A Common Autoimmunity Predisposing Signal Peptide Variant of the Cytotoxic T-lymphocyte Antigen 4 Results in Inefficient Glycosylation of the Susceptibility Allele

Abstract

A common T17A polymorphism in the signal peptide of the cytotoxic T-lymphocyte antigen 4 (CTLA-4), a T-cell receptor that negatively regulates immune responses, is associated with risk for autoimmune disease. Because the polymorphism is absent from the mature protein, we hypothesized that its biological effect must involve early stages of protein processing, prior to signal peptide cleavage. Constructs representing the two alleles were compared by in vitro translation, in the presence of endoplasmic reticulum membranes. We studied glycosylation by endoglycosidase H digestion and glycosylation mutant constructs, cleavage of peptide with inhibitors, and membrane integration by ultracentrifugation and proteinase K sensitivity. A major cleaved and glycosylated product was seen for both alleles of the protein but a band representing incomplete glycosylation was markedly more abundant in the predisposing Ala allele (32.7 ؎ 1.0 versus 10.6% ؎ 1.2 for Thr, p < 10 ؊9 ). In addition, differential intracellular/surface partitioning was studied with co-transfection of the alleles fused to distinct fluorescent proteins in COS-1 cells. By quantitative confocal microscopy we found a higher ratio of cell surface/total CTLAThr 17 versus CTLAAla 17 (p ‫؍‬ 0.01). Our findings corroborate observations, in other proteins, that the signal peptide can determine the efficiency of post-translational modifications other than cleavage and suggest inefficient processing of the autoimmunity predisposing Ala allele as the explanation for the genetic effect.

Allelic effects on gene regulation at the autoimmunity-predisposing CTLA4 locus: a re-evaluation of the 3′ +6230G>A polymorphism

“…This diseaseassociated coding polymorphism has been linked with increased proliferation of T cells 11 through a mechanism that may involve inefficient N-linked glycosylation leading to less mature CTLA-4 at the cell surface. 12 Although coding polymorphisms are likely to have functional implications, there is an obvious bias towards their identification for practical reasons, as regulatory regions are often ill-defined and can extend far beyond the open-reading frame. Their effect cannot be underestimated as cis-acting variation is thought to account for 25 to 35% of the interindividual differences, which determine susceptibility to or protection from most complex diseases and have already been identified in several diseases.…”

Cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) gene polymorphism and susceptibility to non‐Hodgkin's lymphoma

“…Transition from A to G in the CTLA-4 exon 1 results in an amino acid change (Thr to Ala) that seems altering early intracellular trafficking of CTLA-4 receptor and/or its expression on cell surface [22]. Correct functional expression at cellular surface is demonstrated in homozygotes for the Thr allele, which is common in the general population, while the Ala/Ala genotype has been correlated with reduced function of CTLA-4 in patients with Graves' disease [22,23]. The allele ''A'' that we found associated to NHL might have a subtle functional consequence as expected of common alleles predisposing to complex disorders.…”

Association analysis of cytotoxic T-lymphocyte antigen 4 gene polymorphisms with primary biliary cirrhosis in Japanese patients

“…This coding polymorphism is located in a signal peptide that is cleaved from the functional protein, and has been shown to affect glycosylation of the autoimmune susceptibility G allele, resulting in diminished processing efficiency and thus decreased trafficking to the cell surface [43]. It will be necessary to confirm the functional difference between patients with these SNPs and T-cell activation in a future study.…”