N-glycosylation at one rabies virus glycoprotein sequon influences N-glycan processing at a distant sequon on the same molecule

Abstract

Rabies glycoprotein (RGP(WT)) contains N-glycosylation sequons at Asn 37, Asn247, and Asn319, although Asn 37 is not efficiently glycosylated. To examine N-glycan processing at Asn247 and Asn319, full-length glycosylation mutants, RGP(-2-) and RGP(--3), were expressed, and Endo H sensitivity was compared. When the Asn247 sequon is present alone in RGP(-2-), 90% of its N-glycans are high-mannose type, whereas only 35% of the N-glycans at Asn319 in RGP(--3) are high-mannose. When both sequons are present in RGP(-23), 87% of the N-glycans are of complex type. The differing patterns of Endo H sensitivity at sequons present individually or together suggests that glycosylation of one sequon affects glycosylation at another, distant sequon. To explore this further, we constructed soluble forms of RGP: RGP(WT)T441His and RGP(--3)T441His. Tryptic glycopeptides from these purified secreted proteins were isolated by HPLC and characterized by a 3D oligosaccharide mapping technique. RGP(WT)T441His had fucosylated, bi- and triantennary complex type glycans at Asn247 and Asn319. However, Asn247 had half as many neutral glycans, more monosialylated glycans, and fewer disialylated glycans when compared with Asn319. Moreover, when comparing the N-glycans at Asn319 on RGP(--3)T441His and RGP(WT)T441His, the former had 30% more neutral, 28% more monosialylated, and 33% fewer disialylated glycans. This suggests that the N-glycan at Asn 247 allows additional N-glycan processing to occur at Asn 319, yielding more heavily sialylated bi- and triantennary forms. The mechanism(s) by which glycosylation at one sequon influences N-glycan processing at a distant sequon on the same glycoprotein remains to be determined. © Oxford University Press 2005; all rights reserved.