Isolation and characterization of recombinant Drosophila Copia aspartic proteinase

Abstract

The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe-Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 degrees C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.

Biochemical characterization of Ty1 retrotransposon protease

“…Similarly to the proteases of HIV-1 and human foamy virus (HFV) [40,41], the enzyme activity was boosted by high ionic strength, and higher activity was observed for Ty1 PR at > 1 M NaCl concentration. Copia transposon protease of D. melanogaster also showed highest activity at high (2 M) NaCl concentration; activity was significantly lower at < 2 M NaCl concentration, but higher ionic strengths also decreased activity [17].…”

“…For instance, the optimal pH of HFV protease is 6.6-6.8 [40] while that of HIV-1 PR is between 4 and 6 [41]. Interestingly, the optimal pH required for D. melanogaster Copia transposon protease was found to be similar to that of HIV-1, with synthetic substrate cleaved most efficiently at pH 4.0 [17]. Temperature optimum was found to be close to 30˚C, lower enzyme activities were measured at higher temperatures ( Fig 4C).…”

“…Despite the expanding knowledge about retroviral-like proteases, the information about the biochemical, enzymatic, and structural characteristics of retrotransposon proteases are still limited. The only retrotransposon protease of which the recombinant form has been purified and characterized in detail is the protease of Drosophila melanogaster: Copia Gag precursor protein [17]. Both Copia of D. melanogaster and Ty1 of S. cerevisiae belong to the Copia transposon endopeptidase family (family A11) based on the MEROPS database [18], but they are distantly related members within this family [19].…”

Structure and function studies on enzymes with a catalytic carboxyl group(s): from ribonuclease T₁ to carboxyl peptidases

“…We extended our structure-function studies on pepsinogens and pepsins to those on other homologous aspartic peptidases, including human pepsinogens and pepsins 126,127) and non-mammalian pepsinogens and pepsins, 128–133) cathepsin D, 92,134–138) cathepsin E, 139–149) filarial parasite aspartic peptidases (from Brugia malayi ), 150) HIV-1 protease, 151,152) Drosophyla copia protease, 153) rhizopuspepsin (from Rhizopus chinensis ), 154,155) and aspergillopepsin I (from Aspergillus niger ). 156,157) …”

“…As a result, the major aspartic peptidase groups (MEROPS family A1) are now classified into two large groups, pepsin type (subfamily A1A) and nepenthesin type (subfamily A1B). It should be noted that in addition to family A1, several other minor aspartic peptidase families are known to date including Copia transposon peptidase ( Drosophila melanogaster ) 153) (family A11/subfamily A11A).…”