title: Ligand binding by the immunoglobulin superfamily recognition molecule CD2 is glycosylation-independent.
abstract: 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-A 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.
authors.affiliation: MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, United Kingdom.
authors.forename: S J
authors.initials: SJ
authors.lastname: Davis
authors.forename: E A
authors.initials: EA
authors.lastname: Davies
authors.forename: A N
authors.initials: AN
authors.lastname: Barclay
authors.forename: S
authors.initials: S
authors.lastname: Daenke
authors.forename: D L
authors.initials: DL
authors.lastname: Bodian
authors.forename: E Y
authors.initials: EY
authors.lastname: Jones
authors.forename: D I
authors.initials: DI
authors.lastname: Stuart
authors.forename: T D
authors.initials: TD
authors.lastname: Butters
authors.forename: R A
authors.initials: RA
authors.lastname: Dwek
authors.forename: P A
authors.initials: PA
authors.lastname: van der Merwe
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000378:metabolism*; D018801:CD2 Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D018968:CD58 Antigens; D016466:CHO Cells; D006224:Cricetinae; D018360:Crystallography, X-Ray; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology / Q000378:metabolism*; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism*; D015394:Molecular Structure; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018801:CD2 Antigens; D018968:CD58 Antigens; D007136:Immunoglobulins; D008024:Ligands; D008562:Membrane Glycoproteins; D017485:1-Deoxynojirimycin; C059896:miglustat; D006596:Hexosaminidases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase