Proteolytic Activation of Recombinant Pro-memapsin 2 (Pro--secretase) Studied with New Fluorogenic Substrates

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• 作者：Jacques Ermolieff ; Jeffrey A. Loy ; Gerald Koelsch ; and Jordan Tang
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：October 10, 2000
• 年：2000
• 卷：39
• 期：40
• 页码：12450 - 12456
• 全文大小：139K
• 年卷期：v.39,no.40(October 10, 2000)
• ISSN：1520-4995

文摘

Memapsin 2 (-secretase), a membrane-anchored aspartic protease, is involved in the cleavageof -amyloid precursor protein to form -amyloid peptide. The primary structure of memapsin 2 suggeststhat it is synthesized in vivo as pro-memapsin 2 and converted to memapsin 2 by an activating protease[Lin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1456-1460]. To simulate this activation mechanismand to produce stable mature memapsin 2 for kinetic/specificity studies, we have investigated the activationof recombinant pro-memapsin 2 by several proteases with trypsin-like specificity. Clostripain, kallikrein,and trypsin increased the activity of pro-memapsin 2. Clostripain activation was accompanied by thecleavage of the pro region to form mainly two activation products, Leu^30p- and Gly^45p-memapsin 2. Anotheractivation product, Leu^28p-memapsin 2, was also purified. Kinetics of the activated memapsin 2 werecompared with pro-memapsin 2 using two new fluorogenic substrates, Arg-Glu(5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS))-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(4-(4-dimethylaminophenylazo)benzoic acid (DABCYL))-Arg and (7-methoxycoumarin-4-yl)acetyl (MCA))-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(2,4-dinitrophenyl (DNP)). These results establish that the activity of pro-memapsin 2 stems from a part-time and reversible uncovering of its active site by its pro region. Proteolyticremoval of part of the pro-peptide at Leu^28p or Gly^45p, which diminishes the affinity of the shortenedpro-peptide to the active site, results in activated memapsin 2. These results also suggest that Glu^33p-memapsin 2 observed in the cells expressing this enzyme [Vassar et al. (1999) Science 286, 735-741;Yan et al. (1999) Nature 402, 533-537] is an active intermediate of in vivo activation, or that the peptideGlu^33p-Arg^44p may serve a regulatory role.