Domain-specific N-glycosylation of the membrane glycoprotein dipeptidylpeptidase IV (CD26) influences its subcellular trafficking, biological stability, enzyme activity and protein folding.
Dipeptidyl peptidase IV (DPPIV, CD26) is an N-glycosylated type II plasma membrane protein. The primary structure of rat wild-type DPPIV contains eight potential N-glycosylation sites. To investigate the role of N-glycosylation in the function of DPPIV, three of its asparagine residues were separately converted to glutamine by site-directed mutagenesis. The resulting N-glycosylation mutants of rat DPPIV were studied in stable transfected Chinese hamster ovary cells. All three N-glycosylation mutants of DPPIV showed a reduced half-life, as well as differing degrees of inhibition of the processing of their N-glycans. Mutation of the first (Asn83-->Gln) or eighth (Asn686-->Gln) N-glycosylation site had only a small effect on its enzymatic activity, cell-surface expression and dimer formation, whereas the mutation of the sixth N-glycosylation site (Asn319-->Gln) abolished the enzymatic activity, eliminated cell-surface expression and prevented the dimerization of the DPPIV protein. The mutant [Gln319]DPPIV is retained in the cytoplasm and its degradation was drastically increased. Our data suggest that the N-glycosylation at Asn319 is involved in protein trafficking and correct protein folding.
Fan
H
H
Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Germany. fanzheng@zedat.fu-berlin.de
Meng
W
W
Kilian
C
C
Grams
S
S
Reutter
W
W
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D019281:Dimerization; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014162:Transfection
D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D018819:Dipeptidyl Peptidase 4