Cloning and Sequence Analysis of a Rat Liver cDNA Encoding Acyl-peptide Hydrolase

• Published in: The Journal of biological chemistry Vol. 264; no. 15; pp. 8892 - 8899
• Main Authors: Kobayashi, K, Lin, L W, Yeadon, J E, Klickstein, L B, Smith, J A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25-05-1989; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Aminopeptidases - genetics; Animals; Base Sequence; Biological and medical sciences; Biotechnology; Cloning, Molecular; DNA - genetics; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Genes; Genes. Genome; Genetic engineering; Genetic technics; Host Cell Factor C1; Humans; liver; Liver - enzymology; Methods. Procedures. Technologies; Molecular and cellular biology; Molecular cloning; Molecular genetics; Molecular Sequence Data; Nuclear Proteins - metabolism; Octamer Transcription Factor-1; Organ Specificity; Peptide Fragments - isolation & purification; Peptide Hydrolases - genetics; Protein Conformation; Rats; Restriction Mapping; RNA, Messenger - genetics; Transcription Factors; Trypsin; Vertebrata; Mammalia; Complementary DNA; Hydrolase; Rat; Nucleotide sequence; Enzyme; Liver; Rodentia; Primary structure; Molecular cloning
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)81877-4

Acyl-peptide hydrolase catalyzes the removal of an Nα-acetylated amino acid residue from an Nα-acetylated peptide. Two overlapping degenerate oligonucleotide probes based on the sequence of a CNBr tryptic peptide, derived from purified rat acyl-peptide hydrolase, were synthesized and used to screen a rat liver λgt11 cDNA library. A 2.5-kilobase cDNA was cloned and sequenced. This clone contained 2364 base pairs of rat acyl-peptide hydrolase sequence but lacked a translational initiation codon. Using a 220-base pair probe derived from near the 5′-end of this almost full-length cDNA to rescreen the library, full-length clones were isolated, which contained an in-frame ATG codon at nucleotides 6–8 and encoded the NH2-terminal sequence, Met-Glu-Arg-Gln…. The DNA sequence encoded a protein of 732 amino acid residues, 40% of which were confirmed by protein sequence data from 19 CNBr or CNBr tryptic peptides. The isolated enzyme is NH2-terminally blocked (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435–11445), and based on the NH2-terminal protein sequence deduced from the DNA sequence and the sequence of the most NH2-terminal CNBr peptide, it is likely that the NH2-terminal residue is an acetylated methionine residue, since such residues are frequently juxtaposed to glutamyl residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523–527). The RNA blot analysis revealed a single message of 2.7 kilobases in various rat tissues examined. Although this enzyme is known to be inhibited by diisopropyl fluorophosphate and acetylalanine chloromethyl ketone (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435–11445), no strong similarity in protein sequence has been found with other serine proteases. This result suggests that acyl-peptide hydrolase may be a unique serine protease.