Cell surface glycoproteins undergo postbiosynthetic modification of their N-glycans by stepwise demannosylation

Abstract

Primary rat hepatocytes and two hepatoma cell lines have been used to study whether high mannose-type N-glycans of plasma membrane glycoproteins may be modified by the removal of mannose residues even after transport to the cell surface. To examine glycan remodeling of cell surface glycoproteins, high mannose-type glycoforms were generated by adding the reversible mannosidase I inhibitor deoxymannojirimycin during metabolic labeling with [3H]mannose, thereby preventing further processing of high mannose-type N- glycans to complex structures. Upon transport to the cell surface, glycoproteins were additionally labeled with sulfosuccinimidyl-2- (biotinamido) ethyl-1,3-dithiopropionate. This strategy allowed us to follow selectively the fate of cell surface glycoproteins. Postbiosynthetic demannosylation was monitored by determining the conversion of Mans8-9GlcNAc2 to smaller structures during reculture of cells in the absence of deoxymannojirimycin. The results show that high mannose-type N-glycans of selected cell surface glycoproteins are trimmed from Man8-9GlcNA2, to Man5GlcNAc2 with Man7GlcNAc2 and Man6GlcNAc2 formed as intermediates. It could be clearly shown in MH 7777 as well as in HepG2 cells that demannosylation affects plasma membrane glycoproteins after they are routed to the cell surface. As was determined for total cell surface glycoproteins in Hepg2 cells, this process occurs with a half-time of 6.7 h. By analyzing the size of high mannose-type glycans of glycoproteins isolated from the cell surface at the end of the reculture period, i.e. after trimming had occurred, we were able to demonstrate that glycoproteins carrying trimmed high mannose glycans become exposed at the cell surface. From these data we conclude that cell surface glycoproteins can be trimmed by mannosidases at sites peripheral to N-acetylglucosaminyltransferase I without further processing of their glycans to the complex form. This glycan remodeling may occur at the cell surface or during endocytosis and recycling back to the cell surface.