The p68 autoantigen characteristic of rheumatoid arthritis is reactive with carbohydrate epitope specific autoantibodies

Abstract

Objective-The autoantigen p68 is a target of autoantibodies as well as autoreactive T cells with a high specificity in rheumatoid arthritis (RA). The binding characteristics of the autoantibodies to their antigen were now analysed biochemically and cytologically. Methods-Deglycosylation techniques as well as lectin and sugar competition experiments were performed to p68 to discover if the antibodies detected a glycoepitope. Its antigenicity was investigated applying anti-p68 antibodies derived from RA patients in comparison with polyclonal rabbit anti-p68 antibodies. Results-p68 specific antibodies from RA patients did not to bind to p68 that had been deglycosylated by alkaline -elimination, O-glycosidase or periodate treatment. In contrast, binding of p68 specific antibodies raised in rabbit was unaVected by either deglycosylation protocol. Furthermore, lectins specific for the carbohydrate N-acetylglucosamine competed with p68 specific antibodies from RA patients for antigen binding. N-acetylglucosamine by itself also competed with patient derived anti-p68 antibodies for p68 binding. Again, rabbit anti-p68 antibodies did not elicit these competitive eVects. Applying cytoimmunofluorescence, p68 was present in the cytoplasm or endoplasmic reticulum and also in low abundance on the cell surface. Under heatshock conditions, p68 was detectable in the nucleus. Conclusions-Autoimmunity to p68 during RA is carried by anti-carbohydrate autoantibodies. The carbohydrate modification of p68 appears to be N-acetylglucosamine, which may reflect the regulation of intracellular localisation of the antigen. It is hypothesised that a shift in glycosylation pattern accompanied by an unphysiological localisation of the antigen could trigger antigenicity of p68 during the pathogenesis of RA.

ER Stress Proteins in Autoimmune and Inflammatory Diseases

“…The 68-kDa glycoprotein was identified through exploration of autoantigens that predominantly immunoreact with sera from rheumatoid arthritis (RA) patients and thus expected to be associated with RA pathogenesis (Blass et al, 1995 , 1997 , 1998 ). Subsequently, the 68-kDa antigen was demonstrated to be identical to the ER resident hsp70 family chaperone Bip (Blass et al, 2001 ; Corrigall et al, 2001 ).…”

In Vivo Selection Against Human Colorectal Cancer Xenografts Identifies an Aptamer That Targets RNA Helicase Protein DHX9

“…Our previous in vivo selection yielded an aptamer that targets another helicase, p68, which shuttles between the nucleus and cytoplasm as well and has been shown to be present on the cell surface of HeLa cells by other group. 15 Moreover, nucleolin, another RNA helicase involved in ribosome biogenesis, has been shown to function as a cell surface receptor and act as a shuttling protein to help coordinate extracellular and nuclear events. 16 Based on the above observations, we speculate that aptamer S-1 is taken up into cancer cells by binding to DHX9 protein that is known to shuttle to the cytoplasm and—similar to nucleolin and p68—may also be transiently shuttling to the cell surface in cancer cells.…”

The Role of Posttranslational Protein Modifications in Rheumatological Diseases: Focus on Rheumatoid Arthritis

“…These results are in accordance with the previous observation that collagen from human and rats with active arthritis contains glycosylated and unglycosylated zones; on the contrary, in healthy subjects collagen is uniformly glycosylated [ 16 ]. Besides collagen, other proteins—for example, p68—show a glycosylation-dependent T cell recognition [ 17 ]; hence, an aberrant glycosylation might be one of the triggers for the onset and progression of RA.…”