Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity.
In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms of the glycoprotein aminopeptidase N can be synthesized and the effects of altered glycosylation can be studied. It is demonstrated that aminopeptidase N carries "mucin-type" O-glycans and that this is predominantly located in the stalk, which connects the catalytic headgroup to the membrane anchor. Normally glycosylated aminopeptidase N is present in the plasma membrane of the ldl(D) cells. This is also the case for the non-O-glycosylated and defectively N-glycosylated forms. This is in line with the finding that the intracellular transport APN is unaffected by the absence of O-glycosylation or by changes in N-glycosylation as the various glycosylated forms of aminopeptidase N are normally converted from the high-mannose form to the complex glycosylated form. Enzymatic activity is not influenced by the changes in glycosylation.
Norén
K
K
Biochemistry Laboratory C, Department of Oral Pathology, The Panum Institute, Blegdamsvej 3, Copenhagen N, DK-2200, Denmark.
Hansen
G H
GH
Clausen
H
H
Norén
O
O
Sjöström
H
H
Vogel
L K
LK
D000818:Animals; D001692:Biological Transport; D018826:CD13 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D018076:DNA, Complementary / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000032:analysis; D013552:Swine; D014162:Transfection
D018076:DNA, Complementary; D011134:Polysaccharides; D018826:CD13 Antigens