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

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

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

the Center for Cancer Research Macromolecular Crystallography Laboratory NCI National Institutes of Health Frederick Maryland 21702 1201

the Institute of Biotechnology Academy of Sciences of the Czech Republic Vídeňská 1083 Prague 4 Czech Republic and

Zobrazit více v PubMed

Pangalos M. N., Neefs J. M., Somers M., Verhasselt P., Bekkers M., van der Helm L., Fraiponts E., Ashton D., Gordon R. D. (1999) Isolation and expression of novel human glutamate carboxypeptidases with N-acetylated α-linked acidic dipeptidase and dipeptidyl peptidase IV activity. J. Biol. Chem. 274, 8470–8483 PubMed

Shneider B. L., Thevananther S., Moyer M. S., Walters H. C., Rinaldo P., Devarajan P., Sun A. Q., Dawson P. A., Ananthanarayanan M. (1997) Cloning and characterization of a novel peptidase from rat and human ileum. J. Biol. Chem. 272, 31006–31015 PubMed

Barinka C., Rinnová M., Sácha P., Rojas C., Majer P., Slusher B. S., Konvalinka J. (2002) Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II. J. Neurochem. 80, 477–487 PubMed

Hlouchová K., Barinka C., Klusák V., Sácha P., Mlcochová P., Majer P., Rulísek L., Konvalinka J. (2007) Biochemical characterization of human glutamate carboxypeptidase III. J. Neurochem. 101, 682–696 PubMed

Tykvart J., Sácha P., Bařinka C., Knedlík T., Starková J., Lubkowski J., Konvalinka J. (2012) Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II. Protein Expr. Purif. 82, 106–115 PubMed PMC

Kozísek M., Sasková K. G., Rezácová P., Brynda J., van Maarseveen N. M., De Jong D., Boucher C. A., Kagan R. M., Nijhuis M., Konvalinka J. (2008) Ninety-nine is not enough: molecular characterization of inhibitor-resistant human immunodeficiency virus type 1 protease mutants with insertions in the flap region. J. Virol. 82, 5869–5878 PubMed PMC

Langone J. J., Van Vunakis H. (1986) Immunological techniques. Part I. Hybridoma technology and monoclonal antibodies. Methods Enzymol. 121, 1–947 PubMed

Otwinowski Z., Minor W. (1997) Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 PubMed

McCoy A. J., Grosse-Kunstleve R. W., Adams P. D., Winn M. D., Storoni L. C., Read R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 PubMed PMC

Murshudov G. N., Vagin A. A., Dodson E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 PubMed

Emsley P., Cowtan K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 PubMed

Chen V. B., Arendall W. B., 3rd, Headd J. J., Keedy D. A., Immormino R. M., Kapral G. J., Murray L. W., Richardson J. S., Richardson D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 PubMed PMC

Rovenská M., Hlouchová K., Sácha P., Mlcochová P., Horák V., Zámecník J., Barinka C., Konvalinka J. (2008) Tissue expression and enzymologic characterization of human prostate specific membrane antigen and its rat and pig orthologs. Prostate 68, 171–182 PubMed

Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 PubMed

Barlos K., Chatzi O., Gatos D., Stavropoulos G. (1991) 2-Chlorotrityl chloride resin: studies on anchoring of Fmoc-amino acids and peptide cleavage. Int. J. Pept. Protein Res. 37, 513–520 PubMed

Kaiser E., Colescott R. L., Bossinger C. D., Cook P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595–598 PubMed

Woodward C., Henderson J. K., Wielgos T. (2007) High-speed Amino Acid Analysis (AAA) on 1.8 μm Reversed-phase (RP) Columns. Agilent Technologies Application Note 5989-6297EN, Agilent Technologies, Inc., Wilmington, DE

Schilling O., Huesgen P. F., Barré O., Auf dem Keller U., Overall C. M. (2011) Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry. Nat. Protoc. 6, 111–120 PubMed

Krissinel E. (2012) Enhanced fold recognition using efficient short fragment clustering. J. Mol. Biochem. 1, 76–85 PubMed PMC

Mesters J. R., Barinka C., Li W., Tsukamoto T., Majer P., Slusher B. S., Konvalinka J., Hilgenfeld R. (2006) Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer. EMBO J. 25, 1375–1384 PubMed PMC

Pavlícek J., Ptácek J., Barinka C. (2012) Glutamate carboxypeptidase II: an overview of structural studies and their importance for structure-based drug design and deciphering the reaction mechanism of the enzyme. Curr. Med. Chem. 19, 1300–1309 PubMed

Lawrence C. M., Ray S., Babyonyshev M., Galluser R., Borhani D. W., Harrison S. C. (1999) Crystal structure of the ectodomain of human transferrin receptor. Science 286, 779–782 PubMed

Zheng H., Chruszcz M., Lasota P., Lebioda L., Minor W. (2008) Data mining of metal ion environments present in protein structures. J. Inorg. Biochem. 102, 1765–1776 PubMed PMC

Klusák V., Barinka C., Plechanovová A., Mlcochová P., Konvalinka J., Rulísek L., Lubkowski J. (2009) Reaction mechanism of glutamate carboxypeptidase II revealed by mutagenesis, x-ray crystallography, and computational methods. Biochemistry 48, 4126–4138 PubMed PMC

Navrátil M., Ptáček J., Šácha P., Starková J., Lubkowski J., Bařinka C., Konvalinka J. (2014) Structural and biochemical characterization of the folyl-poly-γ-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II. FEBS J. 281, 3228–3242 PubMed PMC

Lindner H. A., Lunin V. V., Alary A., Hecker R., Cygler M., Ménard R. (2003) Essential roles of zinc ligation and enzyme dimerization for catalysis in the aminoacylase-1/M20 family. J. Biol. Chem. 278, 44496–44504 PubMed

Thierry-Mieg D., Thierry-Mieg J. (2006) AceView: a comprehensive cDNA-supported gene and transcripts annotation. Genome Biol. 7, S12.1–14 PubMed PMC

Sedo A., Malík R. (2001) Dipeptidyl peptidase IV-like molecules: homologous proteins or homologous activities? Biochim. Biophys. Acta 1550, 107–116 PubMed

Taylor A. (1996) in The Aminopeptidases (Taylor A., ed) pp. 1–20, Landes Bioscience Publishers, Austin, TX

Weiss M. S. (2001) Global indicators of x-ray data quality. J. Appl. Crystallogr. 34, 130–135

Cheng Y., Prusoff W. H. (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 PubMed

DDI2 protease controls embryonic development and inflammation via TCF11/NRF1

Uncovering the essential roles of glutamate carboxypeptidase 2 orthologs in Caenorhabditis elegans

Characterization of glutamate carboxypeptidase 2 orthologs in trematodes

A New Approach for the Diagnosis of Myelodysplastic Syndrome Subtypes Based on Protein Interaction Analysis

The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity

Mouse glutamate carboxypeptidase II (GCPII) has a similar enzyme activity and inhibition profile but a different tissue distribution to human GCPII

Zobrazit více v PubMed

PDB
2PVW, 3BXM, 4TWE