N-glycosylation is required for human CD2 immunoadhesion functions.

The T-lymphocyte glycoprotein receptor, CD2, mediates cell-cell adhesion by binding to the surface molecule CD58 (LFA-3) on many cell types including antigen presenting cells. Two domains comprise the CD2 extracellular segment, with all adhesion functions localized to the amino-terminal domain that contains a single N-glycosylation site at Asn65. We have defined an important role for the N-linked glycans attached to Asn65 of this domain in mediating CD2-CD58 interactions and also characterize its N-glycotype structure. Analysis of deglycosylated soluble recombinant CD2 as well as a mutant transmembrane CD2 molecule containing a single Asn65-Gln65 substitution demonstrates that neither deglycosylated CD2 nor the mutant CD2 transmembrane receptor binds CD58 or monoclonal antibodies directed at native CD2 adhesion domain epitopes. Electrospray ionization-mass spectrometry demonstrates that high mannose oligosaccharides ((Man)nGlcNAc2, n = 5-9) are the only N-glycotypes occupying Asn65 when soluble CD2 is expressed in Chinese hamster ovary cells. Based on a model of human CD2 secondary structure, we propose that N-glycosylation is required for stabilizing domain 1 in the human receptor. Thus, N-glycosylation is essential for human CD2 adhesion functions.