cDNA cloning and molecular characterization of human brain metalloprotease MP100: a beta-secretase candidate?
G Huber1, A Thompson, F Grüninger
1Pharma Division, Preclinical CNS Research, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Abstract:
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.
Insights
Metalloprotease MP100, initially a beta-secretase candidate, is identified as puromycin-sensitive aminopeptidase (PSA). While MP100 cleaves some amyloid beta peptides, it doesn't show true beta-secretase function in cells.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Metalloprotease MP100 was initially investigated as a beta-secretase candidate for its role in processing beta-amyloid precursor protein (beta-APP).
- Initial isolation utilized a beta-APP-derived peptide substrate for identification.
Purpose of the Study:
- To clarify the identity and function of Metalloprotease MP100.
- To determine if MP100 possesses true beta-secretase activity in cellular models.
Main Methods:
- Peptide sequencing of purified MP100 and subsequent cDNA cloning.
- Expression of recombinant human MP100 and testing its enzymatic activity on various substrates.
- Double immunoimaging and co-immunoprecipitation to assess protein localization and interaction in vivo.
- Co-expression of MP100 and beta-APP695 in HEK cells to measure amyloid beta (A beta) levels.
Main Results:
- MP100 showed high homology to puromycin-sensitive aminopeptidase (PSA), with minor sequence variations.
- Recombinant MP100 cleaved specific synthetic peptides, including an A beta peptide fragment, but not purified beta-APP.
- MP100 and beta-APP showed partial colocalization and co-immunoprecipitation in cellular and brain extracts.
- Co-expression of MP100 and beta-APP did not increase A beta levels in HEK cells.
Conclusions:
- MP100 is identified as a brain-enriched puromycin-sensitive aminopeptidase (PSA), not a primary beta-secretase.
- MP100 exhibits limited enzymatic activity on specific substrates but lacks true beta-secretase function in cellular contexts.
- MP100 may function as a secondary exoprotease involved in beta-APP/A beta metabolism.
More Related Videos
07:08A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status
Published on: October 20, 2016
09:00Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain
Published on: April 28, 2022