LIGAND-BINDING BY THE IMMUNOGLOBULIN SUPERFAMILY RECOGNITION MOLECULECD2 IS GLYCOSYLATION-INDEPENDENT

The evolutionary success of the immunoglobulin superfamily (IgSF) is t hought to reflect the ability of IgSF protein domains to form stable s tructural units. The role of glycosylation in stabilizing these domain s is controversial, however. In this study a systematic analysis of th e effect of glycosylation on the ligand-binding properties of the cell cell recognition molecule CD2, which consists of two IgSF domains, wa s undertaken. A form of human soluble CD2 (hsCD2) with single N-acetyl glucosamine residues at each glycosylation site was produced by inhibi ting glucosidase I with N-butyldeoxynojirimycin during expression in C hinese hamster ovary cells and digesting the expressed hsCD2 with endo glycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD 2 as determined by surface plasmon resonance analyses. The protein als o formed diffraction quality crystals and analysis of the 2.5-Angstrom resolution crystal structure indicated that the single N-acetylglucos amine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 al so bound the ligand and antibodies although this form of the protein t ended to aggregate. In contrast to the results of previous studies, th e current data indicate that the structural integrity and ligand bindi ng function of human CD2 are glycosylation-independent.