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 mo difications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the Idl(D) cel l line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficien cy which blocks the conversion of glucose into galactose derivatives. Thus it is possible in the Idl(D) cell line to selectively block O-gly cosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose, In t his way selectively altered glycosylated forms of the glycoprotein ami nopeptidase N can be synthesized and the effects of altered glycosylat ion 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 membra ne of the Idl(D) cells. This is also the case for the non-O-glycosylat ed and defectively N-glycosylated forms. This is in line with the find ing that the intracellular transport APN is unaffected by the absence of O-glycosylation or by changes in N-glycosylation as the various gly cosylated forms of aminopeptidase N are normally converted from the hi gh-mannose form to the complex glycosylated form. Enzymatic activity i s not influenced by the changes in glycosylation. (C) 1997 Academic Pr ess.