DIFFERENTIAL FATE OF GLYCOPROTEINS CARRYING A MONOGLUCOSYLATED FORM OF TRUNCATED N-GLYCAN IN A NEW CHO LINE, MADIA214, SELECTED FOR A THERMOSENSITIVE SECRETORY DEFECT

摘要

A temperature sensitive secretory line, MadIA214, was selected from mutagenized Chinese hamster ovary cells that express two heterologous export marker proteins: a secretory form of the human placental alkaline phosphatase (SeAP), and the K-d heavy chain of mouse MHC class I, SeAP secretion in MadIA214 was extremely reduced at elevated temperature (40 degrees C), while the export of functional H-2K(d) molecules to the plasma membrane was only slightly affected, This mutant constitutively transferred onto newly synthesized proteins a truncated oligosaccharide core, Man(5)GlcNAc(2), which was monoglucosylated in the protein-bound form, Nevertheless, the final oligosaccharide-structures associated to mature SeAP and H-2K(d) were similar in mutant and wild-type glycoproteins. The inaccessibility in MadIA214 endoplasmic reticulum (ER) of one or more components required for oligosaccharide chain elongation is supported by the reconstitution of a correct core structure, obtained after disruption of cellular compartments, but not after cell permeabilisation or blocking ER-to-Golgi transport, The increased association of the ER-chaperone BiP with immature SeAP correlated with the thermodependent decrease in SeAP secretion, The retention of incompletely folded polypeptides in MadIA214 parallels both a marked ER-dilation and an important glycoprotein degradation documented by the formation of soluble oligomannosides with one GlcNAc residue, Our data provide the first in vivo evidence that the initial step in N-glycosylation differentially governs glycoprotein maturation, transport and degradation. [References: 57]