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

Journal Article · · Journal of Biological Chemistry
 [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
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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).

Research Organization:
National Inst. of Health (NIH), Frederick, MD (United States); Academy of Sciences of the Czech Republic (ASCR), Prague (Czech Republic)
Sponsoring Organization:
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 Organization:
Charles Univ., Prague (Czech Republic)
Grant/Contract Number:
W-31-109-Eng38; P304–12-0847; LO 1302; CZ.1.05/1.1.00/02.0109
OSTI ID:
1251224
Journal Information:
Journal of Biological Chemistry, Vol. 290, Issue 18; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 12 works
Citation information provided by
Web of Science

References (28)

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Cited By (1)

The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity: Calcium is Required for GCPII Function journal September 2018

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Related Subjects

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