Ligand binding by the immunoglobulin superfamily recognition molecule CD2 is glycosylation-independent
Davis SJ, Davies EA, Barclay AN, Daenke S, Bodian DL, Jones EY, Stuart DI, Butters TD, Dwek RA, van der Merwe PA. (1995), J Biol Chem. 270, 369-75
The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-Å resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.
Key figure: Binding of anti-CD2 mAbs to untreated, endo H-treated, and PNGase F-treated hsCD2
Panel A, ascites (1 in 20 dilution) containing the indicated human CD2 mAbs was injected for 6 min through flow cells with immobilized untreated (2678 RU) or endo H-treated (3502 RU) hsCD2. Bound mAbs were eluted with 10 mM HCl (arrow) prior to injection of the subsequent mAb. The flow rate was 5 μl•min-1. Panel B, ascites (1 in 50 dilution) containing the indicated human CD2 mAb was injected for 10 min through flow cells in which untreated hsCD2 (4689 RU), endo H-treated hsCD2 (5955 RU), and PNGase F-treated hsCD2 (5568 RU) were immobilized. The flow rate was 3 μl•min-1.