? Three N-linked glycosylation sites in CD2:. One in each of the immunoglobulin-type domainsand one in the interdomain linker sequence: Essential role of glycosylation for CD2:: Treatment of CD2 with PNGase to remove all N-linked carbohydrate results in complete loss ofbinding to CD58: Mutation of the glycosylation site in the N-terminal domain, either by changing theglycosylated Asn-65 to Glu or by changing Thr-67to Ala, also eliminates binding activity
• Three'NJlinked'glycosyla3on'sites'in'CD2:' • One'in'each'of'the'immunoglobulinJtype'domains' and'one'in'the'interdomain'linker'sequence' • Essen3al'role'of'glycosyla3on'for'CD2:' • Treatment'of'CD2'with'PNGase'to'remove'all'NJ linked'carbohydrate'results'in'complete'loss'of' binding'to'CD58' • Muta3on'of'the'glycosyla3on'site'in'the'NJ terminal'domain,'either'by'changing'the' glycosylated'AsnJ65'to'Glu'or'by'changing'ThrJ67' to'Ala,'also'eliminates'binding'ac3vity
Indicating an essential role for the N-linked carbohydrate at position 65.structural analysis shows its occupationby high-mannose oligosaccharidesbearing five to nine mannose units
Indicating an essential role for the Nlinked carbohydrate at position 65. structural analysis shows its occupation by high-mannose oligosaccharides bearing five to nine mannose units
BindingsiteforCD58RRRRRRRRRRuCD48/CD58onStarget cellAsn-65C2Lys-61SC2CD2onSkillerT cellRRRRRSRRRRRLys-69LyS-7s
Comparison of rat and human CD2: Rat CD2 is not glycosylated at the positioncorresponding to Asn-65 in human CD2: In human protein, lysine at 61, 69, and 71 allproject from the same side the molecule· In rat, this cluster is interrupted by a glutamicacidin position 61: Role of carbohydrate: to stabilize the cluster ofpositive charges by making hydrogen bonds withtheaminogroupsofthelysineresidues: Human CD2 lysine at 61 replaced by glutamic acid
Comparison'of'rat'and'human'CD2' • Rat'CD2'is'not'glycosylated'at'the'posi3on' corresponding'to'AsnJ65'in'human'CD2' • In'human'protein,'lysine'at'61,'69,'and'71'all' project'from'the'same'side'the'molecule'' • In'rat,'this'cluster'is'interrupted'by'a'glutamic' acid'in'posi3on'61' • Role'of'carbohydrate:'to'stabilize'the'cluster'of' posi3ve'charges'by'making'hydrogen'bonds'with' the'amino'groups'of'the'lysine'residues' • Human'CD2'lysine'at'61'replaced'by'glutamic'acid