Browsing by Subject "Peptidyl-Dipeptidase A"

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    A Point Mutation in the Juxtamembrane Stalk of Human Angiotensin I-converting Enzyme Invokes the Action of a Distinct Secretase
    (2001) Alfalah, Marwan; Parkin, Edward T; Jacob, Ralf; Sturrock, Edward D; Mentele, Reinhard; Turner, Anthony J; HOOPER, Nigel M; Naim, Hassan Y
    Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn(631) to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE(NQ)) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE(NQ) was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 degrees C revealed that ACE(NQ) was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn(635)-Ser(636) bond, three residues N-terminal to the normal secretase cleavage site at Arg(638)-Ser(639). These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.
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    Identification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form
    (1997) Yu, X Christopher; Sturrock, Edward D; Wu, Zhuchun; Biemann, Klaus; EHLERS, Mario R W; Riordan, James F
    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 N-linked glycosylation sites, Asn90 and Asn109, 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, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. 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 Ser625. When expressed in the presence of the alpha-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.