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.