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

A temperature sensitive secretory line, MadIA214, was selected from mu tagenized Chinese hamster ovary cells that express two heterologous ex port marker proteins: a secretory form of the human placental alkaline phosphatase (SeAP), and the K-d heavy chain of mouse MHC class I, SeA P secretion in MadIA214 was extremely reduced at elevated temperature (40 degrees C), while the export of functional H-2K(d) molecules to th e plasma membrane was only slightly affected, This mutant constitutive ly transferred onto newly synthesized proteins a truncated oligosaccha ride core, Man(5)GlcNAc(2), which was monoglucosylated in the protein- bound form, Nevertheless, the final oligosaccharide-structures associa ted to mature SeAP and H-2K(d) were similar in mutant and wild-type gl ycoproteins. The inaccessibility in MadIA214 endoplasmic reticulum (ER ) of one or more components required for oligosaccharide chain elongat ion is supported by the reconstitution of a correct core structure, ob tained after disruption of cellular compartments, but not after cell p ermeabilisation or blocking ER-to-Golgi transport, The increased assoc iation of the ER-chaperone BiP with immature SeAP correlated with the thermodependent decrease in SeAP secretion, The retention of incomplet ely folded polypeptides in MadIA214 parallels both a marked ER-dilatio n and an important glycoprotein degradation documented by the formatio n of soluble oligomannosides with one GlcNAc residue, Our data provide the first in vivo evidence that the initial step in N-glycosylation d ifferentially governs glycoprotein maturation, transport and degradati on.