title: Sequence and conformational specificity in substrate recognition: several human Kunitz protease inhibitor domains are specific substrates of mesotrypsin.
abstract: Mesotrypsin is an isoform of trypsin that is uniquely resistant to polypeptide trypsin inhibitors and can cleave some inhibitors rapidly. Previous studies have shown that the amyloid precursor protein Kunitz protease inhibitor domain (APPI) is a specific substrate of mesotrypsin and that stabilization of the APPI cleavage site in a canonical conformation contributes to recognition by mesotrypsin. We hypothesized that other proteins possessing potential cleavage sites stabilized in a similar conformation might also be mesotrypsin substrates. Here we evaluated a series of candidate substrates, including human Kunitz protease inhibitor domains from amyloid precursor-like protein 2 (APLP2), bikunin, hepatocyte growth factor activator inhibitor type 2 (HAI2), tissue factor pathway inhibitor-1 (TFPI1), and tissue factor pathway inhibitor-2 (TFPI2), as well as E-selectin, an unrelated protein possessing a potential cleavage site displaying canonical conformation. We find that Kunitz domains within APLP2, bikunin, and HAI2 are cleaved by mesotrypsin with kinetic profiles of specific substrates. TFPI1 and TFPI2 Kunitz domains are cleaved less efficiently by mesotrypsin, and E-selectin is not cleaved at the anticipated site. Cocrystal structures of mesotrypsin with HAI2 and bikunin Kunitz domains reveal the mode of mesotrypsin interaction with its canonical substrates. Our data suggest that major determinants of mesotrypsin substrate specificity include sequence preferences at the P1 and P'2 positions along with conformational stabilization of the cleavage site in the canonical conformation. Mesotrypsin up-regulation has been implicated previously in cancer progression, and proteolytic clearance of Kunitz protease inhibitors offers potential mechanisms by which mesotrypsin may mediate pathological effects in cancer.
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224.
authors.forename: Devon
authors.initials: D
authors.lastname: Pendlebury
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224.
authors.forename: Ruiying
authors.initials: R
authors.lastname: Wang
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224.
authors.forename: Rachel D
authors.initials: RD
authors.lastname: Henin
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Hockla
authors.affiliation: the Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
authors.forename: Alexei S
authors.initials: AS
authors.lastname: Soares
authors.affiliation: the Medical Genome Facility Proteomics Core, Mayo Clinic, Rochester, Minnesota 55905, and.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Madden
authors.affiliation: the A. A. Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow 127994, Russia.
authors.forename: Marat D
authors.initials: MD
authors.lastname: Kazanov
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224, radisky.evette@mayo.edu.
authors.forename: Evette S
authors.initials: ES
authors.lastname: Radisky
mesh_terms: D000510:Alpha-Globulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007611:Aprotinin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D019040:E-Selectin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C084104:APLP2 protein, human; D000510:Alpha-Globulins; D016564:Amyloid beta-Protein Precursor; D019040:E-Selectin; D006023:Glycoproteins; D008074:Lipoproteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011480:Protease Inhibitors; C109148:SPINT2 protein, human; C016001:alpha-1-microglobulin; C051928:lipoprotein-associated coagulation inhibitor; C021240:tissue-factor-pathway inhibitor 2; D007611:Aprotinin; C040749:PRSS3 protein, human; D014357:Trypsin
keywords: Crystal Structure; Enzyme Kinetics; Protease Inhibitor; Protein Degradation; Protein Structure; Protein-Protein Interaction; Proteolysis; Serine Protease; Substrate Specificity; Trypsin