J Biol Chem 2002 Sep 19
Anjos S, Nguyen A, Ounissi-Benkalha H, Tessier MC, Polychronakos C.
Pediatrics, McGill University Health Center, Montral, Qubec H3H 1P3.
A common Thr17Ala polymorphism in the signal peptide of the cytotoxic T-lymphocyte anti-gen 4 (CTLA-4), a T-cell receptor that negatively regulates immune responses, is associated with risk for autoimmune disease.
Since the polymorphism is absent from the mature protein, we hy-pothesized that its biological effect must involve early stages of protein processing, prior to sig-nal peptide cleavage.
Constructs representing the two alleles were compared by in vitro transla-tion, in the presence of endoplasmic reticulum (ER) membranes.
We studied glycosylation by endoglycosidase-H digestion and glycosylation mutant constructs, cleavage of peptide with in-hibitors, 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 vs. 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.
By quanti-tative confocal microscopy we found a higher ratio of cell-surface/total CTLAThr17 versus CTLAAla17 (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.