The identification of N-linked oligosaccharides on the human CR2/Epstein-Barr virus receptor and their function in receptor metabolism, plasma membrane expression, and ligand binding.
Human complement receptor type 2 (CR2) was biosynthetically labeled by pulsing SB B lymphoblastoid cells for 25 min with [35S]methionine followed by chase in the presence of excess unlabeled methionine. An Mr 134,000 polypeptide represented the major form of the receptor at the end of the pulse period, and within 1 h of chase this disappeared coincident with the appearance of the Mr 145,000 mature form of CR2. Precursor, but not mature, CR2 was sensitive to endoglycosidase H, indicating that maturation of CR2 represented processing of N-linked high mannose oligosaccharides to the complex type. The processing of precursor CR2 was impaired by monensin. In the presence of tunicamycin an Mr 111,000 form of CR2 was synthesized by SB cells, and this did not chase into either precursor or mature CR2. This Mr 111,000 form of CR2 did not incorporate [3H]glucosamine, indicating that it lacked both N- and O-linked oligosaccharide. The half-lives of mature CR2 and nonglycosylated CR2 pulse-labeled in the presence of tunicamycin were 13.8 and 2.8 h, respectively; the turnover rate of B1, a membrane protein normally lacking carbohydrate, was unaffected by the presence of the antibiotic. The percentage of pulse-labeled, nonglycosylated CR2 that was expressed at the cell surface after 1 h of chase in the presence of tunicamycin was 30%, identical to that of mature CR2 in cells chased in the absence of the antibiotic. However, after 6 h of chase there was no additional net accumulation of nonglycosylated CR2 at the plasma membrane, while the proportion of pulse-labeled mature CR2 at this site had risen to 81%. Therefore, N-linked oligosaccharides are essential for the stability of CR2 and have some role in its plasma membrane expression. In contrast, the observation that all three forms of CR2 bound to Sepharose C3 indicates that oligosaccharides are not necessary for the interaction between CR2 and its complement ligand.
D002462:Cell Membrane / Q000032:analysis; D002478:Cells, Cultured; D003176:Complement C3 / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis* / Q000502:physiology; D011951:Receptors, Complement / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D017464:Receptors, Complement 3d; D011991:Receptors, Virus / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D003176:Complement C3; D008024:Ligands; D009844:Oligosaccharides; D011951:Receptors, Complement; D017464:Receptors, Complement 3d; D011991:Receptors, Virus; D014415:Tunicamycin; D005944:Glucosamine