250ecac367376a1d456e9e9bb7510fc9 intjoumolsci_a2020v21n22p8687.pdf 0d9818de7cddf513e3773ebac8882e9cb2a3406b intjoumolsci_a2020v21n22p8687.pdf 50f578e894537967af7b06fd94c21f03a23ece468179f69ee16f28073cdc6ed9 intjoumolsci_a2020v21n22p8687.pdf Title: Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain Subject: 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. Keywords: carboxypeptidase Z; metallocarboxypeptidase; substrate specificity; Wnt signaling; frizzled; cysteine rich domain; growth factor Author: Javier Garcia-Pardo, Sebastian Tanco, Maria C. Garcia-Guerrero, Sayani Dasgupta, Francesc Xavier Avilés, Julia Lorenzo and Lloyd D. Fricker Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Nov 18 07:17:28 2020 CET ModDate: Fri Nov 20 05:43:26 2020 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 24 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4090252 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- BTFGED+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 XHDYZV+VnURWPalladioL Type 1 Custom yes yes no 15 0 DFHPVC+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 TVRMKT+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 PTMWDV+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 OFLXAT+Rpxr Type 1 Builtin yes yes no 32 0 STWKGX+Pxsy Type 1 Builtin yes yes no 36 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 40 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 48 0 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yes no 296 0 BDIOMF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 299 0 BDIONH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 302 0 BDIPJD+TimesNewRoman CID TrueType Identity-H yes yes yes 305 0 BDIOKE+PalatinoLinotype TrueType WinAnsi yes yes no 322 0 BDIOMF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 325 0 BDIONH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 328 0 BDIPJD+TimesNewRoman CID TrueType Identity-H yes yes yes 331 0 BDIOJD+PalatinoLinotype CID TrueType Identity-H yes yes yes 354 0 BDIOKE+PalatinoLinotype TrueType WinAnsi yes yes no 360 0 BDIOMF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 363 0 BDIONH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 366 0 BDIPJD+TimesNewRoman CID TrueType Identity-H yes yes yes 369 0 YWGLGW+Rpxi Type 1 Builtin yes yes no 380 0 TGMEOR+Rpcxr Type 1 Builtin yes yes no 384 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 04:24:08 CEST RepresentationInformation: intjoumolsci_a2020v21n22p8687.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 11:39:44 CEST Size: 4090252 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 651 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Recombinant Protein Expression and Purification Destination: subsection.2.1 Item: Title: Enzymatic Characterization of CPZ Using Fluorescent Synthetic Substrates Destination: subsection.2.2 Item: Title: Substrate Specificity Profiling of Human CPZ by Quantitative Peptidomics Appproaches Destination: subsection.2.3 Item: Title: Structural Modeling of the Catalytic and TTL Domains of Human CPZ Destination: subsection.2.4 Item: Title: Structural Modeling of the CPZ Fz Domain: Insights into Its Structure and Wnt Recognition Destination: subsection.2.5 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Cell Culture Destination: subsection.4.1 Item: Title: Recombinant Protein Production and Purification Destination: subsection.4.2 Item: Title: HEK293T Bortezomib Treatment and Peptide Extraction Destination: subsection.4.3 Item: Title: Generation of the Tryptic Peptide Library Destination: subsection.4.4 Item: Title: Kinetic Measurements Using Fluorescent Synthetic Substrates Destination: subsection.4.5 Item: Title: Quantitative Peptidomics Analyses Destination: subsection.4.6 Item: Title: MALDI-TOF Mass Spectrometry Experiments Destination: subsection.4.7 Item: Title: Sequence Alignment and Structural Modeling Destination: subsection.4.8 Item: Title: References Destination: subsection.4.8 Info: Title: Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain Author: Javier Garcia-Pardo, Sebastian Tanco, Maria C. Garcia-Guerrero, Sayani Dasgupta, Francesc Xavier Avilés, Julia Lorenzo and Lloyd D. Fricker Subject: 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. Keywords: carboxypeptidase Z; metallocarboxypeptidase; substrate specificity; Wnt signaling; frizzled; cysteine rich domain; growth factor Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Nov 18 07:17:28 CET 2020 ModDate: Fri Nov 20 05:43:26 CET 2020 ID: 0xba1da18592948122daa5baf46cc07763, 0xba1da18592948122daa5baf46cc07763 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: OFLXAT+Rpxr FontSubset: true FirstChar: 11 LastChar: 62 FontDescriptor: FontName: OFLXAT+Rpxr Flags: Symbolic FontBBox: -40, -283, 904, 728 FontFile: true Font: BaseFont: TGMEOR+Rpcxr FontSubset: true FirstChar: 169 LastChar: 174 FontDescriptor: FontName: TGMEOR+Rpcxr Flags: Symbolic FontBBox: -212, -286, 1344, 973 FontFile: true Font: BaseFont: XIRVNA+EURB10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: XIRVNA+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true Font: BaseFont: STWKGX+Pxsy FontSubset: true FirstChar: 0 LastChar: 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Link Rect: 425, 456, 463, 467 Annotation: Subtype: Link Rect: 426, 430, 464, 441 Annotation: Subtype: Link Rect: 76, 345, 166, 376 Annotation: Subtype: Link Rect: 234, 340, 407, 351 Checksum: 4caf42b2 Type: CRC32 Checksum: 250ecac367376a1d456e9e9bb7510fc9 Type: MD5 Checksum: 0d9818de7cddf513e3773ebac8882e9cb2a3406b Type: SHA-1 Checksum: 50f578e894537967af7b06fd94c21f03a23ece468179f69ee16f28073cdc6ed9 Type: SHA-256