A β-cell glycoprotein of Mr 40 000 is the major rat islet cell immunogen following xenogenic immunisation

Diabetologia, Jul 1984

An antiserum (R2) was raised in a rabbit against dispersed Sprague Dawley rat islet cells. The R2 antiserum contains islet cell surface antibodies, which mediate complement-dependent cytotoxicity against islet cells resulting in a block of glucose induced insulin release. Immunoprecipitation and gel electrophoretic analysis showed that R2 specifically recognizes an Mr 40 000 glycoprotein present in both rat islet and rat insulinoma cells. This glycoprotein is amphiphilic in character and probably represents a pancreatic β cell specific plasma membrane component. The results support previous observations in mouse β cells that a plasma membrane glycoprotein of Mr 40K constitutes a major islet cell immunogen.

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A β-cell glycoprotein of Mr 40 000 is the major rat islet cell immunogen following xenogenic immunisation

M A/1-cell glycoprotein of Mr 40000 is the major rat islet cell immunogen following xenogenic immunisation S. B~ekkeskov a n d A. L e m m a r k 0 1 0 Hagedorn Research Laboratory , Gentofte , Denmark 1 Dr. S. B~ekkeskov Hagedoru Research Laboratory Niels Steensensvej 6 DK-2820 Gentofte Denmark Summary. An antiserum (R2) was raised in a rabbit against dispersed Sprague Dawley rat islet cells. The 1t2 antiserum contains islet cell surface antibodies, which mediate complement-dependent cytotoxicity against islet cells resulting in a block o f glucose induced insulin release. Immunoprecipitation and gel electrophoretic analysis showed that R2 specifically recognizes an Mr 40 000 glycoprotein present in both rat islet and rat insulinoma cells. This glycoprotein is amphiphilic in character and probably represents a pancreatic/3 cell specific plasma membrane component. The results support previous observations in m o u s e / 3 cells that a plasma membrane glycoprotein o f Mr 40K constitutes a major islet cell immunogen. Glycoprotein Mr 40000; islet cell immunogen - 9 Springer-Verlag 1984 Studies o f islet cell s u r f a c e a n t i b o d i e s ( I C S A ) , b o t h in a n t i s e r a r a i s e d e x p e r i m e n t a l l y a n d in s e r a f r o m insulind e p e n d e n t d i a b e t i c p a t i e n t s , i n d i c a t e t h a t t h e fl cell m a y e x p r e s s cell specific s u r f a c e d e t e r m i n a n t s [ 1-3 ]. W e h a v e p r e v i o u s l y d e m o n s t r a t e d t h a t a m o u s e / 3 cell plasm a m e m b r a n e protein o f Mr 40 000 ( M r 4 0 K ) w a s t h e m a j o r a n t i g e n r e c o g n i z e d b y a r a b b i t a n t i s e r u m (R4) r a i s e d a g a i n s t o b / o b m o u s e islet cells [ 1 ]. A n o t h e r antis e r u m (R2) w a s r a i s e d in rabbits a g a i n s t d i s p e r s e d S p r a g u e D a w l e y rat islets [ 4 ]. I n t h e p r e s e n c e o f c o m p l e m e n t I C S A in this s e r u m m e d i a t e c y t o t o x i c i t y a g a i n s t rat islet cells in vitro a n d b l o c k g l u c o s e i n d u c e d i n s u l i n release [ 5, 6 ]. I n this s t u d y w e d e s c r i b e t h e c h a r a c t e r i z a t i o n o f t h e antigen(s) in rat islets a n d i n s u l i n o m a cells r e c o g n i s e d b y t h e R 2 a n t i s e r u m . Materials and methods Antiserum A rat islet cell antiserum was raised by immunizing a rabbit with living dispersed rat islet cells as described previously [ 4 ]. Insulin antibodies have not been detected in this antiserum [ 4 ]. Isolation o f islets, insulinoma cells and lymphocytes Rat insulinoma cell tumours were propagated in the New England Deaconess Hospital (NEDH) strain of rats using a tumour arising from an original X-ray induced insulinoma [ 8 ] (RIN-tumour). The insulin producing RIN-5AH cell line developed from the X-ray induced insulinoma [ 9 ] (kindly donated by Dr. H. Oie, National Cancer Institute, Washington, DC, USA) was also propagated as tumours in NEDH rats (RIN-5AH tumour). Tumours, 0.5-5 g wet weight, were removed and the cells dispersed in Hanks' balanced salt solution containing 1% bovine serum albumin. Insulinoma cells were separated from erythrocytes by centrifugation (450 g, 5 rain) on Ficoll Paque (Pharmacia, Uppsala, Sweden) and incubated in RPMI 1640 with 10% fetal calf serum for 3 h before radioactive labelling. Labelling o f cells Islets, insulinoma cells and lymphocytes were biosynthetically labelled with 35Smethionine at 250 lxCi/ml (islets) or 100 ~tCi/ml (insulinoma cells and lymphocytes) as described [ 10 ]. Islets (250/ml) were incubated at 37~ for 16h, insulinoma cells and lymphocytes (1 x 106/ml) for 6h. Lysis o f cells and immunoprecipitation Labelled islets (103/ml), insulinoma cells and lymphocytes (107/ml) were lysed in 20 mmol/1 Tris HC1 (pH 7.4) containing 150 mmol/l NaC1, 5 mmol/1 methionine, 2 mmol/1 phenylmethyl-sulphonylfluoride (PMSF) and either 1000 KIE trasylol/ml and 1% NP-40 (NP40 buffer) or 15000 KIE trasylol/ml and 1% Triton X-114 (Triton X-114 buffer). In some experiments cells were lysed by boiling in 0.1-0.2 ml 20 mmol/1 Tris HC1 (pH 7.4) containing 2% SDS followed by immediate dilution in 10 volumes of NP-40 buffer. Insoluble material was removed by centrifugation at 100,000g for 30 min and the supernatants used for immunoprecipitation either after adsorption to normal rabbit serum [ 10 ], after purification of glycoproteins on a lentil lectin-Sepharose 4B affinity column (NP-40 lysate) [ 11 ] or after purification ofamphiphilic membrane proteins by phase separation in Triton X-114 (Triton X-114 lysate) [ 12 ]. Aliquots (100 pJ) were incubated with 5 ~tl of R2 or normal rabbit serum for 16 h and the immune complexes were adsorbed to 100 p.1of pre-swollen protein A-Sepharose CL-4B (Pharmacia). The protein A-Sepharose was washed four times in 0.5% NP40 or Triton X-114 buffer and once with ice-cold water. The immunecomplexes were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5%-15% gradient or 10% uniform slab gels using the discontinuous buffer system of Laemmli [ 14 ]. The gels were stained and processed for autoradiography as described [ 10 ] except that Enlighming (New England Nuclear) was used as an autoradiographic enhancer. Results Analyses ofproteins recognised by the R2 antiserum in whole lysates of rat islets, insulinoma cells and lymphocytes The R2 antiserum specifically immunoprecipitated a major protein of Mr 40K from 35S methionine labelled rat islets, RIN-tumour and RIN-5AH tumour cells (Fig. 1). Several additional components were precipitated from whole NP-40 lysates of rat islet cells (not shown) while few weak additional bands were observed in immunoprecipitates of RIN-tumour cells lysed in NP-40 (Fig. 1B, before lentil lectin chromatography). To reduce the background of non-specific or low affinity binding rat islet cell proteins, rat islets were first lysed in 2% SDS and then diluted 10 times in NP-40 buffer. The major protein immunoprecipitated specifically from such lysates was an Mr 40K component (Fig. 1A). Additional components of Mr 75K, 32K, and 14K were also observed (Fig.lA). No proteins were specifically immunoprecipitated from lysates of rat spleen lymphocytes analysed in parallel (not shown). Accordingly extensive adsorption of the R2 antiserum to rat spleen cells did not affect the ability of this antiserum to detect the M, 40K component in islet cells (data not shown). Analyses of glycoproteins recognized by the R2 antiserum NP-40 lysates of RIN-tumour cells were subjected to Lens culinaris lectin affinity column chromatography. A glycoprotein fraction, eluted by 3% a-methyl mannoside was used for immunoprecipitation. The R2 antiserum immunoprecipitated glycoprotein components of M, 14K, 40K, 50K and a smear of bands at Mr > 90K (Fig. 1 B). The 50K and 14K proteins were tentatively identified as major histocompatibility (MHC) class I antigen heavy chain and/32 microglobulin, respectively, since components of similar M, were immunoprecipitated by a rabbit antiserum raised against purified mouse MHC class I antigens (kindly donated by Dr. P. Peterson, Department of Cell Research, The Wallenberg Laboratory, Uppsala, Sweden). This antiserum cross-reacts with rat M H C class I components [ 10 ]. Analyses of amphiphilicproteins recognized by the R2-antiserum The phase separation technique of Bordier [ 12 ] was used to separate amphiphilic and hydrophilic islet cell proteins in Triton X-114 lysates. Integral membrane proteins with an amphiphilic nature are concentrated at 30 ~ in a confluent detergent phase which is isolated by centrifugation allowing hydrophilic proteins to remain in the aqueous phase. Certain bulky transmembrane proteins have been found to remain in the aqueous phase together with the hydrophilic proteins, while hydrophilic proteins are not found in the detergent phase [ 13 ]. The R2 antiserum specifically immunoprecipitated a Mr 40K protein from the detergent phase of RIN5AH tumour cells (Fig.lC) and rat islet cells (not shown). Additional components at Mr 14K and 50K (probably M H C class I components) and a smear of bands at Mr > 80K were also specifically precipitated. In the aqueous phase no proteins except a very faint band at Mr 40K were specifically immunoprecipitated by the R2 antiserum. Discussion The major protein detected irf immunoprecipitates of rat islet cell lysates with the R2 antiserum, but not with normal rabbit serum had a M r of 40K as determined by SDS-PAGE and autoradiography. The detection of the Mr 40K amphiphilic glycoprotein as a major rat fl cell antigen recognized by an antiserum raised in a rabbit is in agreement with the earlier demonstration of a fl cell specific plasma membrane protein o f Mr 40K being a major antigen in a xenogenic immunisation with fl cell rich mouse o b / o b islet cells [ 1 ]. The detection of this protein in rat islet tumour cells whether passaged from the original tumour [ 8 ] or a cloned insulin-producing cell line [ 9 ] indicates that it is expressed in pancreatic fl cells. The present analysis does not exclude that other endocrine islet cells also express this component; however, the lack of expression of this protein by rat spleen cells was evidenced by both immunoprecipitation and adsorption experiments. The glycoprotein character of the Mr 40K component was indicated by its binding to Lens culinaris lectin affinity column and its amphiphilic nature, characteristic for integral membrane proteins was reflected by its ability to be concentrated in the Triton X-114 detergent phase at temperatures above the clouding point of this detergent. The R2 serum is a xenogenic antiserum raised against dispersed rat islet cells. It is therefore expected to recognize not only antigenic determinants present in the different types of endocrine pancreatic cells, but also determinants common to many rat cells. This is illustrated by the ability of the R2 antiserum to detect rat insulinoma components (Mr 50K and 14K) tentatively identified as M H C class I antigen heavy and light (f12 microglobulin) chain, previously shown to be present in islet cells with specific antisera [ 10 ]. In rat islets the 14K but not the 50K component was specifically detected by the R2 antiserum. However, the heavy chain component may be covered by proteins at a similar M r , immunoprecipitated by both R2 and normal rabbit serum. Some of the components immunoprecipitated by the R2 antiserum from whole rat islet cell lysates, and not detected in immunoprecipitates of insulinoma cells, could either originate from islet cells other than the fl cells or be expressed solely in normal islet cells. In addition to the Mr 40K protein, it was furthermore observed that the R2 antiserum detected a smear of bands at high Mr in the glycoprotein fraction ( > 90K) and amphiphilic fraction ( > 80K) of rat islet turnout cells. Although some degree of homology could be expected between the mouse and rat/3 cell protein of Mr 40K, the cross reactivity of the mouse protein with the R2 antiserum and the rat protein with the R4 antiserum [ 1 ] is weak, judged from immunoprecipitation experiments (unpublished results). The interspecies variations in antigenic epitope structure of this protein may therefore be considerable. Neither the R2 nor the R4 antiserum was found to immunoprecipitate an Mr 40K component in human islet cells (unpublished data). ICSA-positive sera from diabetic BB-rats immunoprecipitate an M r 64K islet cell protein from detergent lysates of rat islets and insulinoma cells [ 15 ]. The Mr 40K component has not been detected in such immunoprecipitates. Thus although the Mr 40K component is a major immunogen evoking a xenogenic immune response it does not seem to play a role in an autoimmune response against islet cells. Acknowledgements.The authors thank Dr. Ole Bjerrumfor valuable discussionsand adviceregarding the phase separationtechnique.The experttechnicalassistanceof H.RichterOlesenand A.Borchis gratefully acknowledged.Steinunn B~ekkeskovis a recipient of a Juvenile Diabetes Foundation ResearchFellowship.This studywas supported by the National Institute of Health (grant AM 26190). 1. DyrbergT, BaekkeskovS, LerumarkA ( 1982 ) Specificpancreatic B cell surface antigens recognized by a xenogenic antiserum . J Cell Bio194 : 472 - 477 2. Van de WinkelM, SmetsG, Gepts W, PipeleersD ( 1982 ) Isletcell surface antibodies from insulin-dependent diabeticsbind specificallyto pancreatic B-cells . J Clin Invest70 : 41 - 49 3. DobersenMJ, ScharffJE ( 1982 ) Preferentiallysisof pancreatic Bcells by isletcellsurfaceantibodies . Diabetes31 : 459 - 462 4. 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Oie HK , Gazdar AF , Minna JD , Weir GC , Naylin SB ( 1983 ) Clonal analysis of insulin and somatostatin secretion and L-dopa decarboxylase expression by a rat islet cell tumor . Endocrinology 112 : 1070 - 1075 10. Baekkeskov S , Kanatsuna T , Klareskog L , Nielson DA , Peterson PA , Rubenstein AH , Steiner DF , Lernmark A ( 1981 ) Expression of major histocnmpatibility antigens on pancreatic islet cells . Proc Natl Acad Sci USA 78 : 6456 - 6460 11. Hayman MJ , Crumpton MJ ( 1972 ) Isolation of glycoproteins from pig lymphocyte plasma membrane using Lens culinaris phytohemaglutinin . Biochem Biophys Res Commun 47 : 923 - 930 12. Bordier C ( 1981 ) Phase separation of integral membrane proteins in Triton X-114 solution . J Biol Chem 256 : 1604 - 1607 13. Bjerrum OJ , Larsen KP ( 1983 ) Some recent developments of the electroimmunochemical analyses of membrane proteins . Application of zwittergent Triton X-114 and Western blotting technique . In: Tschesche H (ed) Modern methods in protein chemistry. Review articles . Walter de Gruyter, Berlin New York, pp 79 - 124 14. Laemmli UK ( 1970 ) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227 : 680 - 685 15. Beekkeskov S , Dyrberg T , Lernmark ,~ ( 1984 ) Autoantibodies to a 64-Kilodalton islet cell protein precede the onset of spontaneous diabetes in the BB rat . Science (in press)


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S. Bækkeskov, Å. Lernmark. A β-cell glycoprotein of Mr 40 000 is the major rat islet cell immunogen following xenogenic immunisation, Diabetologia, 1984, 70-73, DOI: 10.1007/BF00275650