IDENTIFICATION OF N-LINKED GLYCOSYLATION SITES IN HUMAN TESTIS ANGIOTENSIN-CONVERTING ENZYME AND EXPRESSION OF AN ACTIVE DEGLYCOSYLATED FORMS

The sites of glycosylation of Chinese hamster ovary cell expressed tes ticular angiotensin-converting enzyme (tACE) have been determined by m atrix-assisted laser desorption ionization/time of flight/mass spectro metry of peptides generated by proteolytic and cyanogen bromide digest ion. Two of the seven potential N-linked glycosylation sites, Asn(90) and Asn(109), were found to be fully glycosylated by analysis of pepti des before and after treatment with a series of glycosidases and with endoproteinase Asp-N, The mass spectra of the glycopeptides exhibit ch aracteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type, This s tructural information was used to demonstrate that three other sites, Asn(155), Asn(337), and Asn(586) are partially glycosylated, whereas A sn(72) appears to be fully glycosylated, The only potential site that was not modified is Asn(620), Sequence analysis of tryptic peptides ob tained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a c ounterpart in tACE, Comparison of the two proteins reveals a pattern i n which amino-terminal N-linked sites are preferred. The functional si gnificance of glycosylation was examined with a tACE mutant lacking th e O-glycan-rich first amino-terminal 36 residues and truncated at Ser( 625). When expressed in the presence of the alpha-glucosidase I inhibi tor N-butyldeoxynojirimycin and treated with endoglycosidase H to remo ve all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a goo d candidate for crystallographic studies.