inaccessible for their 
suggested MUC2 substrates. Recombinant proteins were purified, analysed 
and used for generation of FCGBP antisera. The murine Fcgbp is smaller but 
highly similar to the human orthologue, making it ideal for functional and 
structural studies. Microscopy was used to study live and fixed tissue from 
mouse colon and airways to investigate its physiological role. Recombinant 
proteins formed C-terminal cysteine dimers with cryogenic electron 
microscopy (cryo-EM) showing a spring feather-like quaternary structure. 
Even larger linear structures were detected in cryo-EM micrographs, and 
electrophoresis showed large complexes in mucus. Immunohistochemistry 
(IHC) also revealed elongated ultrastructures in healthy intestine and airways 
of a murine chronic obstructive pulmonary disease (COPD) model.  Results 
indicated less attached mucus in airways of Fcgbp-/- mice, and further a mucus 
expansion phenotype in colon. An N-terminal sequence linked to helical 
gliding was studied and alignments revealed that the murine sequence had 
partially been genetically lost. The remaining N-terminal sequence shared 
between human and mouse was found to be repeated prior to every vWD in 
the FCGBP sequence. In summary, these results indicate a role for FCGBP in 
mucus structure and attachment. 
Keywords: Mucus, FCGBP, IgG, IBD, COPD, Mucins, Disulphide, GDPH 
iv 
POPULÄRVETENSSKAPLIG SAMMANFATTNING 
FCGBP (IgG Fc-binding protein) hittas i slemmet som hydrerar och skyddar 
tarmens innandöme. Avhandlingen syftade till att etablera grundläggande 
förståelse för detta protein genom kartläggning av struktur, interaktioner och 
funktion. Vidare, att utveckla metoder och verktyg för framtida studier. 
Proteinet namngavs tidigare efter data där bindning till IgG påvisats. Detta 
motbevisas här genom att undersöka interaktioner mellan upprenade proteiner. 
Projektet har genererat flera specifika antikroppar, rekombinanta proteiner och 
lämpliga djurmodeller för studiens genomförande. I syfte att evaluera 
musmodellens lämplighet har skillnader mellan FCGBP hos människa och mus 
kartlagts och verifierats med molekylärbiologiska metoder. Proteinet är 
uppbyggt av von Willebrand D (vWD) domäner som beskrivs som viktiga för 
proteininteraktioner. Aminosyrasekvensen GDPH (Glycin-Aspartat-Prolin-
Histidin), som finns på 11 ställen i humant FCGBP och på 5 ställen i den 
kortare musvarianten, är fullständigt klyvd vilket teoretiskt genererar reaktiva 
grupper som kan binda starkt till MUC2, huvudkomponenten i slemmet. 
Modellering och masspektrometri visar dock att grupperna är gömda inuti 
proteinet och kan spjälkas av vatten. Detta överensstämmer med att inga 
proteiner starkt bundna till FCGBP kunde identifieras i mukus utöver en liten 
fraktion som binder till MUC2, sannolikt inte är fysiologiskt relevant. Data 
från olika metoder visar på kompakt proteinstruktur med disulfidmedierad C-
terminal dimer där flera vWD domäner bildar ett spiralfjäderliknande 
komplex. Icke-kovalenta interaktioner påvisades som medförde ännu större 
komplex, där funktionen sannolikt är att bidra till slemmets organisation och 
mekaniska egenskaper. Genom histologi visades uttryck i alla tarmens 
bägarceller och att FCGBP utsöndras som täta strukturer vilka organiserar 
slemlagrets inre skikt och sannolikt resten av mukustäcket. Försök med 
levande vävnad visar att avsaknad av FCGBP inte gör slemmet mer 
genomsläpplig för partiklar av bakteriestorlek. Infärgning visade dock ett 
mindre strukturerat slemlager och även en förändrad slemtillväxt i 
tjocktarmen. FCGBP är normalt inte uttryckt i lunga men uppregleras 
exempelvis vid kronisk obstruktiv lungsjukdom (KOL) där slemmet blir segare 
och liknar det som hittas i tarm. Avsaknad av FCGBP i djurmodell för KOL 
resulterade i mindre fastsittande slem. Immunfärgningar visade täta 
nätverksliknande strukturer som kapslar in och håller kvar mukus längs 
epitelytan. Sammantaget avslöjas dess funktion som en strukturellt viktig 
komponent i slem för att vid behov bilda ett skyddande mukustäcke på 
kroppens slemhinnor. 
 
v 
LIST OF PAPERS  
This thesis is based on the following studies, referred to in the text by their 
Roman numerals. 
I. Ehrencrona, E. van der Post, S. Gallego, P. Recktenwald, 
C. Rodriguez-Pineiro, A.M. Garcia-Bonete, MJ. Trillo-
Muyo, S. Bäckström, M. Hansson, GC. Johanson, MEV. 
The IgG Fc-binding protein FCGBP is secreted with all 
GDPH sequences cleaved, but maintained by inter-
fragment disulfide Bonds 
Journal of Biochemistry 2021; 293(1):100871. 
 
II. Fakih, D. Ehrencrona, E. Martinez-Abad, B. Arike, L., 
Ermund, A. Trillo-Muyo, S. Gallego, P. Johansson, M.E.V. 
and Hansson, G.C.   The FCGBP Protein Induced at Lung 
Disease Anchors the Mucus Layer to the 
Tracheobronchial Surface 
Manuscript.  
 
III. Ehrencrona, E*. Gallego, P*. Garcia-Bonete, M.J. Trillo-
Muyo, S. van der Post, S.V.P. Recktenwald, C.V., 
Rodriguez-pineiro, A.M. Hansson, G.C. and Johansson, 
M.E.V. The FCGBP Structure Reveals a Convoluted C-
terminal Dimer Stabilised by Cysteine Bonds 
Manuscript. * Equal contribution 
 
IV. Ehrencrona, E. Svensson, F. Gallego, P. Garcia-Bonete, 
M.J. Martinez Abad, B. Hansson, G.C. Johansson, M.E.V. 
Functional Analyses of FCGBP and its Role in 
Organisation of the Colonic Mucus Barrier 
Manuscript. 
 
vi 
CONTENT 
ABBREVIATIONS ........................................................................................... VIII 
1 INTRODUCTION ........................................................................................... 1 
1.1 Mucus is the first line of defense .......................................................... 1 
1.2 Physiology, histology and mucus composition in the upper and lower GI 
tract 3 
1.3 Gastrointestinal mucus and the adaptive immune system ..................... 9 
1.3.1 Mucus-related diseases in the GI tract ........................................ 10 
1.4 Lung .................................................................................................... 11 
1.4.1 Airway mucus and clearance physiology in health and disease .. 11 
1.5 IgG Fc Gamma Binding Protein (FCGBP) ......................................... 14 
1.5.1 Structure, function and evolutionary relatives............................. 14 
1.5.2 Current insight on GDPH motifs – Processing and function ...... 17 
1.5.3 The FCGBP_N sequence and link to helical gliding bacteria ..... 20 
1.5.4 Links between FCGBP and disease............................................. 21 
2 AIM ........................................................................................................... 23 
2.1 General Aims ...................................................................................... 23 
2.2 Specific aims ....................................................................................... 23 
3 CONTRIBUTIONS ....................................................................................... 24 
4 METHODS AND MATERIAL ........................................................................ 25 
4.1 Molecular structure analysis and binding experiments ....................... 25 
4.2 Production of recombinant FCGBP and polyclonal antiserum ........... 27 
4.3 Animal experiments ............................................................................ 29 
4.4 IHC and imaging ................................................................................. 30 
4.5 MS analysis ......................................................................................... 30 
4.6 Statistics .............................................................................................. 31 
5 RESULTS AND DISCUSSION ....................................................................... 32 
5.1 Complete hydrolysis of FCGBP GDPH Motifs and no binding to IgG Fc 
or Muc2 (Paper I) ...................................................................................... 32 
vii 
5.2 Murine Fcgbp polymers drive mucus attachment in an elastase COPD 
model (Paper II) ........................................................................................ 34 
5.3 FCGBP forms a C-terminal disulphide dimer and a highly globular 
quarternary structure involving many D modules (Paper III) ................... 35 
5.4 FCGBP is important for colonic mucus structure and the N-terminal 
region genetically destroyed in mice forms non-covalent dimers (Paper IV)
 37 
6 CONCLUSION ............................................................................................ 40 
7 FUTURE PERSPECTIVES ............................................................................. 41 
8 ACKNOWLEDGEMENTS ............................................................................. 43 
8.1 To collegues and collaborators ............................................................ 43 
8.2 Figures ................................................................................................. 44 
REFERENCES .................................................................................................. 45 
 
  
viii 
ABBREVIATIONS 
GI tract 
FCGBP 
Gastrointestinal tract 
IgG Fc binding protein 
IgG 
MUC2 
MUC5AC 
MUC5B 
Mr 
AMP 
vWF 
vWD 
vWA 
vWC 
CTCK 
C8 
TIL 
CysD 
EGF 
TSP1 
EMI 
LDLRA 
FA58C 
PTPmu  
MAM 
FCGBP_N 
HCl 
IML 
OML 
DC 
PC 
Si8 
ASL 
TECTα 
SSPO 
SCO 
ZAN 
OTOG 
DMBT1 
CLCA1 
ADAMTS-13 
 
MEP1 
MMP 
ZG16 
AGR2 
Immunoglobulin G 
Mucin 2 
Mucin 5AC 
Mucin 5B 
Molecular mass 
Anti-microbial protein or peptide 
von Willeband factor 
von Willeband factor D domain 
von Willeband factor A domain 
von Willeband factor C domain 
Cysteine knot 
Domain with 8 conserved cysteines 
Trypsin inhibitor-like domain 
Cysteine-rich domain 
Epidermal growth factor 
Thrombospondin 1  
Elastin microfibril interfacer domain 
Low density lipoprotein receptor A domain 
Coagulation factor 58 C-terminal domain 
Receptor protein tyrosine phosphatase Mu 
Meprin A-5 protein and PTPmu  
FCGBP N-terminal sequence 
Hydrochloric acid 
Inner mucus layer 
Outer mucus layer 
Distal colon 
Proximal colon 
distal 8th portion of small intestine (Ileum) 
Air surface liquid 
Tectorin alpha 
SCO spondin 
Sub commissural organs 
Zonadhesin 
Otogelin 
Deleted in malignant brain tumors 1 
Chloride channel accessory 1 
A disintegrin and metalloproteinase with a thrombospondin 
type 1 motif, member 13 
Meprin 1 
Matrix metalloproteinase 
Zymogen granule protein 16 
Anterior gradient protein 2 
ix 
TFF3 
NEO1 
PIGR 
IL8 
TNFα 
PTS domain 
GDPH motif 
Trefoil factor 3 
Neogenin-1 
Polymeric immunoglobulin receptor 
Interleukin 8 
Tumour necrosis factor alpha 
Proline-threonine-serine domain 
Glycine-aspartate-proline-histidine motif 
vWF von Willebrand factor 
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis 
BN PAGE Blue native polyacrylamide gel electrophoresis 
SEC Size exclusion chromatography 
SAXS Small angle x-ray scattering 
Cryo-EM 
MS 
GuHCl 
Cryogenic electron microscopy 
Mass spectrometry 
Guanidium hydrochloride 
COPD Chronic obstructive pulmonary disease 
CF 
BALF 
Cystic fibrosis 
Bronchoalveolar lavage fluid 
CRC Colorectal cancer 
TC Thyroid carcinoma 
HNSCC 
MAMP 
Head and neck squamous cell cancer 
Microbe-associated molecular pattern 
PAMP 
SNP 
 
Pathogen-associated molecular pattern 
Single nucleotide polymorphism 
 
Erik Ehrencrona 
1 
1 INTRODUCTION 
1.1 Mucus is the first line of defense 
In order to understand the physiological function and composition of mucus, it 
may be beneficial to discuss the environmental challenges needed to be tackled 
on mucosal surfaces, and how similar issues are dealt with in other places and 
evolutionary contexts. In addition to being subjected to mechanical forces that 
can be injurious, humans are constantly in contact with microorganisms that 
can as well be harmful. As an adaptation, evolution has granted different 
strategies to protect the integrity of mucosal surfaces, with examples including 
physical barriers, an innate and adaptive immune system, along with symbiotic 
relationships with certain microorganisms. The mucosal surface of the 
intestine is a great example. It is lined only by a single layer of epithelial cells, 
yet the luminal content is a source of intense mechanical stress and a home to 
a lush microbiota. Without a more sophisticated shielding, humans would 
likely succumb due to intestinal hemorrhages or invasive infections caused by 
intestinal pathogens.  
The lower GI tract is an incubator of life, containing a warm and nutritious 
microenvironment that constantly promotes bacterial growth. Unsurprisingly, 
the large intestine houses a highly concentrated and diverse microbial 
community not found anywhere else in the human body 1,2. To protect and 
maintain homeostasis at the mucosal surfaces, a highly complex and 
compositionally dynamic film of glycosylated biopolymers is secreted by a 
specialised type of epithelial cell called goblet cells (GCs) 3,4. This secreted 
material is what is known as mucus, and its primary constituents are the large 
glycoproteins called mucins. Mucins assemble into networks resembling a 
sieve where larger structures are trapped 5-8. They have massive O-linked 
glycans stretching from the side chains of the amino acids threonine and serine 
in the PTS (Pro-Thr-Ser) domains. These glycans together with the many 
disulphide bonds that crosslink the protein backbone of the mucins, are thought 
to protect the mucinous networks against proteases and glycosidases 
introduced by the host or colonising bacteria 5,9. Furthermore, O-glycans cause 
the mucins to resemble bottlebrushes. These glycans contain many acidic 
structures such as sulphate and sialic acid groups, causing them to become 
highly water absorbant, subsequently resulting in mucus having the properties 
of a hydrogel. The charges of these glycans likely keep the mucins stretched 
out as they are inherently repulsive. The secreted mucins are also called gel-
forming mucins 5,10-12. There is also a variety of membrane bound mucins found 
The Role of FCGBP in Mucus 
2 
throughout the GI tract 5. These likely contribute to epithelial protection but do 
not extensively contribute to the secreted mucus barrier.  
Mucus structure and properties differs in various locations such as the airways, 
urogenital and GI tract. However, the way the barrier function is maintained is 
essentially the same; the surface is kept hydrated, lubricated, and unwanted 
structures or particles are entrapped and cleared out. This could be bacteria in 
the colon, or dust in the lungs. The complex and not always well studied 
mechanism of mucus clearance is used to control the distance between these 
particles and the mucosa. In general, while bacteria may stick to the mucus, 
smaller molecules should diffuse through the barrier. These systems may 
falter, resulting in disease 5,13. Upon binding the unwanted particle, it is also 
important to rid these by renewing and removing old mucus, otherwise the 
bacterial enzymes may succeed in degrading both protein and glycan 
components of the mucin framework, thereby reaching the epithelial cells.  
Even though the epidermis of the skin deals with a resident microbiota, the 
barrier function is executed in a very different style. In a hypothetical scenario 
were the skin and intestinal barrier systems were swapped, the result would be 
a disaster. On one hand, in a non-aqueous or low humidity environment, a 
terrestrial animal normally having a mucus-coated surface would have 
problems conserving water and keeping the surface hydrated. The tissue would 
likely crack, leading to infection and possibly death. On the other hand, the 
presence of an epidermal-like barrier in the GI tract would lead to starvation. 
Nutrients would not be properly absorbed as the mammalian epidermis is a 
restrictive diffusion barrier. The epidermis has a thick layer of anucleated cells 
and a high content of lipids, creating a mechanically robust diffusion barrier, 
allowing for storage of water and controlling homeostasis in a dry environment 
14,15. However, it is likely that not only epidermal thickness is important for 
barrier function, but also renewal and shedding of the sheets of skin, so that 
damaged tissue may be removed and unwanted microorganisms will struggle 
to get a permanent footing on the surface. 
In the small intestine, the movement of fluid, electrolytes and nutrients such as 
monosaccharides, amino acids and fatty acids, is essential for the intestinal 
physiology and function. The thin intestinal epithelium is therefore shielded 
with a loose hydrous mucus coat that enables diffusion 16. Here, GCs 
differentiate from the stem cells in the crypts and move upwards renewing the 
tissue surface every few days 17. Generally, GCs secrete mucus at a baseline 
rate but can also be stimulated to enhance mucus release 18. The colonic mucus 
barrier is slightly different, having an outer layer housing bacteria, and an inner 
layer that is not normally accessible to colonisation 8. As the airways are a 
Erik Ehrencrona 
3 
location for gas exchange, they must also have a barrier that does not impair 
diffusion. Illustrations showing the first barrier for the epidermis 14,15, 
oropharynx 19, airways 13,20 and colon 8,13 are shown in figure 1 below.  
Figure 1. Histological illustrations of first barriers in different human 
anatomical locations. For the epidermis, the stratum corneum (1) is an important 
barrier for mechanical protection and water consolidation, in addition to the several 
layers of epithelial squamous cells (2). In the oropharynx, the multi-layered 
squamous cells (4), lacking a stratum corneum on top, are lubricated and partly 
protected by saliva (3). The trachea and bronchi are covered with a pseudostratified 
epithelium (5) where columnar cells have stereocilia (6) that transport away liquid 
and mucus (7). The colon has an inner (9) and and outer (8) mucus layer protecting 
a single layer of epith elial cells (10). 
1.2 Physiology, histology and mucus composition 
in the upper and lower GI tract 
There are 21 proteins listed within the human family of mucins. Here, the main 
gel-forming mucins are MUC2, MUC5AC, MUC5B and MUC6. There is also 
a host of transmembrane mucins, with examples including MUC1 and MUC17 
5. While MUC5B is the predominant secreted mucin in the oral cavity and 
airways 21, MUC5AC is the prime component of the gastric mucus layer 22. 
With some exceptions in muscle layer configuration, the traditional 
histological structure remains the same throughout the GI tract. However, due 
to the tissue-like properties of mucus, in some parts of the tract it may be 
considered an additional histological layer. As seen from a cross-sectional 
image of the tube, the GI tract is composed of a set of layers (Fig. 2, Colonic 
section as an example on the next page) 23.  
The Role of FCGBP in Mucus 
4 
Figure 2. The histological layers of the distal colon. In the center of the lumen is the 
fecal pellet (1), covered also by the mucosa-lining mucus layers (2-3) that varies in 
composition throughout the GI tract. The mucosa (4), is covered by varying types of 
epithelial cells. Next, there is the submucosa (5), surrounded by the circular muscularis 
interna (6) and longitudinal muscularis externa (7). The outermost layers is the serosa 
(8), and layers 4-9 are present in all parts of the alimentary tract. 
The GI tract is divided into two main sections; the upper and lower part. 
Whereas the upper GI tract stretches from the tongue to the end of the ileum 
24, the latter reaches from the caecum to the anus 25. At the beginning of the 
upper GI tract, the oropharynx and esophageal region have many layers of 
squamous cells protecting the surface. However, unlike the epidermis, these 
lack anucleated corneocytes 19. Instead, saliva is complementary for the barrier 
function. In a review from 2008 26, de Almeida et Al. presents an overview of 
the general composition and physiology of saliva. More than 90 % of this liquid 
is secreted from the sublingual, submandibular and parotid glands. Although it 
is less gel-like than mucus, saliva contains mucins and mucus components in 
addition to antimicrobial proteins (AMPs) and the digestive enzyme amylase. 
In addition to protection of the teeth, the water-retaining effect of mucins is 
believed to be important in maintaining lubrication. As in other locations, both 
the innate and adaptive immune system work in tandem to detect and react to 
pathogen-associated molecular patterns (PAMPs) and microbe-associated 
molecular patterns (MAMPs) in the mucus secretions. The oral cavity is the 
front door where all new microbes are introduced to the system. Therefore, in 
addition to immunoglobulins, a diverse set of AMPs are needed to prevent 
infection and modulate the microbiota through bactericidal and bacteriostatic 
effects. The protein deleted in malignant brain tumour 1 (DMBT1) is an 
agglutinin found in saliva 26 that has a series of scavenger receptors embedded 
within its sequence. It is highly versatile and has been shown to interact with a 
series of different structures from both viruses and bacteria. DMBT1 can also 
bind to secreted proteins in mucus, including MUC5B and IgA 27. It is found 
also in the airways 28 and throughout the rest of the GI tract 28,29. 
Erik Ehrencrona 
5 
In the stomach, the parenchyma needs protection not only from mechanical 
forces, but also from a hostile acidic environment generated by the secretion 
of hydrochloric acid (HCl) from parietal cells deep within the gastric glands 30. 
A review by Martinsen et al. from 2019 31 details the composition and function 
of gastric juice. The stomach has an alternative approach to handling microbial 
content of the lumen, working as an effective sterilisation chamber 31 with a 
luminal pH of 2 30, in addition to providing the first digestive enzymes targeting 
lipids and proteins. The low pH is further needed to denaturate the 
macromolecules in food 31. The bolus is grinded to pieces by peristaltic 
movements as the ventricle is sealed from both ends by powerful sphincters 32. 
Here, the wall muscle layers differs from the rest of the GI tract, having an 
additional oblique muscle layer that enables the grinding movement 33. How 
the mucus layer is important in the protection against HCl in the stomach is 
summarised by Philipsson  in a review from 2004 30. The GCs, here called 
foveolar cells, produce great amounts of bicarbonate and the main gel-forming 
mucin MUC5AC, in addition to the MUC6 mucin produced by cells inside the 
glands. Gastric mucus is considered to have two mucus layers, with the inner 
being firmly attached to the epithelium. The inner layer forms a pH gradient, 
being most acidic close the luminal content. The gradient is thought to be 
formed by bicarbonate secretion from underneath the mucus and acid from the 
lumen gradually mixing with the mucus as a diffusion barrier 30. Pancreatic 
juice and bile acid is introduced to the chyme upon transition to the duodenum, 
introducing a myriad of enzymes and salts that are needed to digest the 
macromolecules of the chyme and emulsify the lipid content 34,35. One of the 
most important functions is to neutralise the acidic content from the stomach, 
mainly through the secretion of bicarbonate 34. 
In the rest of the GI tract, the main gel-forming mucin is the MUC2 29. 
Recombinant MUC2 forms N-terminal dimers through the von Willebrand 
Factor D3 domain (vWD3) 7 and C-terminal dimers between cysteine knots 
(CTCKs) 6. Consequently, the interactions grant the formation of a polymeric 
network that constitutes the main framework within the mucus barrier of the 
small intestine and colon 13. MUC5B and MUC5AC have a similar domain 
architecture (Fig. 3). The importance of MUC2 in epithelial protection is 
emphasised by studies of Muc2-/- mice which lack a normal mucus layer in the 
colon. These mice develop severe colitis and in some cases cancer 8,36,37. 
MUC2 is densely packed in the GC vesicles, with a sudden drop in Ca2+-levels 
upon secretion being thought to be a driver of mucus expansion 13,38. Next to 
MUC2, IgG Fc- binding protein (FCGBP) is the second main secreted core 
protein in the mucus of the large and small intestine 29. It was initially described 
to be sequestering IgG 39-42, but speculation on its function have shifted 
recently, rather suggesting a structural role 43,44. The amino acid sequence of 
The Role of FCGBP in Mucus 
6 
FCGBP shows many similarities with the family of mucins. However, it lacks 
the central regions rich in proline, serine and threonine, and could thereby be 
considered a naked mucin. As heavy O-glycosylation is suggested to reduce 
the flexibility of mucins 5, FCGBP will likely be a much less rigid molecule. 
Neither does FCGBP have CysDs, CTCKs, or vWCs that are found but not 
limited to MUC2. Here, the main parallel is the D-assemblies (Fig. 3), also 
found in the von Willebrand factor (vWF) protein that binds together collagen 
and blood proteins during the coagulation cascade 45. Both C8 and TIL are 
domains rich in cysteines and therefore shielded against proteolytic digestion. 
 
Figure 3. Illustrative Figures showing domain compositions of important 
mucus proteins. The conserved domains of the human mucins MUC2, MUC5AC 
and MUC5B are compared to those of FCGBP. The figure includes annotations for 
color-coded units representing each type of motif or domain.  
Seen from an evolutionary perspective, the vWF naming scheme is considered 
slightly misleading as these domains originated from mucin-like proteins in 
early eukaryotic organisms that arose long before the vWF 5,46. Currently, there 
are no Fcgbp -/- mice phenotypes described in literature.  
 
Similar to gastric mucus, IHC experiments using Carnoy-fixed tissue has 
revealed an additional inner mucus layer (IML) that is firmly attached to the 
colonic epithelium 8. It is proposed that structures above the IML, as seen in 
sections, is part locally generated and also consist of material gathered by the 
chyme during peristaltic transit through the colon, as mucus remnants still coat 
the surface of extracted fecal pellets 47-49. Specific bacteria can thrive in the 
loose outer mucus layer (OML) 50. In ex vivo experiments with fluorescent 
beads, only the interface area between the IML and OML is seen, as the rest 
has been removed during the preparation of the tissue 51 (Fig. 4).   
Erik Ehrencrona 
7 
Figure 4.  Illustrative figure showing mucus barrier organisation in fixed and 
live colonic tissue. During peristalsis, the colonic fecal pellet gathers layers of 
mucus as it is transported in a distal direction. This is visible in Carnoy fixed 
sections imaged in vivo using immunohistochemistry (IHC). In ex vivo imaging of 
colon using fluorescent beads and a confocal microscopy, preparation of the tissue 
removes most of the OML and other structures resting above it. 
Having a thick compact mucus layer in the small intestine would likely 
negatively impact the diffusion and absorbance of nutrients due to the 
hydrostatic effect of the MUC2 O-glycans. Therefore, AMPs are used to a 
greater extent in order to protect the precious stem cells situated at the base of 
the crypt. In a review from 2011  52, Bevins et al. did a detailed description of 
Paneth cells and AMPs found in the bottom of the small intestinal crypts, 
especially important for controlling the microbiota in the small intestine. 
Among AMPs produced by the host Paneth cells, defensins, lysozymes, 
phosholipases, lectins and ribonucleases are listed in the review. Collectively, 
these are able to shape how close to the epithelium microorganisms such as 
fungi, bacteria and viruses can reach 52. In the the small intestine, there is only 
a loose mucus layer that can be easily detached ex vivo. Compared to that of 
colon, this mucus allows also for increased penetration by bacteria 13,50. The in 
vivo model for mucus organisation and bacterial colonisation in the small 
intestine is shown in figure 5. The model is based on data from both living and 
fixed tissue visualised by microscopy 13. 
 
 
 
 
 
 
 
The Role of FCGBP in Mucus 
8 
Figure 5. In vivo models for mucus organisation in small intestine and colon. 
The ileum has a loose mucus layer where bacteria can reach about a third of the 
length from the tip of the villi to bottom of the crypts. The distal colon has a distinct 
inner mucus layer (IML) firmly attached and not accessible to bacteria, and an outer 
mucus layer (OML), loosely attached and colonised by bacteria. 
Clearance of mucus and chyme is also an important defense against microbes. 
In this context it has been shown that MEP1β is driving detachment of mucus 
in the small intestine by cleaving MUC2. Removal of calcium from MUC2 is 
also thought to be crucial for MEP1β-function 13,53. In the colon, the CLCA1 
metalloprotease is important as an additional modulator of mucus expansion, 
and thereby clearance 54. Fluorescent lectins 54,55, bacteria-sized beads 51, and 
nucleic acid staining reveal the superstructural organisation of the mucus in an 
ex vivo setup. Strangely, the IML becomes penetrable to these beads in germ 
free (GF) mice, implicating the microbiota in shaping the mucus properties 13. 
It has also been shown that a western style diet in mice, being low in fiber and 
high in fat, alters the microbial composition and increases the penetrability of 
the mucus, in addition to reducing the thickness of the IML. Bifidobacterium 
is an example of bacteria can directly affect mucus thickness and growth. The 
colonisation and expansion of these is promoted by dietary fibers 56.  
 
It is important to stress that bacterial colonisation and digestion of mucin 
glycans is not a threat under normal conditions. The symbiotic relationship 
between the host and the gut microbiota is explored in depth in a review by 
Koropatkin et al. 9 The host benefits greatly from commensals such as the 
Bacteroides thetaiotaomicron digesting glycans in the OML. Short chain fatty 
acids (SCFAs) are metabolites of this process, which can in turn be absorbed 
back by the host, thereby recycling the mucus in a symbiotic fashion. Among 
the normal commensal bacteria, the Bacteroidetes, Firmicutes, Actinobacteria 
and Proteobacteria are the main phyla in adults and infants, although infants 
have certain adaptions correlating with the intake of maternal milk 9.  
 
The protective mucus barrier can sometimes fail resulting in injury or infection 
by allowing bacteria to get closer to the epithelium. This triggers a massive 
reaction of mucus release from the upper crypt controlled by a specific sentinel 
Erik Ehrencrona 
9 
goblet cell 57. In addition to mucins and FCGBP, the proteins anterior gradient 
protein 2 (AGR2), trefoil factor 3 (TFF3), zymogen granule protein 16 (ZG16) 
and transglutaminase 2 and 3 (TGM2/TGM3) are other important components 
of mucus in the small and large intestine 44. ZG16 is a small 16 kDa protein 
that binds and aggregates Gram positive bacteria. Zg16-/- mice develop large 
deposits of visceral fat and 16S-sequencing shows bacterial infiltration of the 
spleen. It was therefore suggested that a dysfunctional barrier leads to chronic 
inflammation and weight gain, as possibly as a result of bacterial translocation 
58. AGR2 is thought to function as a protein disulphide isomerase with a 
specific role in biosynthesis of mucus proteins 59. In addition, it has been shown 
to be secreted as a component of intestinal mucus 60. TFF3 is a small protein 
believed to be important for tissue regeneration 61, protection against helminths 
62, and suggested to be involved in stabilising mucus through covalent 
interactions with FCGBP and indirectly with mucins 43,63. While O-glycans 
protect the protein core of MUC2, and disulphide bonds link the protein 
horizontally, the mucin framework is suggested to be stabilised vertically in 
the same way as the epidermal layer of the skin by isopeptide bonds catalysed 
by transglutamination 64,65. 
1.3 Gastrointestinal mucus and the adaptive 
immune system 
As part of the acquired immunity, mucosal plasma cells secrete dimeric 
immunoglobulin A (IgA) as the main antibody. The subject of IgA in mucosal 
immunity is well explained in a review by Brandtzaeg from 2011 66. It is 
translocated through epithelial cells to the intestinal lumen with the help of 
Polymeric IgG Receptor (PIGR).  PIGR is a membrane protein which binds 
the IgA heavy chains. The complex undergoes cleavage along with bound IgA. 
This allows for the secretory component bound to IgA, forming secretory IgA, 
to be released into the mucus. The main function of secretory IgA is 
agglutination of bacteria to control the microbiota 66. Pentameric IgM is not 
normally found in mucus of the small intestine and lower GI tract, or is at least 
it is secreted in very low amounts. However, in saliva it is secreted following 
the same pathway as IgA 66,67 In neonates with a premature adaptive immune 
system, IgA and smaller amounts of IgG is delivered to the GI tract through 
maternal milk 68. This is especially important for IgA as it cannot pass through 
placenta to the fetus during development. With IgG being monomeric 69 , it 
would likely not be useful for agglutination of bacteria unless a mechanism of 
tethering IgG to mucus was available. FCGBP is indeed suggested to be such 
a sequesterer of IgG through binding to its many vWDs 41,42. However, a link 
between FCGBP and IgA has never been detected. FCGBP has also been 
The Role of FCGBP in Mucus 
10 
suggested to be sequestering IgG in cervical mucus, trapping HIV particles 70. 
The massive MUC16 mucin has also been suggested to act as a sequesterer of 
IgG in the female urogenital tract 71, but there is no other known potential Ig-
binding site in intestinal mucus. However, IgG is likely secreted in very low 
concentrations in GI mucus, as it does not have a known dedicated 
translocation mechanism as seen with IgA and PIGR.   
1.3.1 Mucus-related diseases in the GI tract 
Although studies directly linking mucus to disease are scarce, it is widely 
regarded that an inadequate barrier function is one of the key drivers in the 
development of inflammatory bowel disease (IBD), including ulcerative colitis 
(UC) and Crohn’s disease (CD). This inadequacy is suggested to arise from a 
more permeable mucus layer and an increased mucosal reaction and/or 
penetrability to bacteria (Fig. 6) 36,72-74, with increased levels of MAMPs such 
as LPS reaching the mucosa. Alternatively these factors acting in unison can 
result in parenchymal damage 74. Long-term inflammation in UC can drive the 
development of colorectal cancer (CRC), mainly resulting in adenocarcinoma 
75. The link to mucus is clear as Muc2-/-mice with a disrupted mucus are more 
prone to develop cancer 37, and mucinous colorectal adenocarcinoma is known 
to show overexpression not only of MUC2, but also MUC5AC 76.  
Figure 6. Relationship between colonic mucus and bacteria in health and 
IBD illustrated. Under normal conditions, bacterial colonization is limited to 
the OML. However, In IBD the barrier function of the IML is disrupted, 
allowing for bacteria to reach the surface of the epithelial cells. 
General alterations of the proteomic profile 5,73 and mucin glycan composition 
5,77,78 is associated with the disease. Just like Muc2-/- mice, the colonic IML of 
UC patients with active disease is penetrable to bacteria-sized fluorescent 
beads implicating alterations in mucus homeostasis 72. In addition to being 
caused by an out of control immune responses due to penetrant antigens, UC 
is a complex disease with everything from ER stress to a dysbiosis and altered 
immune regulation being suggested to drive the condition 79. Episodes of UC 
are limited to the colon, in contrast, CD can develop at virtually any place in 
Erik Ehrencrona 
11 
the GI tract. However, it is most commonly located to the terminal ileum. 
Unlike UC, the parenchymal damage in CD is not limited to the mucosa and 
submucosa. Here, all the histological layers can be compromised, and even 
fistulas can be formed between the intestine and nearby organs 80.  
Cystic fibrosis (CF) is also related to GI mucus as it can lead to highly viscous 
attached plugs of mucus and fecal material, called meconium, forming in the 
intestine. This leads to blockage of fecal transit 81. As Cftr-/- mice have firmly 
attached mucus in the small intestine, and this process cannot be reversed by 
recombinant MEP1β, but instead through the addition of bicarbonate, it is 
proposed that impaired calcium homeostasis is part of the pathology 53,82. 
Calcium and low pH drive intracellular packing of MUC2 7,38, and it was 
therefore suggested that mucin expansion by calcium chelation reveals the 
MEP1β cleavage sites 53,82. The molecular details of the links between CF and 
the many mucus core proteins are otherwise scarcely studied. Mucus plugging 
can at least be associated with the CLCA1 gene, as patients with the 
pAsn357Ser single nucleotide polymorphism (SNP) variant shows increased 
risk of plugging 83. In concurrence with the SNP driven pathology, studies of 
a murine Cftr-/- model showed lower expression of the murine CLCA1 ortholog 
(Clca1, previously called Clca3) 84. Transgenic Cftr-/- mice not being able to 
express Cftr, but overexpressing Clca1, in turn showed less damage to the villi 
and normalised mucus release. This effect on mucus retention was suggested 
to be partly mediated by its function as a metalloprotease 85, being able to aid 
in dissolving the accumulated mucus.  
1.4 Lung  
1.4.1 Airway mucus and clearance physiology in health 
and disease 
Compared to the GI tract, the physiology pertaining to mucus is different in 
the lung under healthy conditions. The tracheal surface is lined by 
pseudostratified epithelium with embedded goblet cells which secrete mucus. 
Mucinous material is also secreted by submucosal glands. Under healthy 
circumstances, this glandular mucus mainly contains MUC5B. The glands are 
only found in the submucosa of the cervical part of murine trachea, and unlike 
human and pig, not at all present in the bronchi 5,13,20. In human and pig, 
MUC5B forms bundles that extend horizontally in relation to the trachea 5,86,87, 
whereas the murine mucus has a mainly cloud-like appearance. Muc5ac is 
thought to be important for the formation of these clouds in mice 5,88. Epithelial 
cells are apically covered by vertically projecting stereocilia continuously 
The Role of FCGBP in Mucus 
12 
beating overlaying material in a cephalic direction, moving it through the 
epiglottis into the esophagus where it swallowed together with the bolus. This 
mediates clearance of the mucus separated from epithelium by the ASL. The 
cilia help remove microbes stuck to the mucus by interacting directly with the 
mucin or components of the innate or adaptive immune system, and also 
eliminate particles such as dust inhaled from the environment 89. It has been 
suggested that these MUC5B bundles or clouds are used to rake the surface, 
depending on the species (Fig. 7) 5.  
Figure 7. Illustrative Figures showing tracheal mucus transport in mouse, 
humans and pigs. The psuedostratified and ciliated epithelial cells (1) with 
embedded GCs (2) are shared histological features of human, pig and murine 
tracheas. As well the layer of air surface liquid (ASL) resting on top of the 
epithelium is shared (3). However, unique to the murine mucus organisation is 
cloud-like structures sweeping the surface (4) being transported in a cephalic 
direction, whereas human and pig airways have strands and bundles (5).  
Chronic inflammatory conditions of the lung are most often associated with 
dysregulation or impairment of this mucus clearence system. After many years 
of chronic inflammation of the airways driven by tobacco smoke, a person may 
have developed COPD. Fundamental pathological traits of COPD are 
bronchitis, fibrosis and emphysema, making it difficult for the patient not only 
to inhale, but also disrupts the gas exchange in the alveoli 90. In a review from 
2017 91, Barnes  summarises the known molecular details underlying COPD. 
Toxic gasses trigger macrophages to release pro-inflammatory cytokines such 
as IL-8 and TNFα mainly through a reactive oxygen species (ROS)-induced 
pathway. Epithelial cells are as well affected by ROS, and further take part in 
secretion of cytokines. Altogether, this leads to a subsequent release of 
proteases such as elastase and matrix metalloproteinases (MMPs) by 
neutrophils and alveolar macrophages, causing mucus hypersecretion and 
airway damage, with the latter resulting in emphysema. As the epithelium is 
damaged, the ciliary function is also compromised. This results in impaired 
mucociliary clearance, with the patient having to cough in order to remove 
Erik Ehrencrona 
13 
mucus. Activation of latent TGF-β appears to be one of the more important 
pathways for induction of fibrosis, being linked mainly to epithelial damage 91. 
Recent findings indicate that the mucus of COPD airways become more similar 
in organisation to that normally found in the colon (Fig. 8) 5,92.  
Figure 8. An illustraion of tracheal mucus organisation in health and under 
chronic inflammatory conditions. Under healthy conditions, the mucus bundles 
(1) are separated from the stereocilia by the ASL (2). In COPD, a stratified attached 
mucus layer (3) is formed, resembling that of the colon. 
In COPD, levels of MUC5AC increase 92,93, and proteins normally associated 
with GI mucus appear at high concentrations, resulting in the formation of a 
firmly attached mucus layer. Examples of these proteins include CLCA1, 
FCGBP and TFF3 92. While MUC5B is produced in the submucosal glands, 
MUC5AC is produced by the goblet cells embedded within the 
pseudostratified epithelial surface 93. It has therefore been suggested that 
similarly to the stomach, MUC5AC is used to mediate mucus attachment, here 
by providing an interface between the epithelial surface and MUC5B bundles 
that rake the epithelial surface 5.  
Chronic inflammation, static mucus and parenchymal damage are traits 
particular also to CF 94. Contemporary literature on CF is summarised in a 
review by Elborn CBE published in The Lancet in 2016 95. Mucus becomes 
highly static and the clearance system is impaired, generally believed to be 
mediated by increased mucus production and attachment to the epithelium. As 
well, further thickening of mucus by hyposecretion of water and thereby 
dehydration. Destruction of the lung is caused by chronic inflammation driven 
by microbes such as bacteria and fungi. The cystic fibrosis transmembrane 
receptor (CFTR) is a chloride channel important for the homeostasis of 
chloride and bicarbonate  95. A disrupted secretion of bicarbonate has in turn 
been linked to a reduced pH 95,96, and the speculation that absence of HCO3- 
could actually be the main factor resulting in a firmly attached mucus layer 5,97. 
The Role of FCGBP in Mucus 
14 
Similarly, asthma shows hypersecretion and retention of mucus, however 
parenchymal damage is not a hallmark of the disease 98.  
1.5 IgG Fc Gamma Binding Protein (FCGBP)  
1.5.1 Structure, function and evolutionary relatives 
The FCGBP gene is found on chromosome 19 in Homo sapiens and 7 in Mus 
musculus. First described by Kobayshi et al., it was found in the GCs of the 
colon 39,40. In addition to the larger airways, cystobiliar apparatus and cervix, 
IHC stainings revealed it to be expressed in submandibular glands and 
throughout the small intestine 40,42 . More recently, mass spectrometry (MS) 
analysis has showed that FCGBP can be found in the mucus of the small 
intestine and lower GI tract 29. It has also been found in saliva 63, parts of the 
reproductive tract, including seminal plasma 99 and cervical mucus under 
normal conditions 100. Peptides of FCGBP were also detected in the viscous 
cervical mucus plug generated during pregnancy 101. In addition to being 
expressed with the mucins, CLCA1 and TFF3, it is usually expressed alongside 
DMBT1 29,92. MUC2, CLCA1 and FCGBP expression levels seem to be 
equally important in mucus as label free quantitative MS shows almost 
equimolar concentrations of the proteins expressed and secreted in mucus 
throughout the small intestine and lower GI tract 29. Paralogues that are even 
more similar to FCGBP than mucins are the proteins Otogelin (OTOG) and α-
Tectorin 46, both being components of the tectorial membrane of the inner ear. 
Thus, evolutionary related proteins are found in various extracellular matrices 
throughout the body. Mutations in the vWDs of α-Tectorin usually results in 
partial or complete hearing impairment, meaning that it is important for the 
structure and transduction of kinetic energy in the tectorial membrane 102. In 
Xenopus tropicalis, the mucus coating of the tadpole epidermis contains 
Otogelin-like protein, Muc5E and possibly more than one version of FCGBP 
103, depending on reliability of the available sequence data. The Otogelin-like 
protein of Xenopus is instead richly O-glycosylated and seems to function as 
the main mucin, with the suggested name MucXS 104. The tadpoles have an 
adhesive organ called the cement gland that mainly secretes Muc5E, allowing 
the tadpoles to stick to surfaces. Due to similarities with the lung epithelium, 
as a result of the types of secreted proteins and function of the tissue as a gas 
exchange zone, Xenopus tropicalis is used as a model to study the lung mucus 
103.  
Another protein with domain composition similar to FCGBP is zonadhesin 
(ZAN), found in the acromion of spermatocytes, and believed to mediate 
binding to the zona pellucida during impregnation 105. Furthermore, there is 
Erik Ehrencrona 
15 
also SCO spondin (SSPO), a fiber forming protein that is secreted from 
subcommissural organs (SCO) into the cerebrospinal fluid of the ventricles of 
the brain 106. In Danio rerio, gene knockout of the SSPO orthologue leads to 
total absence of embryotic assembly of Reisner´s fiber in the central spinal 
canal, resulting in the body axis becoming bent and crooked during 
morphogenesis 107. Of all these proteins, FCGBP contains the highest number 
of vWDs without interruption of any other type of domain. Excluding FCGBP 
orthologues, there are actually no other known proteins containing this many 
repeating vWDs 108. Domain composition of FCGBP orthologues and 
paralogues is shown in figure 3. The X-ray crystal structure of von Willebrand 
factor D´D3 domain 109 and the MUC2 N-terminal cryo-EM structure 7 were 
recently published. These may prove to be useful tools for in silico predictions 
and aid other methods to solve the 3D-structure of FCGBP and related proteins. 
In general, other than protein-protein interactions, functions are not known for 
any of the FCGBP vWD domains. However, they have been shown to be 
involved in the biosynthesis and vesicle storage of the vWF 110,111 and assembly 
of MUC2 N-terminal polymers 7. vWD-assemblies (D-assemblies), are with 
some exceptions, primarily constituted by a vWD domain, a cysteine rich 
domain (C8), trypsin inhibitor-like domain (TIL) and an E domain (E). Human 
FCGBP has 12 D-assemblies and a free von Willebrand D domain at the C-
terminal end. Assemblies D3 to D11 are essentially 3 tandem repeats, or 
triplets, at the center of the human protein. Murine Fcgbp is truncated, having 
only one repeat, meaning a total 6 D-assemblies and a free C-terminal vWD 
domain (Fig. 9).  
 
 
The Role of FCGBP in Mucus 
16 
 
Figure 9. Domain compositions of FCGBP orthologues and paralogues.  FCGBP 
domain structure in human and mouse compared to the paralogous proteins SCO 
spondin, otogelin and zonadhesin. The architecture of the von Willebrand factor is 
included for comparison. Color-coded motifs or domain structures are also included in 
the figure, along with annotation for each.  
The domain composition of vWF and many of its interactions are summarised 
in a review by Haberichter in 2015 112. The first two vWDs together constitute 
a propeptide important for intracellular packing. D´D3 is site of covalent 
dimerisation and sequestering of factor VIII. This is followed by 3 vWAs, 
which together constitute a region that is susceptible to regulation by 
proteolysis and binding to endothelial collagen and platelets. The vWA#2 is 
cleaved by the ADAMTS-13 metalloprotease 113, but only when the protein is 
stretched by sheer stress, resulting in the target sequence being exposed, 
modulating the hemodynamics. The sheer-dependent A2-binding and cleavage 
is suggested to prevent clotting 114-116. vWA#1 and #3 can bind collagen, and 
the former can also bind the platelet surface glycoprotein 1Iβ (GPIIβ) 117. 
Erik Ehrencrona 
17 
Interestingly, the link between vWA#1 and Platelet GPIβ is also shear 
dependent, where increased flow instead promotes clotting 113. The vWAs are 
followed by the D4 assembly, later vWC#1-6, according the latest domain 
architecture consensus 117. vWC#4, called vWC#1 in older references, 
mediates additional binding to the platelet surface glycoproteins GPIIβ and 
GPIIIα. Finally, at the C-terminus there is a cysteine knot 112 that results in 
dimerisation via 3 disulphide bonds 118. As only interactions between vWD 
domains have been observed 7,109 it would be likely that FCGBP only interacts 
with itself in an intra- or intermolecular fashion or with other vWD containing 
molecules such as mucins. As calcium binding is important for mucin and vWF 
D-assembly binding and expansion 7,38,109, it is likely that a similar mechanism 
is used by FCGBP.  
1.5.2 Current insight on GDPH motifs – Processing and 
function 
In addition to being secreted together into mucus and having vWDs, a link 
between FCGBP and mucins, such as MUC2, MUC5AC and the membrane 
mucin MUC4, is the post-translational cleavage of vWDs 119-121. In humans, 
MUC2 and MUC5AC have one self-cleaving GDPH (Gly-Asp-Pro-His) motif 
in the most C-terminal vWD. Cleavage of MUC2 is mediated by a low pH 120, 
which also drives but is not necessary for autocatalytic processing of 
MUC5AC 119. MUC4 is as well GDPH cleaved, and as there are no cysteines 
crosslinking the domain, 121 the protein backbone is thought not to be 
covalently stable, allowing for the MUC4α peptide to dissociate from the β-
peptide, resulting in its release from the cell membrane 122. A similar  
mechanism has been detailed in MUC1, which is autocatalytically cleaved 
instead at a SEA domain, and not through a GDPH motif 123. Here, as the 
cleaved fragments lack disulphide bond tethering, mechanical forces can 
detach MUC1 from the membrane. This is thought to function as a clearance 
mechanism used against penetrating bacteria, and as a means for sensing of 
mechanical stresses 124. FCGBP carries a GDPH motif in all but the last 2 
terminal vWDs, making a total of 11 motifs. This is a hallmark of FCGBP, 
with no other protein, other than orthologues, having more than 2 motifs. Given 
the large number of motifs, these are likely very important for structure and 
function of FCGBP. 
 
Literature on GDPH motifs encompass many different areas of biology. For 
paralogues with vWDs more similar to those of FCGBP, ZAN has 2 GDPH 
motifs, whereas OTOG and SSPO have none. The motif is suggested to drive 
covalent crosslinking between FCGBP and other proteins through reactive 
Asp-anhydrides which are a result of the autocatalytic cleavage event. The 
described link between MUC2 and FCGBP was sensitive to neither a 
The Role of FCGBP in Mucus 
18 
chaotropic or reducing agents, suggesting a covalent isopeptide bond 44. 
Thuveson et al., has shown that the Asp-anhydrides generated from GDPH 
cleavage could be used to form a stable link between the H3 heavy chain and 
the pre-alpha inhibitor and the chondroitin sulfate of Bikunin 125,126. Similarly, 
for bacterial proteins, the FrpC toxin of Neisseria meningitidis, the ApxIVA of 
Actinobacillus pleuropneumoniae and the Nope1 protein of Bradyrhizobium 
japonicum, all have been shown to cleave through the GDPH motifs in the 
presence of calcium 127,128. For Nope1, the cleavage took place under both 
acidic and alkaline conditions and was not affected by the addition of protease 
inhibitors 127. In the case of FrpC and ApxIVA, it has been shown that the 
proteins assemble into covalently cross-linked multimers through isopeptide 
bonds between the Asp-anhydrides and lysine residues. It was also shown for 
FrpC that cleavage occurred in a wide pH range of 5.5 to 8.5 and was not 
affected by protease inhibitors 128. High molecular mass (Mr) complexes that 
were not present upon mutation of the GDPH motif were also reported for 
Nope1, but these were not shown to be covalent in nature as they were only 
seen in Native-PAGE and not SDS-PAGE  127. This could indicate the cleavage 
is important for correct conformation. 
 
The repulsive guidance molecule (RGM) family of proteins induce a diverse 
set of effects upon regulatory signaling through interaction with Neogenin-1 
(NEO1) at the cells surface 128,129, consequently, with mutations also leading to 
impairment of many different systems 129. A published crystal structure of the 
NEO1-RGMB complex revealed that the GDPH motif constitutes a loop 
between beta strands in the vWD domain of RGMB, and that upon cleavage 
the aspartate was accessible on the surface, whereas the proline was hidden 
inside the vWD domain. Bell et al. predicted that half of the, at the time 14, 
documented mutations in RGMC that caused loss of function, leading to iron 
retention in Juvenile Hemochromatosis (JHH), were located in proximity to 
the GDPH motif, further stressing the functional importance of this motif 130. 
Furthermore, the function of NEO1 is tightly linked to bone morphogenetic 
protein (BMP) signaling. NEO1 binds BMP, competing with BMP Endothelial 
cell precursor-derived regulators (BMP-ER) for binding the ligand. Therefore, 
NEO1 and BMP-ER together modulate BMP signalling. BMP-ER binds to the 
same BMP site as NEO1, and they together regulate iron metabolism 129. 
Interestingly, BMP-ER has its own GDPH motif. Here, the GDPH motif is 
found in a C-terminal vWD domain, and upon cleavage, most of these two 
fragments are still held together by a single disulphide bridge. Lockhart-Cairns 
et al. stressed the difference between BMP-ER and RGM, where RGM was 
not secreted due to misfolding upon mutation of the GDPH motif, but this was 
not the case for BMP-ER 131.  
 
Erik Ehrencrona 
19 
A protein BLAST (Basic Local Alignment Search Tool) search of the GDPH 
motif in Homo sapiens showed that both intra and extracellular proteins have 
this motif (unpublished). A strange example in this search is the 
unconventional Myosin-15 (MYO15) where the GDPH is found in the MyTH4 
1 tail region at position 2097-2100 (uniprot ID: Q9UKN7-1). Mutations of this 
protein have been shown to cause hearing loss. Mutations and deletions 
causing hearing loss without truncating the protein seem to be spread between 
amino acids 1253 and 3420. The described mutations closest to the motif can 
be found at position 2073, 2011, 2013 and 2014 132. However, it is unclear if 
the protein is even cleaved and what conditions could possibly drive cleavage. 
Current literature mentions no mutations associated with the motif. 
Furthermore, as the crystal structure of this protein has not been solved, not 
much can be said about the spatial localisation of the mutations in relation to 
the GDPH motif.  
Another example of GDPH loss of function is the sushi domain containing 2 
(SUSD2) protein which has been shown to be necessary for translocating 
Galectin-1 to the cell membrane 133, where it has a wide range of functions, 
involved in many different cellular functions 134. Galectin-1 must be in a 
reduced state in order to exert its extracellular effect on T-cells 135. Galectin-1 
lacks a signal sequence and it could be speculated that it is not to going through 
the ER and Golgi because these compartments promote an oxidised state in 
contrast to the cytosol that is mainly a reductive 136. The cleaved SUSD2 
peptides were also tethered by a single disulphide bridge, with mutation of 
these cysteines inhibiting cleavage, and thereby surface transport. pH did not 
affect GDPH cleavage of SUSD2, instead, it was suggested to be cleaved by 
serine proteases 133. 
All verified examples of cleaved GDPH motifs seem to be associated with 
proteins that reside in the extracellular environment. As Asp-anhydrides 
resulting from GDPH hydrolysis can react both with amino acids 128 and sugars 
125, it was suggested that FCGBP was covalently bound to the MUC2 
framework 44 through amide or ester bonds. The cleaved fragment of 
recombinant MUC5AC with a C-terminal aspartate showed reactivity with a 
primary amine through biotinylated ethylenediamine hydrobromide (B-EDA), 
indicating that the reactive group is actually preserved after cleavage 119. 
However, a GAPH mutant negative control was not used. Hypothetically, this 
reactive group should be highly unstable and could theoretically react with 
water, thereby being hydrolysed. Therefore, binding to other molecules should 
happen early during biosynthesis and protein maturation as is the case for the 
pre-alpha inhibitor with H3125 and FrpC toxin 128. In addition, it has also been 
shown that FCGBP forms reducible high Mr complexes not only with MUC2 
The Role of FCGBP in Mucus 
20 
44, but also TFF3 where heteromer formation is suggested to be mediated by 
disulphide bonds 43,63.  
1.5.3 The FCGBP_N sequence and link to helical gliding 
bacteria  
FCGBP in species ranging from vertebrates such as Homo sapiens to 
amphibians like Xenopus tropicalis contains a conserved N-terminal domain 
(FCGBP_N) described by phylogenetic and position-specific iterative basic 
local alignment search tool (PSI-BLAST) analysis by Lang et al. Most of this 
sequence cannot be found in Mus musculus or Rattus norvegicus, but it is found 
in closer relatives such as Mesocricetus auratus 108. Currently, the PFAM 
database lists this region as IgG-binding, even though the initial publication on 
the subject shows that the D-assemblies mediate IgG sequestering 41. Lang et 
al. showed that this sequence could be found early in evolution, long before 
the first antibodies emerged in cartilaginous fish 108,137. The earliest known 
FCGBP_N-vWD-C8 composition emerged in the Ctenophores but TIL 
domains are not included until the emergence of the Chordata phylum. 
Therefore, mucins are considered older than FCGBP with complete D- 
assemblies already found in Ctenophora 108. Furthermore, with the adaptive 
immune system being limited to vertebrates, it is further unlikely that 
FCGBP_N is involved in IgG sequestering as these sequences can also be 
found in several proteins produced in bacteria that utilise helical binding 
motility for movement, albeit without vWD, C8 or TIL domains. An example 
of this motility can be seen in Myxococcus xhantus, a widely studied biofilm-
producing bacteria which possesses the FCGBP_N domain 108.   
With the FCGBP and similar proteins are only found in eukaryotes 108, the link 
to prokaryotes through Myxococcus xhantus is interesting since researchers 
have compared them to multicellular organisms. The habitats and survival 
strategies are discussed by Muñoz-Dorado et al. in a review from 2016 138. The 
extracellular matrix (ECM), is considered a mesh-like network mainly 
composed of carbohydrates called exopolysaccharides (EPS), but there is also 
a protein component. In a biofilm, clusters of these bacteria form specialised 
regions that work symbiotically, much like cells in higher organisms 138. 
Interactions with EPS and between cells is believed to be partly mediated by 
type 4 pili, and the ECM is also thought to be littered with protein-filled 
vesicles 138,139. An organised system of movement, called social motility is 
coordinated by chemotaxis, with the kinetic pattern of the colony being 
compared to the rippling effect of water. However, when a single bacterium 
travels independently, called adventurous movement, a system of helical 
locomotion combined with focal adhesion, propels the bacteria forward 
Erik Ehrencrona 
21 
through rotational movement 138. There are no known pathogenic or 
opportunistic strains of the myxobacteria mentioned in modern literature. 
However, they are found in the fecal material of some animal, mainly 
herbivores 140,141. The FCGBP_N sequence is not linked to the biofilm 
formation itself. However, Lang et al. further noted that these domains were 
found together with conserved domain sequences associated with surface-
bound proteins, including domain of unknown function 11 (DUF11) and 
laminin_G3 of Algibacter lectus. Interestingly, Strongylocentrotus purpur has 
an autoproteolytic motif between the FCGBP_N and a transmembrane domain 
suggesting that the FCGBP_N sequence could be released to the environment 
upon membrane incorporation. It was also found that the sequence exists in a 
protein containing a SprB domain. Flavobacterium johnsoniae uses a helical 
gliding motility system were the SprB forms filaments on the surface that 
mediate focal adhesion to surfaces 108,142. The adoption the FCGBP_N 
sequence in higher organisms might be linked to shaping and controlling 
mucosal colonisation of myxococcales or similar types of bacteria encountered 
in soil-based food sources. This structure could be binding EPS found within 
the biofilm and tether it the protective mucus layers of the gut, resulting in the 
bacterial films being cleared out with the stool. Contrarily, it is also possible 
that this sequence emerged in higher organisms but was adopted by the lower 
organisms. 
1.5.4 Links between FCGBP and disease 
Currently function of FCGBP upregulation in disease is not well understood. 
As was previously presented, FCGBP is expressed at high levels during COPD 
where airway mucus function and morphology become more similar to colonic 
mucus, showing a firmly attached mucus layer 92. In human bronchoalveolar 
lavage (BALF) from CFTR patients, there are increased levels of FCGBP but 
it is not contained within the 99 % confidence interval 143. FCGBP is also one 
of the main constituents in the highly viscous mucus material found in the 
gallbladders of dogs with mucocele, which further highlights its importance 
for the rheological properties of the mucus 144. In an article from 2014 70, 
Schwartz suggested that copy-number variations is linked to FCGBP 
protection against human immunodeficiency virus 1 (HIV-1) in cervical 
mucosa of a specific group of seronegative HIV-exposed women, with the 
argument that additional IgG binding units in FCGBP enhances the IgG 
sequestering and immobilisation of HIV virus particles within the mucus 
network 70. In relation to cancer, malignancies can be associated with both 
down and upregulation of FCGBP. Downregulation is observed in CRC 145 and 
thyroid cancer (TC) 146. In contrast, ovarian cancer (OC) and head and neck 
squamous cell cancer (HNSCC) show upregulation of FCGBP 147,148. The 
The Role of FCGBP in Mucus 
22 
upregulation in HNSCC is hypothesised to be caused by Human 
Papillomavirus (HPV) infection-associated gain of chromosome 19, the 
chromosome where the FCGBP gene is encoded 147. Many of the published 
studies utilise transcriptomic data, however a small number of proteomic 
studies have been published. A study combining label-free proteomics with 
transcriptomic data from a public database reports that stage 2 CRC has a 
strong down-regulation of FCGBP on both protein and mRNA-level 145. 
Another proteomic study suggests that FCGBP could be used as a marker to 
differentiate non-mucinous neoplastic cysts from other cysts in the pancreas 
149.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Erik Ehrencrona 
23 
2 AIM 
2.1 General Aims 
The aim of this thesis is to study the structural features of FCGBP, find possible 
interactors, and determine the importance of FCGBP for the integrity of the 
mucus barrier found in small and large intestine, and airways during disease. 
Furthermore, this thesis aims to generate new models for studying FCGBP, 
and map which cell types secrete FCGBP and the mechanisms of secretion. 
2.2 Specific aims 
Paper I – Map the extent of GDPH cleavage in FCGBP and determine its 
effect on the integrity of the protein. Study FCGBP expression in vivo and 
perform quantitative analysis of the covalent interaction with MUC2 to assess 
its physiological significance. Reproduce and reassess IgG Fc binding in vitro 
using purified proteins. 
Paper II – Study FCGBP function in a murine COPD model, correlating 
molecular and ultrastructural data from both in vitro and in vivo experiments, 
to ex vivo and in vivo phenotypes. 
Paper III – Utilise in vitro approaches to map the interactions driving 
homomeric oligomerisation of FCGBP and study its 3D-structure using the 
murine orthologue as a model. 
Paper IV – Combine in vitro, in vivo and ex vivo approaches to study FCGBP 
ultrastructures and function in the GI tract focusing mainly on the colon. In 
addition, explore the function of the FCGBP N-terminal sequence in human, 
and uncover why parts of the sequence is missing in murine Fcgbp. 
 
 
 
The Role of FCGBP in Mucus 
24 
3 CONTRIBUTIONS 
Paper I – I generated clones for recombinant proteins used for anti-serum 
generation. I was strongly involved in setting up, analysing the experiments 
and writing the paper. I was also highly involved in obtaining and setting up 
the Fcgbp-/- mouse strain, in addition to extracting and analysing the samples. 
I set up and performed purification of proteins and generated stable clones, 
performed electrophoresis, Western blot and analysed FCGBP content of 
bands through in gel digestion and MS, with data analysis. Furthermore, I 
detected peptides verifying the predicted signal sequences along with peptides 
from GDPH cleaved fragments. I did EndoH treatment of lysates from stable 
clones and performed sample preparation and label-free MS quantification of 
mucus proteins to assess covalent binding between FCGBP and MUC2. I also 
set up and performed binding assays with FCGBP and IgG. Finally, I 
performed the IHC experiments and analysis. 
Paper II – I was involved in intranasal treatment of mice with elastase and 
extraction of tissue. I performed biochemical studies of in vivo and in vitro 
protein, including protein purification. I took part in setting up IHC staining of 
FCGBP and image acquisition.  
Paper III – I cloned recombinant proteins and performed most protein 
purifications. I discovered the C-terminal cysteine dimer of FCGBP, 
performed the electrophoresis and size exclusion chromatography (SEC) 
experiments in addition to MS sample preparation and analysis. I did not 
perform the cryogenic electron microscopy (cryo-EM) and small angle x-ray 
scattering (SAXS) methods, but I got to observe and learn when my collegues 
performed these.  My in silico sequence analyses and predictions helped in the 
workflow for elucidating the Fcgbp structure.  
Paper IV – I designed and performed all experiments. I participated in 
measuring mucus thickness and designed the primer sequences. I also 
performed cDNA/DNA amplifications in addition to analysing the sequencing 
data.  
 
Erik Ehrencrona 
25 
4 METHODS AND MATERIAL 
4.1 Molecular structure analysis and binding experiments 
In this thesis, experiments were designed and analysed based on the uniprot 
FCGBP consensus sequences Q9Y6R7 and E9Q9C6, for human and mouse 
respectively. Structures, processing and Mr of in vitro and in vivo proteins were 
analysed by multiple approaches, including in silico methods. Both native and 
denaturing conditions were used for these studies. In silico sequence analysis 
was important for driving the project forward, as alignments and phylogenetic 
analyses enabled prediction of the vWD, C8 and TIL domain borders, along 
with determination of similarities between FCGBP orthologues and 
paralogues. The project pivoted towards in vitro analysis of FCGBP using the 
murine orthologue as a cornerstone, due to it being highly homogenous to the 
human variant (papers I-III), even though it is shorter and lacks tandem 
repeats at the center. N-terminals regions differed between species, and 
therefore a set of truncated N-terminal constructs based on both orthologues 
were used to find and study differences (papers I, IV).  
Laemmli SDS-PAGE gels were used to anticipate Mr and study post-
translational modifications (PTMs) both under reducing and non-reducing 
conditions 150 (papers I-IV). For analysis in absence of denaturing agents, 
Native-PAGE, developed by Schägger et al.151, was used to study non-covalent 
interactions (papers I-IV), both homomeric (papers II-IV) and heteromeric 
interactions as seen in the case of FCGBP and IgG Fc (paper I). 
Electrophoresis and Western blot analysis was performed on both in vivo and 
in vitro material, comparing electrophoretic migration of recombinant protein 
(papers I-IV) and protein expressed in colonic mucus (papers I, III), as well 
as in lung BALF (paper II). This was valuable also for insight to whether or 
not in vitro protein was processed and folded in a comparable means to in vivo 
FCGBP.  
Working with large proteins, heavily crosslinked by disulphides, and studying 
differences between oligomeric states under different oxidative states is 
challenging as the oxidised protein could be expected to migrate faster than 
reduced protein through polyacrylamide gels, due to lower friction arising from 
a more compact structure. As well, disulphide bonds can shuffle, forming new 
links within or between proteins, which can reduce the size of individual 
cleaved proteins, or create larger polymers by cross-linking. At a high pH, the 
disulphides will lose their protons, becoming reactive thiolates that can attack 
other disulphide bonds forming new links 152. Mucins are thought to be stored 
The Role of FCGBP in Mucus 
26 
at a low pH inside the vesicles of GCs 38. Such a pH would also be beneficial 
in preventing unwanted shuffling of cysteines. 
The Native-PAGE method seeks to study protein structures in absence of 
denaturants. Here, the issue of protein charge which varies greatly between 
proteins depending on isoelectric points (pIs) and pH is mitigated by adding 
negatively charged Coomassie G-250 dye 151. Using the NativeMark ladder 
from Invitrogen, complexes between 20 (soybean trypsin inhibitor) to 1236 
(pentameric IgM) kDa can be resolved in a 4-16% gradient gel. Hypothetically, 
Coomassie G-250 could interfere with electrostatic interactions, thereby 
masking some binding events and conformations. In support, SEC was also 
used for size anticipation and interpolation for recombinant proteins. Using the 
low and high Mr gel filtration calibration kits from General Electric 
Healthcare, sizes within the range of 6.5 (aprotinin) to 669 (thyroglobulin) kDa 
could be interpolated by linear regression analysis (papers II-III). The method 
grants full control of the solvent used for the proteins and thereby simplifies 
studies of molecular dynamics. Here, charge is not normally a factor but just 
as is the case for Native-PAGE, the shape of the molecule will affect the 
results. Both Native-PAGE and SEC methods are based on molecular mass 
markers that are globular, meaning that more fibrillar proteins will likely show 
an overestimation of the Mr. To study IgG sequestering in vitro, all these 
methods were combined to screen for co-migration or co-elution of IgG and 
recombinant FCGBP (paper I). 
In a collaborative study, SAXS (paper III) and cryo-EM (papers II-III) were 
used to study full-length recombinant murine Fcgbp. SAXS is useful as it 
provides information not only on the radius of gyration (Rg) and Mr, but as 
well the number of subunits in the complex. An envelope for the volume of the 
molecule can further give information on structural organisation and folding. 
It is also possible to perform the analysis using any buffer, allowing for studies 
of molecular dynamics 153. Cryo-EM allowed for structural determination of 
less flexible regions of the molecule (paper III), as well as analysis of 
polymeric structures in micrographs (papers II-III). This technique is 
generally great for studying structure of large proteins. Proteins can be plunge 
frozen using any buffer, therefore also allowing for characterisation of 
structural dynamics depending on solvent. From imaged cryo-EM 
micrographs, a vast amount of molecules from different angles are found. Even 
though many regions of a molecule might be flexible, which would make 
structural determination by X-ray crystallography impossible, many particles 
in the Cryo-EM micrographs will share a specific conformation at a given time, 
allowing for structure to be determined by software by specifically picking 
these out and constructing an envelope 154.  Early studies of Fcgbp ultra 
Erik Ehrencrona 
27 
structure was performed with a low-resolution Talos L120C (paper II), 
whereas the further analysis was performed using a Titan Krios G2 (paper 
III), both from Thermo Fisher Scientific. Results from all methods were 
combined to draw conclusions on the structure of FCGBP. 
4.2 Production of recombinant FCGBP and polyclonal 
antiserum 
The main method used for cloning of N and C-terminal human FCGBP fusion 
proteins was through restriction enzyme linearisation of vectors and PCR 
amplification of inserts with up to 15 bp overhangs complementary to target 
vector (papers I, III-IV). Inserts and vectors were fused together by 
isothermal assembly, using the Gibson assembly master mix (New England 
Biolabs) 155. For generating a D9-D11 vector, being the most similar to the one 
triplet found in the center of murine Fcgbp, two inserts were synthesised by 
GenScript and assembled into the psecTAG vector. The D9-D11 construct was 
not included in manuscripts as we did not manage to successfully express the 
protein in CHO cells.  
Due to the sequences being highly unique and differing the most from 
paralogous sequences, the FCGBP_N and C-terminal FCGBPD13 regions 
were recombinantly expressed, purified by histidine affinity chromatography 
and used to immunise rabbits for generation of a highly specific polyclonal 
antiserum [paper I]. After comparing MUC2 peptides used for antisera, it was 
found that the MUC2C3 antiserum 8, would likely cross-react with GDPH 
cleavage products from FCGBP. This MUC2 antiserum was therefore also 
used to study FCGBP processing in paper I. There existed also two 
commercial polyclonal antibodies from Atlas Antibodies. These are the 
HPA003564 and HPA003517. The first of these targeted the human N-terminal 
sequence shared with helical gliding bacteria, whereas the latter was less 
specific, targeting the D5, 8 and 11 assemblies of human FCGBP. A full 
description of these and the rest of the antibodies used to study human and 
murine FCGBP is found in paper I. 
A full-length murine Fcgbp expression vector was purchased from Origene as 
its sequence is in many parts similar to that of human FCGBP, and its 
expression easier to achieve in mammalian cell-lines due to a smaller size. 
However, in order to express the protein in adherent CHO cells, stable clones 
had to be generated. Transient expression in suspension-grown CHO-cells 
eventually resulted in good yields (paper I). Either affinity purification or ion 
exchange chromatography, sometimes followed by SEC were used to purify 
The Role of FCGBP in Mucus 
28 
the recombinant FCGBP proteins (papers I-IV). The low pIs of the FCGBP 
proteins facilitated anionic exchange chromatography, resulting in the proteins 
being found in the fractions eluted using high salt concentration. Bearing in 
mind that reactive internal anhydrides generated from putative GDPH cleavage 
should bind amine groups, HEPES and PBS were generally buffers used for 
storing the proteins, as Tris-HCl would potentially bind to the anhydrides. All 
FCGBP vectors used in this thesis are described in table 1 below. 
Table 1: Recombinant FCGBP expression vectors  
Vector Supplier Species Domains Sequence Tag Antiserum CHO 
Expression  
pcDNA3.
1 
Invitrogen Human N Q9Y6R7 
(#1-471) 
Myc, 
His 
Rabbit + 
psec-
TAG 
Invitrogen Human N-D2 Q9Y6R7 
(#1-1251) 
Myc, 
His 
No + 
psec-
TAG 
Invitrogen Human D9-D11 Q9Y6R7 
(#3604-
4865) 
Myc, 
His 
No - 
pcDNA3.
1 
Genescript Human D11-D12 Q9Y6R7 
(#4466-
5234) 
Myc, 
His 
No + 
psec-
TAG 
Invitrogen Human D12-D13 Q9Y6R7 
(#4856-
5405) 
Myc, 
His 
No + 
psec-
TAG 
Invitrogen Human D13 Q9Y6R7 
(#5235-
5405) 
Myc, 
His 
Rabbit + 
pCMV Origene Human N-D13 Q9Y6R7 
(#1-5405) 
Myc, 
DDK 
No - 
pcDNA3.
1 
Genescript Mouse N-D2 E9Q9C6 
(#27-835) 
Myc, 
His 
No + 
pCMV Origene Mouse N-D7 E9Q9C6 
(#1-2583) 
Myc, 
His 
No + 
 
 
Erik Ehrencrona 
29 
4.3 Animal experiments 
A major issue encountered in beginning of my work was that the initial murine 
knockout model available, the TF0744 from Taconic, had a gene-trapping 
cassette in intron 18, meaning a stop sequence prior to exon 19. With the Fcgbp 
gene having 20 exons, amino acids #1-2490 out of 2583 could theoretically 
still be expressed, producing a 262 out of 272 kDa protein. This was a misstake 
as it had been generated before the Fcgbp gene had been properly sequenced. 
Thus, it was deemed unsuitable for studies as Fcgbp consists of repeats of many 
similar domains, therefore greatly stalling the project. Instead, an Fcgbp-/- 
variant with a LacZ insertion found after exon 3 was purchased from the 
EUCOMM library (EM05780) [paper I]. This model can be regarded as a full 
Fcgbp-/- as exon 2 corresponds to the 1st vWD domain (visualised in Fig. 10), 
theoretically only resulting in a 12 kDa peptide. The EM05780 is the model 
referred to as Fcgbp-/- in papers I-IV of this thesis. Data from the truncated 
TF0744 variant is not included. 
Figure 10. An overview of the translated product length in murine Fcgbp-/- 
variants. The original TF0744 ends translation in the 7th vWD, whereas the 
EM05780 from the EUCOMM library ends translation in the first vWD, resulting 
a substantially truncated product. 
An ex vivo explant system for analysis of colonic mucus was used for studies 
(papers I, III-IV). Looking at barrier defects, unchallenged non-cohoused 
non-littermate WT and Fcgbp-/- mice were screened for phenotypes pertaining 
to mucus structure and organisation in the distal colon (DC). First introduced 
by Gustafsson et al. 51 micrometer-sized fluorescent carboxylate beads were 
added apically on the mucus to compare bead penetrability between Fcgbp-/- 
and WT controls. These beads are negatively charged to avoid binding to the 
acidic glycan structures of mucins. As well, fluorophore-labelled wheat germ 
agglutinin (WGA) and Ulex Europeus Agglutinin-1 (UEA1) lectins were used 
to stain and compare ultrastructures of mucus as described previously by 
Nyström et al. 55,156. Mucus expansion was tested over-time through 
measurement of the thickness between mucus coated by 10 µm polystyrene 
beads and the epithelium, using a conventional stereo microscope and a 45° 
tilted glass capillary needle, performed at 37 °C with fresh Krebs’s glucose 
buffer pumped basally (paper IV) 51.  
The Role of FCGBP in Mucus 
30 
Murine mucus for biochemical and MS analysis was extracted by directly 
scraping off the apical side of the tissue into PBS on a silicon-coated petri dish, 
whereas human mucus was extracted after 1 h growth in the mucus 
measurement chamber (papers I, III).  
Studies pertaining to lung was done in collaboration. Intranasal administration 
of elastase was used to induce a COPD-like condition 92 in both Fcgbp-/- and 
C57BL/6 mice, comparing them to naïve mice. Bronchoaleolar lavage fluid 
(BALF) was collected for MS and biochemical analyses. Airways were fixed 
and used for IHC experiments. Some mice were used for testing ciliary mucus 
transport velocity by a method previously described by Fakih et al. using an 
explant system 88 (paper II).  
4.4 IHC and imaging 
For in vivo IHC, paraffin-embedded sections from both co-housed littermates 
and non-cohoused non-littermates fixed in either Carnoy’s solution (papers I-
IV) or formaldehyde (papers II, IV) were studied. Images of lung (paper II) 
and GI tissue sections were obtained using a Nikon Eclipe E1000 (Nikon) 
epifluorescence microscope or a LSM-700 confocal system (Zeiss). The latter 
granted the ability to acquire z-stacks tracking FCGBP ultrastructures in mucus 
(papers II, IV).  
4.5 MS analysis 
In literature, proteomic analyses of secretions from different areas are often 
based on SDS-PAGE electrophoresis separation. However, this methodology 
is likely not always optimal for detecting mucins and other large proteins, 
depending also on oxidative state, with too large complexes not being able to 
enter gels with high polyacrylamide concentrations. Here, we relied on a mix 
of methods to study FCGBP and other mucus component (papers I-III). Three 
different instruments were used to analyse lysate and mucus from colon or lung 
BALF through in-gel digestions or total protein content analysis by filter-aided 
sample preparation (FASP) protocols and label-free quantification.  
The instruments used here included LTQ Orbitrap XL, Q-Exactive HF and Q-
Exactive HFX, all from Thermofisher Scientific. Mascot V. 2.6.0. (Matrix 
Science) (papers I, III), PEAKS 2017 (Bioinformtics Solution Inc) (paper I) 
and MaxQuant V. 1.5.7.4 157 (papers I-II) were programs used to analyse the 
data. In order to assess the portion total FCGBP peptides coming from the 
different GDPH cleavage products, thereby assessing which fragment the 
Erik Ehrencrona 
31 
peptides belonged to, manual processing was required (papers I, III). When 
studying covalent cross-linking between Fcgbp and Muc2 in presence of 
GuHCl at pH 8, as was done in paper I, N-Ethylmalemide (NEM) was added 
to avoid shuffling of disulphides by binding and neutrallising free thiolate 
groups 64. 
4.6  Statistics 
All statistical test and presented diagrams were generated using GraphPad 
Prism version 8 (Graphpad). Both parametric and non-parametric tests were 
performed for independent groups. The former included students T-test (paper 
IV) and the latter Mann Whitney-U test (papers I-II) or Kruskal-Wallis 
(paper II). A p < 0.05 was set as a threshold for statistical significance. 
Parametric tests were used for comparing mucus thickness as data follows 
normal distribution 54 (Paper IV). However, for the rest of the statistical 
analyses, we used non-parametric tests because of small sample sizes and lack 
of knowledge on variance and normal distribution (Papers I, II).  
 
 
 
 
 
 
 
 
 
 
 
 
The Role of FCGBP in Mucus 
32 
 
5 RESULTS AND DISCUSSION 
5.1 Complete hydrolysis of FCGBP GDPH Motifs 
and no binding to IgG Fc or Muc2 (Paper I) 
To set the direction for exploratory studies of the FCGBP protein, descriptive 
work examining and further scrutinising known and hypothetical aspects of 
function, expression and structure, was performed as summarised in this paper. 
A main focus of this paper was to study the many GDPH motifs which are the 
hallmark of the FCGBP protein. Although potential cleavages have been 
described previously 43,44, no extensive study has been made where the 
cleavage of the full protein has been examined. It was found that all GDPH 
motifs were cleaved in vivo, studying both human and murine FCGBP 
orthologues by electrophoresis and MS analyses of both cellular and secreted 
material. Mutating the GDPH sequence to GAPH in recombinant truncated 
human N-D2 also showed that the Asp was necessary for cleavage, but not for 
secretion of the protein. In contrast, absence of GDPH cleavage blocks 
extracellular translocation of RGM 129 and SSDO2 133.  Working with 
recombinant murine Fcgbp revealed that intracellular GDPH cleaved 
fragments were sensitive to EndoH treatment, showing that the cleavage occurs 
in the endoplasmic reticulum. Furthermore, in silico modelling and MS 
analysis of recombinant proteins suggested that the reactive Asp-anhydrides 
resulting from GDPH cleavage can be hydrolysed. The fact that the GDPH 
motif is located within a non-exposed region of the domain structure, argues 
against the hypothesis that anhydrides could be exposed following cleavage 
and be involved in crosslinking FCGBP to other proteins such as MUC2 44. In 
support, quantitative in vivo approaches revealed murine Fcgbp to be mostly 
soluble in GuHCl, whereas Muc2 was highly insoluble. Hardly any Fcgbp was 
found upon MS-analysis of the insoluble Muc2 band separated in AgPAGE, 
arguing that a covalent link between MUC2 and FCGBP is not physiologically 
relevant. 
For both the human and mouse FCGBP orthologue, GDPH cleaved fragments 
were still tethered by disulphide bonds. While MUC1 is cleaved in the SEA 
domain 123 and MUC4 by GDPH 121, none of these are covalently stable. In 
contrast, the disulphide bonds of FCGBP stabilised the protein backbone 
otherwise thought to be held together by two beta strands. However, these 
bonds could still be the weakest covalent links within the FCGBP structure, as 
Erik Ehrencrona 
33 
disulphides can be placed in different angles and are subjected to varying 
degrees of axial tension within different molecules. This affects how much 
energy is needed to break the bonds 158, which could be of relevance for 
FCGBP, resulting in a controlled dissociation and detachment of mucus in 
response to heavy sheer stress. In silico alignments and modelling showed that 
only a disulphide bridge stabilises each GDPH cleaved vWD of FCGBP, 
similar to what has been described for SUSD2 133, and BMPER 131. One of the 
cysteines in each disulphide holding together GDPH cleaved fragments is also 
part of a CXXC motif. CXXC motifs are mostly found in redox proteins such 
as thioredoxin, and they are thought to lower the redox potential, allowing for 
reducing agents to attack and reduce disulphide bonds 159. It is possible that 
having this motif would allow for the disulphides to be more sensitive to a 
reducing environment. The human defensin HD1B is suggested to be activated 
by bacteria producing a reducing environment inside the intestinal lumen 160, 
and it is thought that the mucin networks also can be dissolved by sulphide-
producing bacteria, not only by breaking the intermolecular links, but also the 
intramolecular bonds that protect against proteolytic degradation 161,162. It 
could therefore be speculated that a reductive environment generated by a high 
bacterial load in the OML of the colon could also cause the FCGBP to break, 
thereby driving dissociation and detachment of mucus.  
In vitro, the described binding between IgG and FCGBP 39-42 was tested under 
native conditions. IgG sequestering by FCGBP could not be detected in either 
Native-PAGE or SEC, arguing against this function, touted in literature as 
being of primary importance, by which the protein has also been named. 
However, with recent progress in studies on structure and function of similar 
proteins containing vWDs, a clear link to IgG sequestering has not really been 
indicated. 
Additionally, MS analyses revealed the peptides for the signal sequences of 
murine and human orthologues, which would prove useful in future cloning 
endeavors.  
 
 
 
 
 
The Role of FCGBP in Mucus 
34 
 
5.2 Murine Fcgbp polymers drive mucus 
attachment in an elastase COPD model 
(Paper II) 
With Fcgbp not normally being expressed in the murine lung, the use of an 
elastase induced model of inflammation helped partially elucidate its function 
in the organisation of the mucus layer covering the tracheal surface under 
COPD-like conditions 92. It was found that for elastase treated mice with Fcgbp 
absent in BALF, there was less retention of mucus after washing and 
performing BALF extractions. Epithelial surfaces in the airways were less 
covered by attached mucus, and there was less airway obstruction in general. 
IHC stainings using the αFCGBP-D13 serum revealed dense trabecular Fcgbp 
structures in the mucus that did not mix with UEA1-stained material, thereby 
possibly devoid of Muc5b and Muc5ac mucins. However, Fcgbp trabeculi 
were still embedded within the mucinous materials, and they also formed a 
characteristic cap-like formation covering the stratified mucus. Such large 
structures could suggest that FCGBP is able to form linear polymers, and that 
there are also lateral interactions. Ex vivo assays revealed that elastase treated 
Fcgbp-/- mice had significantly higher mucus clearance transport velocity 
mediated by beating cilia. It has been speculated that MUC5AC is an important 
mediator of mucus attachment in the lungs, however Muc5ac-/- mice still 
displayed mucus attachment in the same ex vivo setup 88. This suggests that 
rather than MUC5AC, FCGBP could be the key mediator of mucus attachment 
in the airways during disease, and thereby drive mucus plugging in COPD, 
asthma and CF. Elastase treated Fcgbp-/- mice also showed less parecnhymal 
inflammation, suggsting that abberant FCGBP production leading to mucus 
retention drives the inflammation in COPD, possibly indirectly by 
accumulation of bacteria.  
The molecular details of the FCGBP superstructures were investigated. Size 
analyses using recombinant full-length murine Fcgbp showed a band close to 
460 kDa in electrophoresis with SDS but without reducing agent. Proteins 
crosslinked by disulphides tend to migrate faster in SDS-PAGE in an oxidised 
states, and as the Fcgbp has a predicted size of 275 kDa, it could be argued that 
the band detected is covalent dimer. This band came from protein fractions 
separated by a Superose 6 increase column in a peak that had lower column-
retention than thyroglobulin.  The thyroglobulin molecular mass standard is 
669 kDa, meaning that the size of the native protein might correspond to 
complexes even larger than a dimer, where non-covalent interactions drive 
Erik Ehrencrona 
35 
further polymerisation. Native-PAGE analysis as well revealed a size of at 
least 720 kDa, strengthening previous findings. Both the SDS-PAGE and 
Native-PAGE bands detected in vitro, were confirmed in vivo when analysing 
BALF from elastase treated mice using Western blot and the αFCGBP-D13 
serum from paper I. This further suggested that the recombinant protein was 
folded and processed similarly to the in vivo protein expressed in the airways. 
Low-resolution cryo-EM images of the recombinant murine orthologue 
revealed linear polymers of varying length, likely mediated by transient 
interactions as such complexes were not indicated by neither Native-PAGE nor 
SEC.  
In all, findings in this paper suggest that FCGBP drives retention of airway 
mucus through a combination of homo and heteromeric interactions, with the 
former having a covalent component. Pharmacological compounds targeting 
FCGBP synthesis or function could therefore decrease mucus retention and 
inflammation. 
5.3 FCGBP forms a C-terminal disulphide dimer 
and a highly globular quarternary structure 
involving many D modules (Paper III) 
In paper III, the homomeric polymerisation of FCGBP was further studied. 
The base model for various interactions mapped by recombinant proteins is 
summarised in figure 11 on page 36. The same 720 kDa band observed using 
Native-PAGE in vitro and in BALF from elastase treated mice (paper II) was 
also found in mucus from murine distal colon. SAXS analysis of recombinant 
full-length Fcgbp also showed a Mr in the same range. However, further data 
analysis suggested that the complex was comprised of only two subunits. 
Analysing briefly reduced and alkylated murine protein in Native-PAGE 
showed a band that had migrated faster in the gel than non-reduced material. 
MS analyses of this band suggested is was actually a reduced monomer that 
had not yet been dissociated into individual GDPH fragments. The result 
suggested that the dimer was at least partly mediated by disulphide bonds. 
Expression of a truncated C-terminal construct, human FCGBPD11D12, 
corresponding to D5D6 of murine Fcgbp, did not form a dimer (Fig. 11). 
However, both the human D12D13 and D13 recombinant proteins showed 
reducible dimers using SDS-PAGE (Fig. 11), therefore mediated by the 
terminal D13 domain. In silico alignments showed that the cysteine 
configuration of the terminal vWD was conserved in both the human and 
murine orthologue. Further modelling and MS analysis of the recombinant D13 
The Role of FCGBP in Mucus 
36 
protein suggested that subunits were organised in an antiparallel manner (Fig. 
11). 
Through cryo-EM, using micrographs with full-length recombinant Fcgbp, it 
was possible to determine the structure of the sequence between D4 and D7. It 
formed a convoluted, or globular, antiparallel dimeric interface stabilised by 
inter-molecular bonds between D7´-D7 and D7´ to D4, and vice versa (Fig. 11, 
subunit Fcgbp´ shown in red, other subunit in green), with partially de-
convoluted conformations also observed in micrographs. We chose to call the 
complete quaternary structure of the C-terminal interface the “core complex”. 
The cysteine dimer was also found in the murine structure, and the resolved 
area was fully GDPH-cleaved. Due to flexibility, the structure of the sequence 
spanning from the N-terminus to the end of the D3-assembly was not resolved 
at his time. Superimposing the SAXS envelope with the cryo-EM structure 
suggested that N-terminals were protruding like flexible arms from the core-
complex (Fig. 11). Native-PAGE migration and SEC Mr interpolation further 
suggested a Mr closer to a tetramer for the human D12D13 (Fig. 11).  
Figure 11. An illustrative figure visuallising interactions and structures found 
using recombinant FCGBP proteins. The hD11D12, hD12D13 and hD13 are 
human recombinant truncates of the the FCGBP C-terminus, whereas Fcgbp is the 
recombinant full-length murine protein. The figure includes description of methods 
used for analysis. The motifs, bonds and domains included in the image have a 
description and annotation. The different domains, bonds and and regions of interest 
are color-coded and annotated. When D-assemblies are forming a dimer or tetramer, 
both red and green colors are used to represent the domains of a specific subunit. 
The illustration of the murine Fcgbp 3D-structure down on the right has pink 
triangles marking the flexibility of the N´-D3 sequences. 
Erik Ehrencrona 
37 
It was speculated that partially de-convoluted FCGBP C-terminal interfaces 
could re-assemble into even bigger complexes. Using both conformations of 
murine Fcgbp detected in cryo-EM to search for filaments in high-resolution 
micrographs again revealed linear structures of varying length, as in paper II. 
With mucus attachment being a proposed function for FCGBP, it could further 
be speculated that the core complex can contribute to the elastic properties of 
mucus. The murine Fcgbp structure resembles a spring feather, likely being 
able to store and release kinetic energy, as there are no known covalent 
crosslinks that locks the structure internally. The transient nature of Fcgbp 
polymerisation likely allows for a much more dynamic and shapable material 
than what mucins can provide, as the linear polymers formed by MUC2 are 
stabilised by covalent interfaces at both termini 6,7. However, in addition to not 
having O-glycans causing steric hindrances, great amount of D-assemblies in 
FCGBP, compared to the mucins, likely also contributes to far more 
possibilities in how FCGBP can assemble into larger complexes.  It could be 
that shear stress modulates interactions with other structures in a similar 
manner as vWF, albeit without involvement of vWA domain, similarly 
resulting in binding or susceptibility to proteolytic cleavage 113, thereby 
affecting mucus structural properties and clearance.   
5.4 FCGBP is important for colonic mucus 
structure and the N-terminal region genetically 
destroyed in mice forms non-covalent dimers 
(Paper IV) 
With new knowledge on FCGBP structure (papers I-III), by in silico and in 
vitro work we tried to elucidate all the main differences in the amino acid 
sequences between the human and murine orthologues, as it was important to 
determine how much of the structural information was actually transferable 
between orthologues. Furthermore, we also wanted to closer study the protein 
function in its main sites of expression, the small intestine and lower GI tract 
29. 
The illusive FCGBP_N domain described by Lang et al.108 was further 
investigated. It was found that the sequence unique to humans, here named 
FCGBP_N1 (N1), was limited to only one exon that was missing in mouse. 
cDNA amplification and sequencing did not reveal any new data compared to 
the mouse Fcgbp sequence already published. However, alignments of the 
murine genomic Fcgbp sequence with cDNA from species with complete N-
terminal sequences showed that murine N1 had been genetically destroyed 
through several deletions and insertions. The FCGBP_N2 sequence (N2), 
The Role of FCGBP in Mucus 
38 
being the N-terminal sequence found the D1 assembly still shared between 
humans and mice, was originally called FCGBP_N´ (paper I). This new 
architecture nomenclature was deemed to be better as it is based on N-terminal 
positioning in human FCGBP. Alignments of all FCGBP D-assemblies with 
the N2 sequence showed that this sequence was actually repeated and found 
after the E domain in each D-assembly of both human and mouse FCGBP. In 
relation to the suggested flexible D3 hinge-region (paper III), the N2 sequence 
was located N-terminally. Both the longer human N1 and shorter N2 sequence 
shared with mouse are linked to helical gliding bacteria such as the 
myxococcales 108.  
Another key inter-species difference identified was that vWD1 domain of 
human FCGBP had an unpaired cysteine close to the previously described 
CXXC motif, whereas murine Fcgbp had an extra cysteine in TIL1. The 
proximity to a CXXC motif could indicate involvement in disulphide shuffling. 
As found in paper I, one of the cysteines in the CXXC motif is believed to be 
part of the disulphide bridge tethering the GDPH fragments of the N-terminus 
and D1. Here, an unpaired cysteine seemed to cause a small proportion of the 
shorter recombinant human (FCGBPN-D2) and murine (FcgbpN-D2) N-
terminal proteins to form cysteine dimers in SDS-PAGE. However, mostly 
monomers were observed under native conditions, suggesting that these SDS-
PAGE findings might be an artefact. Interestingly, expressing the complete 
human N-terminal sequence in absence of D-assemblies, composed of N1 and 
N2 (FCGBP_N1-2), showed mostly non-covalent dimers, suggesting that the 
assemblies block an interaction under normal circumstances. 
As was previously found (paper I), murine DC has an Fcgbp expression 
pattern similar to human sigmoid colon where FCGBP expression is seen in all 
GCs of the crypts. Similarly, it was observed that expression of FCGBP could 
be found along most of the ileal crypts of both species. However, expression 
varies when comparing murine proximal colon (PC) and human ascending 
colon. In human aschending colon, FCGBP can be seen expressed at all places 
of the crypts, but it is only found near the top of the crypt in mouse. Co-staining 
of Carnoy-fixed sections using UEA1 lectin, MUC2 and FCGBP antibodies 
suggested that FCGBP is expressed in all goblet cells, but that the proteins are 
segregated upon secretion. Fcgbp forms dense elongated structures in the 
colonic IML, similar to those found in lung (paper II). Stainings of PFA-fixed 
sections suggested that the proteins can be packed in the same vesicles. The 
sections also reveal stronger Muc2 signal in Fcgbp-/- compared to WT, 
indicating that Muc2 could be upregulated in these mice.  
Erik Ehrencrona 
39 
Using the MUC2C3 antibody, Johansson et al. first described a structure in the 
innermost part of the colonic IML, stained differently 8. Our stainings further 
indicate cross-reactivity (paper I) with Fcgbp and that these differently stained 
structures could partly be composed of Fcgbp. Similar formations are not 
present in Fcgbp-/-, and these structures can also be identified using the specific 
αFCGBP-D13 serum on WT sections. Z-stacks of UEA1 and FCGBP co-
stained colons shows dense fiber-like and trabecular structures secreted from 
GCs, similar to those observed in tracheas of elastase treated mice (paper II). 
The Fcgbp and mucinous materials appear, in agreement, segregated and do 
not mix. Yet, Fcgbp structures are still embedded as a mesh within the UEA1 
stained material. These dense structures are long and usually retain their link 
to the cells from which they were secreted suggesting that they provide a focal 
point for attachment.  
The absence of these Fcgbp structures appears to negatively affect the 
organisation of mucus secreted by surface GCs ex vivo, stained by the WGA 
lectin. 1 h mucus growth-rate measurements showed a significantly higher 
expansion rate (p < 0.05) and more fragile mucus in Fcgbp-/-, further suggesting 
that Fcgbp is complicit in mucus organisation and clearance. However, the 
sample size (n) for the mucus measurements is small, consisting of only 4 in 
each group. Using fluorescent micrometer-sized carboxylate beads on top of 
mucus ex vivo did not show a penetrability phenotype for these mice, arguing 
against a disrupted barrier.  
Just like transglutaminases are thought to provide vertical stabilisation of the 
mucus layer by covalent bonds, making the mucus more insoluble 64 and likely 
promoting attachment, FCGBP could be providing another dimension to 
vertical stabilisation and attachment, by means of more dynamic non-covalent 
interactions. 
 
 
 
 
 
 
The Role of FCGBP in Mucus 
40 
 
 
6  CONCLUSION 
In conclusion, the murine Fcgbp orthologue is an excellent model for studying 
FCGBP both in vivo and in vitro as the D-assemblies share a high sequence 
homology (paper I, IV) with the human protein. The murine orthologue is 
shorter, making it ideal for in vitro expression (Paper I-III). As it does not 
have any additional domains compared to human FCGBP, data on structure 
and function should be directly transferable to the human protein. However, 
human FCGBP has a longer N-terminal sequence, here dubbed FCGBP_N1, 
not found in the murine orthologue. This sequence is genetically lost in 
evolution and just like the FCGBP_N2 sequence shared with mouse, it is linked 
to helical gliding bacteria 108.  Therefore, the full human N-terminus must be 
studied by other means.  In vitro analyses suggest that the human N-terminus 
can form non-covalent dimers, a function that is normally blocked or 
modulated by the D-assemblies (paper IV). Fcgbp forms long discrete 
trabecular or network-like super structures in mucus, embedded within mucin 
material but not necessarily mixing. FCGBP structures, most likely 
homopolymers, are sometimes seen directly connected to GCs from which they 
were secreted, thereby resembling mucus anchors. Both in vivo and ex vivo 
data further suggest that FCGBP is important for the structural organisation of 
the mucus layers. Interestingly, it was also found to be an important mediator 
of mucus attachment in lungs under diseased conditions (papers II, IV). In 
vitro studies support the hypothesis that FCGBP assembles into 
homopolymers. The base variant of Fcgbp structure is a C-terminal covalent 
dimer, with D4 to D7 of both subunits forming a non-covalent complex that 
could promote elastic properties. Linear complexes of varying length were also 
detected suggesting even larger dynamic complexes can be formed. These 
were mediated by non-covalent interactions (papers II-III), enabling much 
more dynamic structures in mucus compared to MUC2 polymers crosslinked 
by covalent bonds 6,7.  
Finally, both human and murine FCGBP is fully cleaved through its many 
GDPH motifs. These cleavages were hypothesised to drive covalent 
crosslinking between FCGBP and MUC2, by generating a reactive Asp-
anhydride. However, no physiologically significant covalent links between 
these proteins were found, suggesting that such interactions would, if existed, 
instead likely be mediated by transient non-covalent interactions. Each GDPH 
Erik Ehrencrona 
41 
cleaved fragment is tethered by a disulphide bridge (Paper I), and an altered 
redox environment driven by the microbiota could therefore disassemble 
FCGBP fragments, thereby increasing mucus clearance or mucus detachment.  
7 FUTURE PERSPECTIVES 
Mucus clearance is likely highly important in the relationship between host 
and bacteria as is exemplified in CF were increased mucus retention drives 
inflammation in both the lungs 94 and small intestine 163. Clearence mechanisms 
pertaining to MUC2 have been described. Both MEP1β and CLCA1 can cleave 
the MUC2 protein backbone, thereby modulating mucus clearance 53,54. As 
structural organisation and attachment of mucus are here the proposed 
functions of FCGBP (paper II, IV), the general co-expression of FCGBP and 
CLCA1 29,92 is interesting. It is possible that these two proteins are important 
in modulating the attachment and expansion of intestinal mucus, and that a 
similar relationship might occur in the airways during COPD. The murine 
CLCA1 orthologue is however not upregulated in elastase treated Fcgbp-/- 
mice (paper II). Still, it could be speculated that Clca1-/- mice might show 
increased mucus attachment and reduced mucus transport velocity in airways, 
as a consequence of this relationship. In vitro studies where recombinant 
proteins are mixed could show if FCGBP can directly interact with or be 
cleaved by CLCA1.  
Essentially, Paper III is ready for submission, but it could benefit from more 
experiments that focus on the relationships between structure and function. 
Mixing recombinant FCGBP with growth medium from different anaerobic 
bacteria and analysing the protein processing in SDS-PAGE under non-
reducing conditions would indicate if bacteria are able to break the disulphide 
bonds that hold the GDPH cleaved fragments together. This way, GDPH 
motifs could be linked to structural alterations. Furthermore, similar to what 
has been previously carried out in relation to the MUC1 mucin 124, using atomic 
force spectroscopy on recombinant proteins that are either reduced, non-
reduced or have mutated GDPH motifs or cysteines, could show the axial 
forces needed to mechanically dissociate FCGBP. If a GDPH mutant is more 
stable than the non-mutated protein, then it would be shown that inter-fragment 
disulphide bridges are actually the weakest covalent links stabilising the 
FCGBP protein.  With the murine Fcgbp missing the human FCGBP_N1 
sequence 108 (paper IV), the cryo-EM structure of murine Fcgbp should be 
complemented with studies of recombinant human N-terminal protein. Some 
electrophoresis data from paper IV showing the migration of human and 
murine N-terminal recombinant proteins could instead be moved here, and 
The Role of FCGBP in Mucus 
42 
complemented with additional cryo-EM analysis. Alternatively, a truncated 
human protein lacking the first two of the central D-assembly triplets (D3-D8) 
could be cloned. This would likely be easy to express in CHO cells just like 
the murine variant of the full-length protein (papers I-III). The paper could be 
further complemented with experiments studying the function of the N-
terminal sequence unique to human FCGBP. Using ELISA, the human 
recombinant N-terminus could be used in screening for binding to different 
types of exopolysaccharides or other molecules associated with biofilm 
formation, including biofilms from helical gliding bacteria. 
Paper IV is in need of more work before it is ready to be submitted for 
publication. Here, it would be interesting to further study FCGBP in small 
intestinal mucus. Ex vivo mucus attachment in Cftr-/-, WT and Fcgbp-/- mice 
could be compared, using previously described methods 53,82, also looking at 
protein cleavage by MEP1β and CLCA1, in addition to ion transport. It could 
be that not only MUC2 but also FCGBP is a mediator of mucus attachment. 
As Cftr-/- mice have more attached mucus ex vivo 53, it could be interesting to 
generate a Fcgbp-/- and Cftr-/- double knockout and and comparing the ex vivo 
mucus attachment of these with individual knockouts and WT mice, the double 
knockout model might reveal less mucus retention in the small intestine. 
Reduced attachment of mucus would then be mediate by a lack of Fcgbp. 
Creating such a strain should not be a problem since the Fcgbp gene is located 
on mouse chromosome 7, whereas the Cftr gene is found on chromosome 6. 
Calcium and pH-related molecular dynamics should also be further studied for 
recombinant proteins. This could be done in vitro by SEC, SAXS, cryo-EM, 
and ex vivo by adding bicarbonate to mucus on murine intestinal explants. As 
Fcgbp-/- mice thus far present few phenotypes (paper II, IV), it would be 
interesting to eventually challenge these with either DSS treatment 164 or 
Citrobacter rodentium infection 165 in order to further study the importance of 
FCGBP in inflammation and infection, as a link to inflammation was found in 
the airways (paper II).  
 
 
 
 
 
 
 
 
 
 
 
 
Erik Ehrencrona 
43 
 
8 ACKNOWLEDGEMENTS 
8.1 To collegues and collaborators 
Many people were involved in my PhD and I am very thankful for all of the 
input, guidance and asisstance that I received during this period. 
Some people invested more time in my PhD and I would like to give some 
extra credit to them. Thank you Pablo Gallego Alonso and Maria-José 
Garcia Bonete for teaching me methodology, involving yourselves in the 
FCGBP project and giving input on my writing. Without your involvment in 
the structural biology, experiment planning, and data analysis, my thesis would 
have been much more difficult. Thank you very much Frida Svensson for 
involving yourself in the project in general but especially in planning and 
assisting in animal experiments. Hope you come to Värmland! Thank you very 
much Ana-Maria Rodriguez Piñeiro for involving yourself and teaching me 
how to do mass spec, but also for involvment in the the writing of Paper I. 
Good luck with your new job! Thank you Christian Recktenwald for all 
invaluable input especially on the biochemistry part of my project. I hope you 
will do more teaching in the future as you have a lot of knowledge to share. 
Thank you Dalia Fakih for involving me in lung research. You will have great 
future at Astra! Thanks also to Beatriz Martinez-Abbad for your involvment 
with the animals. Good luck with your expanding familly and I hope we can 
have more dinner nights even though I won’t live in Gothenburg.  Thank you 
Sergio Trillo-Muyo for your involvement in the structural and biochemical 
experiments. You have many interesting stories and anecdotes to tell! Thank 
you also Sjoerd van Der Post for your involvement in GDPH and MS-related 
experiments. I hope we meet at Sunne vattenpark! Thank you very much 
Gunnar Hansson and Malin Johansson for your supervision and giving me 
the opportunity to do my PhD in your prestigious mucin collective. I hope there 
will be many more good years of mucus studies!  
There were many others involved in my PhD that were helpful and kind, 
teaching me methods and giving me input on my research. To Liisa Arike, I 
wish you and your expanding familly the best, hope the familly will keep 
getting bigger! Thank you Elena Layunta for fixing the Brady printer and 
showing me FISH. You are the best at mixing drinks! Jenny Gustafsson thank 
you for teaching me animal work and discussing my project. I wish you the 
best. Thank you Anna Ermund for the many discussions on FCGBP and the 
The Role of FCGBP in Mucus 
44 
many good laughs in the corridor. I wish you good fortune in the future! Thank 
you Brendan Dolan for teaching me methods, discussing experiments and 
proofreading my manuscripts. Good luck with your grand things! Thank you 
Hannah Schneider for helping in the lab. Have fun at Astra! Thanks to 
Thaher Pelasayeed for all the help in the lab and the many good hours of 
cleaning the cell culture lab. I hope you group will continue growing in the 
future! Thank you Sofia Jäverfält for the many good years. I am sure your 
thesis will turn out great! To Grete Raba, thank you for the many parties, hair 
discussions and secret fikas. I hope you will have a great future in Tallin!  
Thank you also Jack Sharpen for the many partys, I hope you wil have a great 
PhD! Thank you Elisabeth Nyström and George Birchenough for teaching 
me animal experiments and discussing the project. Hope you will publish even 
more prestigious papers in the future! Thank you Joakim Bergström, 
Karolina Sjöberg Jabbar and Lisbeth Ekholm Eklund for helping me early 
in the project. It was a pleasure working with you and I hope you are happy 
where you are now. Thank you also to Malin Bäckström, Elizabeth 
Thomson, and Richard Lymer from MPE, not only for producing nice 
batches of protein over the years, but also for teaching me methods and 
discussing results, especially early in my scientific career. It was nice working 
with you all! Thank you Karin Ahlman for your service in the lab and good 
maintenance of laboratory discipline. Very dangerous. I hope your retirement 
years are good! Thank you Suzan Fares for teaching me Danish culture, laws 
and language. Thank you Anandi Raijan for many years of wittiness and 
magic tricks. I hope you will buy a nice house in Malung! Thank you Gustaf 
Hellsén for being the strongest clinical connection maintained throughout my 
PhD years. You have both a great clinical and scientific career ahead of you. 
Thank you Ana Luis for the good music exchange in the lab. Good luck with 
your research! Thank you Melania Giorgetti for teaching me Italian culture, 
hand gestures and animal experiments. Hope you are enjoying a drink on the 
beach! To Mahadevan Venkitasubramani, I wish you a good, productive and 
long PhD! To Franscesco Suriano, I wish you a good time in Sweden and the 
mucin biology group! Thank you Jenny Persson for the good years. To Sarah 
Thulin, Kristina Johansson, Jesper Magnusson and Gudrun 
Ragnarsdottir, it was nice meeting you and I wish you the best for the future. 
8.2 Figures 
The icons cell-column-1 (Fig. 1-8), epithel-squamos-stratiedifed (Fig. 1), and 
gobelet-cell-2 (Fig. 1, 7-8) were altered for usage in the figures. They were 
taken from Servier Medical Arts (https://smart.servier.com/) and are licensed 
for free use under Creative Commons license (CC-BY 3.0 Unported 
https://creativecommons.org/licenses/by/3.0/). 
 45 
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