Transfer of free polymannose-type oligosaccharides from the cytosol to lysosomes in cultured human hepatocellular carcinoma HEPG2 cells

Abstract

Large, free polymannose oligosaccharides generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo processing by a cytosolic endo H-like enzyme and a mannosidase to yield the linear isomer of Man5GlcNAc (Man[α1-2]Man[α1-2]Man[α1-3][Man α1- 6]Man[β1-4]GlcNAc). Here we have examined the fate of these partially trimmed oligosaccharides in intact HepG2 cells. Subsequent to pulse chase incubations with D-[23H]mannose followed by permeabilization of cells with streptolysin O free oligosaccharides were isolated from the resulting cytosolic and membrane-bound compartments. Control pulse-chase experiments revealed that total cellular free oligosaccharides are lost from HcpG2 cells with a half-life of 3-4 h. In contrast use of the vacuolar H+/ATPase inhibitor, concanamycin A, stabilized total cellular free oligosaccharides and enabled us to demonstrate a translocation of partially trimmed oligosaccharidcs from the cytosol into a membrane-bound compartment. This translocation process was unaffected by inhibitors of autophagy but inhibited if cells were treated with either 100 μM swainsonine, which provokes a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or agents known to reduce cellular ATP levels which lead to the accumulation of the linear isomer of Man5GlcNAc in the cytosol. Subcellular fractionation studies on Percoll density gradients revealed that the cytosol- generated linear isomer of Man5GlcNAc is degraded in a membrane-bound compartment that cosediments with lysosomes.