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Title: Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine

Abstract

N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. In this paper, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile. By solving the NAALADase L x-ray structure, we provide the first experimental evidence that it is a zinc-dependent metallopeptidase with a catalytic mechanism similar to that of glutamate carboxypeptidase II yet distinct substrate specificity. A proteome-based assay revealed that the NAALADL1 gene product possesses previously unrecognized aminopeptidase activity but no carboxy- or endopeptidase activity. These findings were corroborated by site-directed mutagenesis and identification of bestatin as a potent inhibitor of the enzyme. Analysis of NAALADL1 gene expression at both the mRNA and protein levels revealed the small intestine as the major site of protein expression and points toward extensive alternative splicing of the NAALADL1 gene transcript. Taken together, our data imply that the NAALADL1more » gene product's primary physiological function is associated with the final stages of protein/peptide digestion and absorption in the human digestive system. Finally, based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP).« less

Authors:
 [1];  [2];  [3];  [1];  [4];  [4];  [4];  [1];  [5];  [1]
  1. Academy of Sciences of the Czech Republic (ASCR), Prague (Czech Republic). Gilead Sciences and IOCB Research Centre. Inst. of Organic Chemistry and Biochemistry; Charles Univ., Prague (Czech Republic). Dept. of Biochemistry
  2. Academy of Sciences of the Czech Republic (ASCR), Prague (Czech Republic). Inst. of Biotechnology
  3. Academy of Sciences of the Czech Republic (ASCR), Prague (Czech Republic). Gilead Sciences and IOCB Research Centre. Inst. of Organic Chemistry and Biochemistry; Charles Univ., Prague (Czech Republic). Dept. of Physical and Macromolecular Chemistry
  4. Academy of Sciences of the Czech Republic (ASCR), Prague (Czech Republic). Gilead Sciences and IOCB Research Centre. Inst. of Organic Chemistry and Biochemistry
  5. National Inst. of Health (NIH), Frederick, MD (United States). Macromolecular Crystallography Lab. Center for Cancer Research
Publication Date:
Research Org.:
National Inst. of Health (NIH), Frederick, MD (United States); Academy of Sciences of the Czech Republic (ASCR), Prague (Czech Republic)
Sponsoring Org.:
USDOE; National Inst. of Health (NIH) (United States); Czech Science Foundation (GACR) (Czech Republic); Ministry of Education (Czech Republic); European Union (EU); European Molecular Biology Organization (EMBO) (Germany)
Contributing Org.:
Charles Univ., Prague (Czech Republic)
OSTI Identifier:
1251224
Grant/Contract Number:  
W-31-109-Eng38; P304–12-0847; LO 1302; CZ.1.05/1.1.00/02.0109
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 290; Journal Issue: 18; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Aminopeptidase; Intestinal Metabolism; Metalloprotease; Molecular Evolution; Protein Degradation; X-ray Crystallography; DPP IV Activity; PICS; Human Ileal Aminopeptidase

Citation Formats

Tykvart, Jan, Bařinka, Cyril, Svoboda, Michal, Navrátil, Václav, Souček, Radko, Hubálek, Martin, Hradilek, Martin, Šácha, Pavel, Lubkowski, Jacek, and Konvalinka, Jan. Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine. United States: N. p., 2015. Web. doi:10.1074/jbc.M114.628149.
Tykvart, Jan, Bařinka, Cyril, Svoboda, Michal, Navrátil, Václav, Souček, Radko, Hubálek, Martin, Hradilek, Martin, Šácha, Pavel, Lubkowski, Jacek, & Konvalinka, Jan. Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine. United States. doi:10.1074/jbc.M114.628149.
Tykvart, Jan, Bařinka, Cyril, Svoboda, Michal, Navrátil, Václav, Souček, Radko, Hubálek, Martin, Hradilek, Martin, Šácha, Pavel, Lubkowski, Jacek, and Konvalinka, Jan. Mon . "Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine". United States. doi:10.1074/jbc.M114.628149. https://www.osti.gov/servlets/purl/1251224.
@article{osti_1251224,
title = {Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine},
author = {Tykvart, Jan and Bařinka, Cyril and Svoboda, Michal and Navrátil, Václav and Souček, Radko and Hubálek, Martin and Hradilek, Martin and Šácha, Pavel and Lubkowski, Jacek and Konvalinka, Jan},
abstractNote = {N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. In this paper, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile. By solving the NAALADase L x-ray structure, we provide the first experimental evidence that it is a zinc-dependent metallopeptidase with a catalytic mechanism similar to that of glutamate carboxypeptidase II yet distinct substrate specificity. A proteome-based assay revealed that the NAALADL1 gene product possesses previously unrecognized aminopeptidase activity but no carboxy- or endopeptidase activity. These findings were corroborated by site-directed mutagenesis and identification of bestatin as a potent inhibitor of the enzyme. Analysis of NAALADL1 gene expression at both the mRNA and protein levels revealed the small intestine as the major site of protein expression and points toward extensive alternative splicing of the NAALADL1 gene transcript. Taken together, our data imply that the NAALADL1 gene product's primary physiological function is associated with the final stages of protein/peptide digestion and absorption in the human digestive system. Finally, based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP).},
doi = {10.1074/jbc.M114.628149},
journal = {Journal of Biological Chemistry},
number = 18,
volume = 290,
place = {United States},
year = {Mon Mar 09 00:00:00 EDT 2015},
month = {Mon Mar 09 00:00:00 EDT 2015}
}

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