CELL-SURFACE GLYCOPROTEINS UNDERGO POSTBIOSYNTHETIC MODIFICATION OF THEIR N-GLYCANS BY STEPWISE DEMANNOSYLATION

Primary rat hepatocytes and two hepatoma cell lines have been used to study whether high mannose-type N-glycans of plasma membrane glycoprot eins may be modified by the removal of mannose residues even after tra nsport to the cell surface. To examine glycan remodeling of cell surfa ce glycoproteins, high mannose-type glycoforms were generated by addin g the reversible mannosidase I inhibitor deoxymannojirimycin during me tabolic labeling with [H-3]mannose, thereby preventing further process ing of high mannose-type N-glycans to complex structures. Upon transpo rt to the cell surface, glycoproteins were additionally labeled with n imidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. This strategy allowe d us to follow selectively the fate of cell surface glycoproteins. Pos tbiosynthetic demannosylation was monitored by determining the convers ion of Man(8-9)GlcNAc(2) to smaller structures during reculture of cel ls in the absence of deoxymannojirimycin. The results show that high m annose-type N-glycans of selected cell surface glycoproteins are trimm ed from Man(8-9)GlcNAc(2) to Man(5)GlcNAc(2) with Man(7)GlcNAc(2) and Man(6)GlcNAc(2) 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 w as determined for total cell surface glycoproteins in HepG2 cells, thi s process occurs with a half-time of 6.7 h, By analyzing the size of h igh mannose-type glycans of glycoproteins isolated from the cell surfa ce at the end of the reculture period, i.e, after trimming had occurre d, we were able to demonstrate that glycoproteins carrying trimmed hig h mannose glycans become exposed at the cell surface. From these data we conclude that cell surface glycoproteins can be trimmed by mannosid ases at sites peripheral to N-acetylglucosaminyltransferase I without further processing of their glycans to the complex form. This glycan r emodeling may occur at the cell surface or during endocytosis and recy cling back to the cell surface.