A common autoimmunity predisposing signal peptide variant of the cytotoxic T-lymphocyte antigen 4 results in inefficient glycosylation of the susceptibility allele.
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.
Anjos
Suzana
S
Endocrine Genetics Laboratory, Department of Pediatrics, Division of Pediatric Endocrinology, McGill University Health Center, 2300 Tupper, Montréal, Québec H3H 1P3, Canada.
Nguyen
Audrey
A
Ounissi-Benkalha
Houria
H
Tessier
Marie-Catherine
MC
Polychronakos
Constantin
C
D000069594:Abatacept; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D019556:COS Cells; D060908:CTLA-4 Antigen; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D018796:Immunoconjugates; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011110:Polymorphism, Genetic; D021382:Protein Sorting Signals; D017386:Sequence Homology, Amino Acid
D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D017931:DNA Primers; D018796:Immunoconjugates; D021382:Protein Sorting Signals; D000069594:Abatacept