Structural and biochemical characterization of a novel aminopeptidase from human intestine.
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. Here, 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. Based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP).
From the Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic, the Departments of Biochemistry and.
Jan
J
Tykvart
the Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 4, Czech Republic, and.
Cyril
C
Bařinka
From the Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic, Physical and Macromolecular Chemistry, Faculty of Natural Science, Charles University, Albertov 6, Prague 2, Czech Republic.
Michal
M
Svoboda
From the Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic, the Departments of Biochemistry and.
Václav
V
Navrátil
From the Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic.
Radko
R
Souček
From the Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic.
Martin
M
Hubálek
From the Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic.
Martin
M
Hradilek
From the Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic, the Departments of Biochemistry and.
Pavel
P
Šácha
the Center for Cancer Research, Macromolecular Crystallography Laboratory, NCI, National Institutes of Health, Frederick, Maryland 21702-1201.
Jacek
J
Lubkowski
From the Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic, the Departments of Biochemistry and jan.konvalinka@uochb.cas.cz.
Jan
J
Konvalinka
D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D043425:Glutamate Carboxypeptidase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007422:Intestines / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats
D010450:Endopeptidases; D018819:Dipeptidyl Peptidase 4; D043425:Glutamate Carboxypeptidase II
Aminopeptidase; DPP IV Activity; Human Ileal Aminopeptidase; Intestinal Metabolism; Metalloprotease; Molecular Evolution; PICS; Protein Degradation; X-ray Crystallography