Substrate specificity and structural modeling of human carboxypeptidase z: A unique protease with a frizzled-like domain

Javier Garcia-Pardo, Sebastian Tanco, Maria C. Garcia-Guerrero, Sayani Dasgupta, Francesc Xavier Avilés, Julia Lorenzo, Lloyd D. Fricker

Research output: Contribution to journalArticlepeer-review

Abstract

Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1 position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.

Original languageEnglish (US)
Article number8687
Pages (from-to)1-24
Number of pages24
JournalInternational Journal of Molecular Sciences
Volume21
Issue number22
DOIs
StatePublished - Nov 2 2020

Keywords

  • Carboxypeptidase Z
  • Cysteine rich domain
  • Frizzled
  • Growth factor
  • Metallocarboxypeptidase
  • Substrate specificity
  • Wnt signaling

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

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