cDNA cloning and molecular characterization of human brain metalloprotease MP100: a beta-secretase candidate?
Metalloprotease MP100 was originally isolated as a beta-secretase candidate from human brain using a beta-amyloid precursor protein (beta-APP)-derived p-nitroanilide (pNA) peptide substrate. Peptide sequences from purified MP100 were now found to resemble sequences reported for a puromycin-sensitive aminopeptidase (PSA) highly enriched in brain, and cDNA cloning revealed nearly complete homology of MP100 to PSA, with only a single bp difference resulting in an amino acid change at position 184. Another MP100 cDNA encoded a protein with a 36-amino acid deletion (positions 180-217) and a two-amino acid insertion after Val533. Purified recombinant human MP100 cleaved the original pNA substrate as well as a free beta-site-spanning amyloid beta (A beta) peptide (A beta(-10/+10)), generating A beta(1-10). The latter substrate, however, remained uncleaved, if N- and C-terminally blocked, and also purified beta-APP was not cleaved. Double immunoimaging revealed partial, patchy, colocalization of beta-APP and MP100 in doubly transfected human embryonic kidney cells (HEK cells) and in normal neuroblastoma cells, and both proteins could be coimmunoprecipitated from rat brain extracts, suggesting their close vicinity in vivo. Coexpression of MP100 and beta-APP695, however, did not boost A beta levels in HEK cells, although active enzyme was produced. Thus, MP100 does not exert true beta-secretase-like function in cells, although it may well act as a secondary exoprotease in a complex beta-APP/A beta metabolism.
Pharma Division, Preclinical CNS Research, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
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G
Huber
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A
Thompson
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F
Grüninger
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H
Mechler
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R
Hochstrasser
H P
HP
Hauri
P
P
Malherbe
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D016564:Amyloid beta-Protein Precursor; D018076:DNA, Complementary; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human