Purification, characterization and immunological studies of rat urinary proteins causing allergy in humans Second edition

Abstract

Rats are among the most frequently used laboratory animals and rat allergy constitutes a common occupational problem. Approximately 20-30% of the personnel engaged in work with laboratory animals show symptoms of allergy. These include rhinitis, conjunctivitis, urticaria and sometimes asthma, symptoms which usually develop during the first three years of exposure. The allergic reaction arises from direct contact with rat urine, or by exposure to airborne dusts originating from dried animal urine. Rat urine contains a complex mixture of proteins some of which are known to be allergenic, for example a2u-globulin. In urine from fertile male rats a2u-globulin is one of the most abundant protein. The primary aim of this thesis was to purify and identify the most potent allergens in rat urine. Knowledge about the biochemical and biological properties of allergenic proteins is of importance for the understanding of allergic diseases. Consequently, two other aims of this thesis were to optimize various assays for evaluation of the allergenicity of the identified allergens and to examine in some detail the IgE binding regions of the major allergen, Rat n 1.02, a2u-globulin. Rat urinary proteins were separated and purified by ultrafiltration through centrifugation and by chromatographic methods. The high resolving chromatographic purification procedures used involve molecular size separation by gel filtration followed by charge separation on an ion exchange column. The two purified dominating proteins were identified as different forms of the same parent protein, a2u-globulin, by amino-acid compositional and sequence analysis as well as mass spectrometry. Electrophoretic methods were also used to analyze the proteins with respect to relative molecular mass by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and isoelectric point was determined by isoelectric focusing. It was further proved that the urinary protein previously named pre-albumin had no amino acid sequence resemblance to prealbumin (transthyretin) present in rat serum. Additionally, the procedures reported here for isolation and studies of allergens can be used for examination of allergens from other sources. The allergenicity of the rat urinary proteins was studied with Western blotting using IgE antibodies in sera from allergic patients. A detection system utilizing chemiluminescence and a luminometer apparatus gave scanning curves of IgE binding and allowed arbitrary measurement of the amount of IgE bound to a certain urine protein. Specific patterns of IgE binding to the electrophoretically separated rat urinary proteins were shown for each individual. All allergic subjects studied had IgE antibodies binding to the major urinary protein, a2u-globulin. In most of the sera examined the IgE antibodies specifically binding to a2u-globulin were more abundant than such binding to other proteins. However, this protein was not always the most dominating allergen. Immunoblotting offered a good overview of the strongly varying individual IgE-binding patterns seen in sera from rat allergic subjects regarding both distribution and intensity of the binding. Overlapping octapeptides corresponding to the amino acid sequence of the major allergen Rat n 1.02 were synthesized on solid support and screened in parallel to pinpoint the IgE-binding regions of the protein using a modified ELISA procedure. Our results indicate the existence of linear IgE binding epitopes, mainly located towards the N-terminal and C-terminal parts of the protein, as recognized by IgE antibodies in the studied sera. The role of these short amino acid sequences in the allergic reaction and their appropriateness for immunotherapy calls for further investigation. In conclusion, this thesis has contributed to the isolation, identification and characterization of the allergens involved in a major occupational disease among people who work with laboratory animals, namely rat allergy.

Upp till 30% av personer som hanterar försöksdjur drabbas av allergiska symtom vilka kan relateras till kontakter med djur. Typiska symtom är ökat tårflöde, urtikaria och astma. Dessa utvecklas ofta inom tre år efter första exponeringen. Råttor är ett av det vanligaste försöksdjuren och deras urin innehåller en komplex blandning av olika proteiner. Speciellt hanråttors urin har visat sig innehålla allergiframkallande proteiner som t.ex. a2u-globulin. Den allergiska reaktionen kan uppkomma genom direkt kontakt med råtturin eller genom inandning av luftburna partiklar i form av damm som härrör från intorkad urin. I urin från unga fertila hanråttor är a2u-globulin det kvantitativt mest förekommande proteinet. Studiens syftet var att rena och identifiera de mest potenta allergenerna i råtturin och sedan studera deras allergenicitet med känsliga delvis nya metoder. Dessutom omfattade studien undersökningar av IgE bindande regioner på huvudallergenet, Rat n 1.02, a2u-globulin. Råtturinproteiner separerades och renades genom ultrafiltrering och högupplösande kromatografiska metoder. Storleksseparation utfördes genom gelfiltrering följt av uppdelning efter laddning på en jonbytarkolonn. Även isoelektrisk fokusering och SDS-polyakrylamidgelelektrofores användes för att analysera proteinerna. Två dominerande proteiner identifierades som olika varianter av samma moderprotein, a2u-globulin, genom aminosyraanalys och sekvenering samt masspektrometrisk bestämning. Härmed bevisades också att det protein som tidigare kallats pre-albumin inte hade någon aminosyrasekvensiell likhet med prealbumin (transthyretin) i råttserum. De beskrivna separations- metoderna kan även användas för rening och studier av andra allergener. IgE antikroppar som finns i sera från råttallergiska patienter studerades med kemiluminiscens och immunoblotting. Individuella reaktivitetsmönster för de elektroforetiskt separerade råtturinproteinerna kunde ses och IgE i alla patientsera band till huvudproteinet, a2u-globulin. Dock var detta protein inte alltid det mest dominanta proteinet som kändes igen i alla sera. Med en specialutvecklad luminometer kunde relativ mängd av IgE som bundit till olika råtturinproteiner mätas i varje sera, samt diagram erhållas som illustrerade relativa mängder bundet IgE. Överlappande peptider av huvudallergenet Rat n 1.02 syntetiserades på fast fas och undersöktes med en modifierad ELISA-metod för att lokalisera IgE bindande regioner. Indikation på linjära IgE bindande epitoper erhölls vid proteinets N- och C-terminala delar. Ytterligare studier av peptidernas roll för den allergiska reaktionen och deras eventuella användbarhet inom immunoterapin bör dock göras. Sammanfattningsvis kan sägas att denna studie har bidragit till isolering och karakterisering av allergener, vilka ger upphov till yrkesrelaterad råttallergi bland dem som arbetar med laboratoriedjur.