Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine

Tykvart, Jan; Bařinka, Cyril; Svoboda, Michal; Navrátil, Václav; Souček, Radko; Hubálek, Martin; Hradilek, Martin; Šácha, Pavel; Lubkowski, Jacek; Konvalinka, Jan

doi:10.1074/jbc.M114.628149

Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine

Journal Article · Mon Mar 09 00:00:00 EDT 2015 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M114.628149· OSTI ID:1251224

Tykvart, Jan ^[1]; Bařinka, Cyril ^[2]; Svoboda, Michal ^[3]; Navrátil, Václav ^[1]; Souček, Radko ^[4]; Hubálek, Martin ^[4]; Hradilek, Martin ^[4]; Šácha, Pavel ^[1]; Lubkowski, Jacek ^[5]; Konvalinka, Jan ^[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
Academy of Sciences of the Czech Republic (ASCR), Prague (Czech Republic). Inst. of Biotechnology
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
Academy of Sciences of the Czech Republic (ASCR), Prague (Czech Republic). Gilead Sciences and IOCB Research Centre. Inst. of Organic Chemistry and Biochemistry
National Inst. of Health (NIH), Frederick, MD (United States). Macromolecular Crystallography Lab. Center for Cancer Research

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).

View Accepted Manuscript (DOE)

Research Organization:: Academy of Sciences of the Czech Republic (ASCR), Prague (Czech Republic); National Inst. of Health (NIH), Frederick, MD (United States)

Sponsoring Organization:: Czech Science Foundation (GACR) (Czech Republic); European Molecular Biology Organization (EMBO) (Germany); European Union (EU) (Belgium); Ministry of Education (Czech Republic); National Inst. of Health (NIH) (United States); USDOE

Contributing Organization:: Charles Univ., Prague (Czech Republic)

Grant/Contract Number:: W-31109-ENG-38

OSTI ID:: 1251224

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 18 Vol. 290; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
Aminopeptidase
DPP IV Activity
Human Ileal Aminopeptidase
Intestinal Metabolism
Metalloprotease
Molecular Evolution
PICS
Protein Degradation
X-ray Crystallography

Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine

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References (28)

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