Cargando…

Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain

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 pro...

Descripción completa

Detalles Bibliográficos
Autores principales: Garcia-Pardo, Javier, Tanco, Sebastian, Garcia-Guerrero, Maria C., Dasgupta, Sayani, Avilés, Francesc Xavier, Lorenzo, Julia, Fricker, Lloyd D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698808/
https://www.ncbi.nlm.nih.gov/pubmed/33217972
http://dx.doi.org/10.3390/ijms21228687
_version_ 1783615914692837376
author Garcia-Pardo, Javier
Tanco, Sebastian
Garcia-Guerrero, Maria C.
Dasgupta, Sayani
Avilés, Francesc Xavier
Lorenzo, Julia
Fricker, Lloyd D.
author_facet Garcia-Pardo, Javier
Tanco, Sebastian
Garcia-Guerrero, Maria C.
Dasgupta, Sayani
Avilés, Francesc Xavier
Lorenzo, Julia
Fricker, Lloyd D.
author_sort Garcia-Pardo, Javier
collection PubMed
description 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.
format Online
Article
Text
id pubmed-7698808
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-76988082020-11-29 Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain Garcia-Pardo, Javier Tanco, Sebastian Garcia-Guerrero, Maria C. Dasgupta, Sayani Avilés, Francesc Xavier Lorenzo, Julia Fricker, Lloyd D. Int J Mol Sci Article 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. MDPI 2020-11-18 /pmc/articles/PMC7698808/ /pubmed/33217972 http://dx.doi.org/10.3390/ijms21228687 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Garcia-Pardo, Javier
Tanco, Sebastian
Garcia-Guerrero, Maria C.
Dasgupta, Sayani
Avilés, Francesc Xavier
Lorenzo, Julia
Fricker, Lloyd D.
Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain
title Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain
title_full Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain
title_fullStr Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain
title_full_unstemmed Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain
title_short Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain
title_sort substrate specificity and structural modeling of human carboxypeptidase z: a unique protease with a frizzled-like domain
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698808/
https://www.ncbi.nlm.nih.gov/pubmed/33217972
http://dx.doi.org/10.3390/ijms21228687
work_keys_str_mv AT garciapardojavier substratespecificityandstructuralmodelingofhumancarboxypeptidasezauniqueproteasewithafrizzledlikedomain
AT tancosebastian substratespecificityandstructuralmodelingofhumancarboxypeptidasezauniqueproteasewithafrizzledlikedomain
AT garciaguerreromariac substratespecificityandstructuralmodelingofhumancarboxypeptidasezauniqueproteasewithafrizzledlikedomain
AT dasguptasayani substratespecificityandstructuralmodelingofhumancarboxypeptidasezauniqueproteasewithafrizzledlikedomain
AT avilesfrancescxavier substratespecificityandstructuralmodelingofhumancarboxypeptidasezauniqueproteasewithafrizzledlikedomain
AT lorenzojulia substratespecificityandstructuralmodelingofhumancarboxypeptidasezauniqueproteasewithafrizzledlikedomain
AT frickerlloydd substratespecificityandstructuralmodelingofhumancarboxypeptidasezauniqueproteasewithafrizzledlikedomain