cDNA Cloning and Molecular Characterization of Human Brain Metalloprotease MP100

: Metalloprotease MP100 was originally isolated as aβ‐secretase candidate from human brain using a β‐amyloid precursorprotein (β‐APP)‐derived p ‐nitroanilide (pNA) peptide substrate.Peptide sequences from purified MP100 were now found to resemble sequencesreported for a puromycin‐sensitive aminopeptidase (PSA) highly enriched inbrain, and cDNA cloning revealed nearly complete homology of MP100 to PSA,with only a single bp difference resulting in an amino acid change at position184. Another MP100 cDNA encoded a protein with a 36‐amino acid deletion(positions 180‐217) and a two‐amino acid insertion after Val 533 .Purified recombinant human MP100 cleaved the original pNA substrate as well asa free β‐site‐spanning amyloid β (Aβ) peptide(Aβ ‐10/+10 ), generating Aβ 1‐10 . The lattersubstrate, however, remained uncleaved, if N‐ and C‐terminally blocked, andalso purified β‐APP was not cleaved. Double immunoimaging revealedpartial, patchy, colocalization of β‐APP and MP100 in doubly transfectedhuman 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 andβ‐APP 695 , however, did not boost Aβ levels in HEK cells, although active enzyme was produced. Thus, MP100 does not exert true β‐secretase‐like function in cells, although it may well act as a secondary exoprotease in a complex β‐APP/Aβ metabolism.