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gp180, a protein that binds duck hepatitis B virus particles, has metallocarboxypeptidase D-like enzymatic activity
F J Eng1, E G Novikova, K Kuroki
1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Abstract:
Duck gp180 was previously identified by its ability to bind to the preS envelope protein of duck hepatitis B virus particles (Kuroki, K. , Cheung, R., Marion, P. L., and Ganem, D. (1994) J. Virol. 68, 2091-2096). Cloning and sequencing of gp180 cDNA revealed that it is a polyprotein with three carboxypeptidase-like domains (Kuroki, K., Eng, F., Ishikawa, T., Turck, C., Harada, F., and Ganem, D. (1995) J. Biol. Chem. 270, 15022-15028). To evaluate enzymatic properties of this protein, a soluble 170-kDa form of the protein (gp170) lacking the C-terminal transmembrane domain and cytoplasmic tail was expressed in a baculovirus system. The purified 170-kDa protein cleaved 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Phe-Ala-Arg with a pH optimum of 5.5-6.5. With this substrate at pH 5.5, the 170-kDa protein displayed a Km of 12 microM and a Kcat of 57 s-1. Dansyl-Pro-Ala-Arg and dansyl-Phe-Phe-Arg were cleaved with Km values of 17 and 21 microM, and Kcat values of 57 and 17 s-1, respectively. Constructs containing only the first or second carboxypeptidase domains also showed enzymatic activity. The effects of inhibitors and ions on enzyme activity of gp170 were generally similar to the effects of these compounds on purified bovine carboxypeptidase D. To evaluate the regions within gp180 necessary for binding preS, a series of deletion mutants were expressed in the 293T human kidney cell line. Deletions of the first and second domains, leaving the third domain intact, eliminated carboxypeptidase activity but retained preS binding. Deletion of the third domain eliminated preS binding but not carboxypeptidase activity. These results indicate that the third domain is responsible for preS binding, and this binding does not require carboxypeptidase activity.
Insights
Duck gp180 protein exhibits carboxypeptidase activity and binds to duck hepatitis B virus preS envelope protein. Its third domain is crucial for preS binding, independent of enzymatic function.
Area of Science:
- Biochemistry
- Virology
- Molecular Biology
Background:
- Duck gp180 protein was identified for its interaction with duck hepatitis B virus preS envelope protein.
- Gene sequencing revealed gp180 as a polyprotein containing three carboxypeptidase-like domains.
Purpose of the Study:
- To characterize the enzymatic properties of a soluble form of duck gp180 (gp170).
- To identify the specific domains of gp180 responsible for binding to the preS protein.
Main Methods:
- Expression and purification of a soluble 170-kDa form of duck gp180 (gp170) using a baculovirus system.
- Enzymatic assays using dansylated peptide substrates to determine kinetic parameters (Km, Kcat) and pH optimum.
- Construction and expression of deletion mutants in 293T cells to map preS binding and enzymatic activity domains.
Main Results:
- Purified gp170 demonstrated carboxypeptidase activity with a pH optimum of 5.5-6.5.
- Enzymatic activity and substrate specificity of gp170 showed similarities to bovine carboxypeptidase D.
- Deletion analysis indicated that the third carboxypeptidase-like domain is essential for preS binding, while the first two domains possess enzymatic activity.
- PreS binding was retained even when enzymatic activity was abolished by domain deletions.
Conclusions:
- The third domain of duck gp180 is solely responsible for binding to the duck hepatitis B virus preS protein.
- Carboxypeptidase activity is not required for the preS binding function of gp180.
- Duck gp180 possesses distinct functional domains for enzymatic activity and viral protein interaction.