Molecular Cloning and Expression of Rat Liver Aminopeptidase B

Abstract

We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A 4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-␤-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl ؊ -dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.Aminopeptidases (Ap 1 ) play critical roles in processes such as protein maturation, protein digestion in its terminal stage, regulation of hormone levels, selective or homeostatic protein turnover, and plasmid stabilization (1).These enzymes generally have broad substrate specificity and occur in several forms. Of such enzymes, Ap-B (EC 3.4.11.6), which was discovered in 1964 from a rat duodenal supernatant fraction (2), catalyzes specifically the removal of unsubstituted, N-terminal Arg and Lys residues from peptides and ␤-naphthylamide (NA) or other synthetic derivatives. The rate of arginine release is typically about twice that of lysine (3). Although there are numerous reports on the enzyme, conclusive evidence on the characterization of the enzyme is restricted to its Cl Ϫ -dependent activation (4) and sensitivity to inhibition by bestatin (5) and Arphamenine B (6). Since earlier reports based on inhibition and restoration experiments utilizing metal chelators and divalent metal ions (4, 7, 8), Ap-B has been regarded as a metalloenzyme; but there is some controversy on this point. Söderling and Mä kinen (9) used atomic absorption spectrophotometry to measure the zinc content of this enzyme, but they detected no zinc. On the other hand, Ocain and Rich (10) reported that this enzyme was inhibited by L-lysine thiol, a compound that would be expected to inhibit the enzyme by interaction with a catalytic zinc ion in the active site. In the current study, we cloned Ap-B cDNA and found that the enzyme belongs to the family of zinc metallopeptidases and contains the zinc-binding domain in its sequence. In addition, by an atomic absorption experiment we demonstrated that the recombinant enzyme contains a zinc ion, which we presume to be catalytic. EXPERIMENTAL PROCEDURESMaterials-Various aminoacyl-NAs (Arg-, Bz-Arg-, Leu-, IIe-, Val-, Ala-, Pro-, Ser-, Tyr-, Phe-, Met-) were obtained from Sigma, and NAs (Lys-, His-, Asp-, Hyp-, Trp-, Gly-) were from Koch-Light Lab., Ltd. (Colnbrook, United Kingdom). Restriction and modifying enzymes were from Toyobo Co

Zinc regulation of aminopeptidase B involved in neuropeptide production

“…AP-B generates mature (Met)enkephalin (ME) from Arg-ME and Lys-ME peptide intermediates [6]. Sequence analyses of bovine and rat AP-B cDNAs [6][7][8] illustrate the presence of the metal-binding HEXXH motif which is typical of members of metalloprotease families [9,10]. Most metalloproteases are regulated by zinc metal ion, but a few metalloproteases, such as methionyl aminopeptidase in Escherichia coli (M24 family of metalloprotease) and aminopeptidase T from thermus aquaticus (M29 family) utilize cobalt for activity [10].…”

Human Leukocyte-derived Arginine Aminopeptidase

“…Gluzincin aminopeptidases share the consensus HEXXH(X) 18 E zinc-binding motif essential for enzymatic activity (10). This growing family of mammalian zinccontaining aminopeptidase includes membrane-bound (P-LAP, aminopeptidase A, aminopeptidase N, and thyrotropin-releasing hormone degrading enzyme) (3,4,6,11,12), cytosolic (puromycin-sensitive aminopeptidase (PSA) and leukotriene A 4 hydrolase) (13,14), secretory (aminopeptidase B) (15), and ER resident (A-LAP/ERAP1) (9,16) proteins. Mutational analyses revealed that essential amino acid residues are well conserved among members of the family (17)(18)(19)(20).…”

PC18, a Specific Aminopeptidase N Inhibitor, Induces Vasopressin Release by Increasing the Half-Life of Brain Angiotensin III

“…The cloning of the cDNA encoding APN [7, 8]revealed, as in the case of APA [9, 10, 11]and aminopeptidase B (APB, EC 3.4.11.6) [12, 13], the presence of the consensus sequence HEXXH...E found in the zinc metalloprotease family classified as gluzincins [14, 15]. Rat APN shares an overall amino acid homology of 34 and 15% with rat APA and APB, respectively [7, 8, 9, 12, 13], and the conservation is higher in the region surrounding the zinc-binding motif.…”