Identification of N-Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

Abstract

The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential Nlinked glycosylation sites, Asn 90 and Asn 109 , were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn 155 , Asn 337 , and Asn 586 , are partially glycosylated, whereas Asn 72 appears to be fully glycosylated. The only potential site that was not modified is Asn 620 . Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser 625 . When expressed in the presence of the ␣-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.Both forms of angiotensin-converting enzyme (ACE 1 ; EC 3.4.15.1 peptidyl-dipeptidase A) are class I transmembrane ectoenzymes (1) that have N-and O-linked oligosaccharides attached to their polypeptide chains (2, 3). Expression of ACE in human HeLa cells in the presence of tunicamycin resulted in complete inhibition of glycosylation, rapidly degraded intracellular ACE, and no enzyme released in the medium (4). An enzymatically active ACE was produced with partial glycosylation in a mutant Chinese hamster ovary (CHO) cell line (ldlD), although it was released to a lesser extent (4). Similarly, it was reported (5) that inhibitors of glucosidases I and II in the endoplasmic reticulum (ER) and mannosidase I in the cis-Golgi reduced the amount of oligosaccharide attached to human intestinal ACE and delayed protein release significantly. These data strongly suggest that glycosylation plays an important role in the membrane targeting and release of ACE, possibly by affecting the folding of the polypeptide and its recognition by a variety of enzymes in the folding and transport machineries. Recently, Sadhukhan and Sen (6) reported that mutations at individual N-linked glycosylation sites (sequons) in rabbit testis ACE (tACE) resulted in varied efficiencies in enzyme release, which suggests that N-linked glycans at each site may make diff...

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

“…A major advance in this area was the successful determination of the crystal structure of human testis ACE. These studies were made possible by work from Riordan, Sturrock, and others who systematically investigated the role of glycosylation in the activity and stability of testis ACE (Yu et al, 1997;Gordon et al, 2003). With the exception of the N-terminal 36 amino acids, mature human testis ACE is identical to the C terminus of somatic ACE, and it was thought to be easier to crystallize than the larger somatic isozyme.…”

“…Information about such differences is available from a variety of sources, including the X-ray crystallographic analyses of both domains and a variety of pharmacologic and physicochemical studies. Both the N-and the C-domains are thought to be glycosylated, 26 with the N-domain containing 10 and the C-domain containing seven potential N-glycosylation sites (Yu et al, 1997;O'Neill et al, 2008). It appears that glycosylation plays a critical role in maintaining the stability of ACE, since the expression of this protein in bacterial cells, which are unable to perform eukaryotic glycosylation, or the tissue culture expression of ACE protein in the presence of tunicamycin, an inhibitor of glycosylation, results in protein that is catalytically inactive and subject to rapid degradation (Sadhukhan and Sen, 1996).…”

Agm1/Pgm3-Mediated Sugar Nucleotide Synthesis Is Essential for Hematopoiesis and Development

“…For example, in the testis, we identified aberrant glycosylation of the testis-specific isoform of ACE in Pgm3 mld1/mld1 mice. Testicular ACE is a heavily glycosylated integral membrane protein containing both Oand N-glycosylation sites (34). Since no overall decrease in the level of glycosylation was observed in the testis, testicular ACE appears to be particularly sensitive to reductions in the UDPGlcNAc level.…”

Crystal Structure of the N Domain of Human Somatic Angiotensin I-converting Enzyme Provides a Structural Basis for Domain-specific Inhibitor Design

“…Geneticin (G418) (Sigma)-resistant clones expressing soluble N domain were further selected for resistance to methionine sulfoximine (MSX). 33 Cells stably expressing N domain were grown in Glasgow Minimum Essential Medium (GMEM) supplemented with 10% (v/v) dialysed foetal calf serum (Gibco BRL) and 20 mM MSX. When confluent, growth medium was changed to 1% dialysed FCS, 0.05% albumax I (Gibco BRL), 20 mM MSX and 1.5 mM N-butyldeoxynojirimycin (NBDNJ) (Toronto Research Chemicals Inc.).…”