title: Proteolytic activation of recombinant pro-memapsin 2 (pro-beta-secretase) studied with new fluorogenic substrates.
abstract: Memapsin 2 (beta-secretase), a membrane-anchored aspartic protease, is involved in the cleavage of beta-amyloid precursor protein to form beta-amyloid peptide. The primary structure of memapsin 2 suggests that 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 mechanism and to produce stable mature memapsin 2 for kinetic/specificity studies, we have investigated the activation of 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 the cleavage of the pro region to form mainly two activation products, Leu(30p)- and Gly(45p)-memapsin 2. Another activation product, Leu(28p)-memapsin 2, was also purified. Kinetics of the activated memapsin 2 were compared 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-dimethylaminophe nyl azo)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. Proteolytic removal of part of the pro-peptide at Leu(28p) or Gly(45p), which diminishes the affinity of the shortened pro-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 peptide Glu(33p)-Arg(44p) may serve a regulatory role.
authors.affiliation: Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
authors.forename: J
authors.initials: J
authors.lastname: Ermolieff
authors.forename: J A
authors.initials: JA
authors.lastname: Loy
authors.forename: G
authors.initials: G
authors.lastname: Koelsch
authors.forename: J
authors.initials: J
authors.lastname: Tang
mesh_terms: D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive; D002863:Chromogenic Compounds / Q000138:chemical synthesis / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D020349:Surface Plasmon Resonance
chemical_list: D002863:Chromogenic Compounds; D004792:Enzyme Precursors; D005456:Fluorescent Dyes; C414614:OM99-2; D009842:Oligopeptides; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human; C509280:BACE1 protein, human; D007930:Leucine; D005998:Glycine