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Proteolytic activation of recombinant pro-memapsin 2 (pro-beta-secretase) studied with new fluorogenic substrates
J Ermolieff1, J A Loy, G Koelsch
1Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
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.
Insights
Memapsin 2 (beta-secretase) activation involves partial pro-peptide removal, uncovering the active site. This process, simulated by trypsin-like proteases, yields active memapsin 2, crucial for beta-amyloid peptide formation.
Area of Science:
- Biochemistry
- Enzymology
- Neuroscience
Background:
- Memapsin 2 (beta-secretase) cleaves beta-amyloid precursor protein, producing beta-amyloid peptide.
- Memapsin 2 is synthesized as an inactive pro-form (pro-memapsin 2) requiring proteolytic activation.
Purpose of the Study:
- To investigate the activation mechanism of recombinant pro-memapsin 2.
- To produce stable, mature memapsin 2 for kinetic and specificity studies.
- To understand the role of the pro-peptide in memapsin 2 activity.
Main Methods:
- Recombinant pro-memapsin 2 activation using proteases with trypsin-like specificity (clostripain, kallikrein, trypsin).
- Purification and characterization of memapsin 2 activation products.
- Kinetic analysis of activated memapsin 2 and pro-memapsin 2 using novel fluorogenic substrates.
Main Results:
- Clostripain, kallikrein, and trypsin activated pro-memapsin 2.
- Clostripain cleavage yielded Leu(30p)-memapsin 2 and Gly(45p)-memapsin 2, with Leu(28p)-memapsin 2 also purified.
- Pro-memapsin 2 activity results from reversible uncovering of the active site by the pro-region; removal of parts of the pro-peptide activates the enzyme.
Conclusions:
- Proteolytic activation of memapsin 2 involves partial removal of the pro-peptide, diminishing pro-peptide affinity to the active site.
- The findings suggest Glu(33p)-memapsin 2 is an active intermediate in vivo or that the Glu(33p)-Arg(44p) peptide has a regulatory function.