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Title: Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues]

Abstract

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamicsmore » that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.« less

Authors:
 [1];  [2];  [2];  [2];  [3];  [2]
  1. Mayo Clinic Comprehensive Cancer Center, Jacksonville, FL (United States); Columbia Univ., New York, NY (United States)
  2. Mayo Clinic Comprehensive Cancer Center, Jacksonville, FL (United States)
  3. Brookhaven National Lab. (BNL), Upton, NY (United States)
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source II (NSLS-II)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1341510
Report Number(s):
BNL-108587-2015-JA
Journal ID: ISSN 0021-9258; R&D Project: LS001
Grant/Contract Number:  
SC00112704
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 290; Journal Issue: 35; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; National Synchrotron Light Source II; crystal structure; enzyme catalysis; protease inhibitor; protein evolution; protein structure; proteolysis; serine protease; site-directed mutagenesis; substrate specificity; trypsin

Citation Formats

Alloy, Alexandre P., Kayode, Olumide, Wang, Ruiying, Hockla, Alexandra, Soares, Alexei S., and Radisky, Evette S. Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues]. United States: N. p., 2015. Web. doi:10.1074/jbc.M115.662429.
Alloy, Alexandre P., Kayode, Olumide, Wang, Ruiying, Hockla, Alexandra, Soares, Alexei S., & Radisky, Evette S. Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues]. United States. doi:10.1074/jbc.M115.662429.
Alloy, Alexandre P., Kayode, Olumide, Wang, Ruiying, Hockla, Alexandra, Soares, Alexei S., and Radisky, Evette S. Tue . "Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues]". United States. doi:10.1074/jbc.M115.662429. https://www.osti.gov/servlets/purl/1341510.
@article{osti_1341510,
title = {Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues]},
author = {Alloy, Alexandre P. and Kayode, Olumide and Wang, Ruiying and Hockla, Alexandra and Soares, Alexei S. and Radisky, Evette S.},
abstractNote = {Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.},
doi = {10.1074/jbc.M115.662429},
journal = {Journal of Biological Chemistry},
number = 35,
volume = 290,
place = {United States},
year = {2015},
month = {7}
}

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