gp180, a Protein That Binds Duck Hepatitis B Virus Particles, Has Metallocarboxypeptidase D-like Enzymatic Activity

Abstract

Duck hepatitis B virus is a member of the hepadnavirus group, which includes human hepatitis B virus (1). These viruses are small enveloped DNA viruses that infect liver cells, causing acute and chronic hepatitis (1). The cellular receptor for human hepatitis B virus has not been identified. A candidate receptor for the duck hepatitis B virus (DHBV) 1 has recently been described (2, 3). This 180-kDa protein, named gp180, was identified based on its ability to co-precipitate with DHBV particles or recombinant DHBV envelope proteins (2). gp180 was found to bind with high affinity and specificity to the preS region of the DHBV large envelope protein (2). The binding domain within the preS region that is responsible for the gp180 interaction overlaps key areas that have been established as important for viral infectivity (4). The cDNA for gp180 has recently been cloned, revealing that gp180 is a member of the metallocarboxypeptidase gene family (3). Interestingly, gp180 contains an N-terminal signal peptide, three metallocarboxypeptidase-like domains, a putative C-terminal transmembrane domain, and a short cytoplasmic tail. Of the known carboxypeptidases, gp180 has the highest homology to carboxypeptidase E (CPE). Specifically, the first and second domains of gp180 have 39% and 43% amino acid identity with mammalian CPE, and the third domain has 29% amino acid identity.CPE functions in the biosynthesis of numerous peptide hormones and transmitters (5, 6). CPE removes C-terminal Arg and Lys residues remaining after endoprotease cleavage of the prohormone. For many years, CPE was thought to be the only carboxypeptidase involved with neuropeptide biosynthesis. However, recent studies on the Cpe fat /Cpe fat mouse suggest that additional CPE-like carboxypeptidases partially compensate for a defective CPE. These mice have a point mutation within the coding region of the CPE gene that renders the protein inactive (7). Despite the absence of active CPE in the Cpe fat /Cpe fat mice, a reduced but detectable amount of peptide processing occurs, implying that additional carboxypeptidases may perform this step in vivo. A novel carboxypeptidase with CPE-like properties, named metallocarboxypeptidase D (CPD), was identified in bovine pituitary and in other tissues (CPD) (8). In bovine tissues, the major form of CPD is 180 kDa (8). In addition to the 180-kDa form, rat tissues also contain several smaller forms ranging from 100 to 180 kDa (9). In contrast, all other known carboxypeptidases are . The size of the major species of CPD is similar to that of gp180, raising the possibility that these proteins are homologs. In addition, the partial N-terminal amino acid sequence of bovine CPD is similar to that of the duck gp180, and both proteins are

Characterization of the Enzymatic Properties of the First and Second Domains of Metallocarboxypeptidase D

“…3, bottom), consistent with previous studies (47). In contrast, the pH optimum of the construct with only the active first domain is 6.3-7.5, and the construct with only the active second domain is 5.0 -6.5 (Fig.…”

“…Previous studies had established that duck CPD (gp180) lacking the C-terminal transmembrane domain was soluble and secreted from cells into the medium (47). The enzymatic properties of this 170-kDa form of duck CPD (termed gp170) were found to be identical to those of full-length gp180, so we chose to use the soluble form for further analysis of the individual domains.…”

“…Proteins were electrophoretically transferred to nitrocellulose from a denaturing polyacrylamide gel. Blots were probed with a 1:1000 dilution of polyclonal rabbit antiserum raised against gp170 (47). Following exposure of the blot to primary antiserum, the enhanced chemiluminescence method (Amersham Pharmacia Biotech) was used to detect bound antiserum.…”

“…The construction of gp170 and gp75 within the baculovirus expression vector pVL1392 (Pharmingen) were previously described (47), resulting in vectors pVL170 and pVL75. For mutagenesis, portions of gp170 were first inserted into the vector pAlter (Promega).…”

“…The purpose of the present study was to investigate whether the various domains perform complementary functions. A previous study investigated the activity of individual domains using a deletion approach and found that only the first two domains possessed enzymatic activity toward standard CPE substrates (47). However, removal of hundreds of residues can potentially cause large changes in the structure of the protein.…”