The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 ± 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed >60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the α-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1992|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology