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.
Recny
M A
MA
PROCEPT, Inc., Cambridge, Massachusetts 02139.
Luther
M A
MA
Knoppers
M H
MH
Neidhardt
E A
EA
Khandekar
S S
SS
Concino
M F
MF
Schimke
P A
PA
Francis
M A
MA
Moebius
U
U
Reinhold
B B
BB
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D018801:CD2 Antigens; D018968:CD58 Antigens; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism; D012397:Rosette Formation; D013329:Structure-Activity Relationship
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D018801:CD2 Antigens; D018968:CD58 Antigens; D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D011971:Receptors, Immunologic