Cloning and Expression of a Novel Hepatitis B Virus-binding Protein from HepG2 Cells(847 views) De Falco S, Ruvoletto MG, Verdoliva A, Ruvo M, Raucci A, Marino M, Senatore S, Cassani G, Alberti A, Pontisso P, Fassina G
Keywords: Amino Acids, Cell Membranes, Cloning, Viruses, Plasma Membranes, Proteins, Cathepsin L, Chymotrypsin A, Cysteine Proteinase, Nucleotide, Papain, Protein Pres1, Squamous Cell Carcinoma Antigen, Unclassified Drug, Virus Protein, Complementary Dna, Hepatitis B Virus Binding Protein, Human, Hepatitis B Virus-Binding Protein, Polyadenylic Acid, Proteinase Inhibitor, Recombinant Protein, Serine Proteinase Inhibitor, Squamous Cell Carcinoma Related Antigen, Squamous Cell Carcinoma-Related Antigen, Tumor Antigen, Virus Receptor, Amino Acid Substitution, Animal Cell, Article, Cho Cell, Concentration Response, Controlled Study, Enzyme Inhibition, Gene Overexpression, Genetic Transfection, Human Cell, Molecular Cloning, Nonhuman, Polymerase Chain Reaction, Priority Journal, Protein Domain, Protein Expression, Protein Isolation, Receptor Intrinsic Activity, Virus Adsorption, Virus Cell Interaction, Virus Particle, Amino Acid Sequence, Binding Competition, Biosynthesis, Cell Culture, Cell Line, Chemistry, Chromatography, Dose Response, Drug Antagonism, Hamster, Kinetics, Liver Cell, Metabolism, Molecular Genetics, Nucleotide Sequence, Protein Binding, Protein Tertiary Structure, Sequence Homology, Cricetinae, Cricetulus Griseus, Dna Viruses, Neoplasm, Base Sequence, Cysteine Endopeptidases, Dose-Response Relationship, Hepatocytes, Molecular Sequence Data, Protease Inhibitors, Protein Structure, Serpins, Time Factors, Tumor Cells,
Affiliations: *** IBB - CNR ***
TECNOGEN S.C.p.A., Parco Scientifico, Piana M. Verna (CE), Caserta 81015, Italy
Universita degli Studi di Padova, Dipto. di Med. Clin. e Sperimentale, Clinica Medica 5, 35128 Padova, Italy
TECNOGEN S. C. p. A., Parco Scientifico, Piana M. Verna (CE), Caserta 81015, Italy
References: Not available.
Cloning and Expression of a Novel Hepatitis B Virus-binding Protein from HepG2 Cells
A direct involvement of the hepatitis B virus (HBV) preS1-(21-47) sequence in virus attachment to cell membrane receptor(s) and the presence on the plasma membranes of HepG2 cells of protein(s) with receptor activity for HBV have been suggested by many previous experiments. In this study, by using a tetravalent derivative of the preS1-(21-47) sequence, we have isolated by affinity chromatography from detergent-solubilized HepG2 plasma membranes a 44-kDa protein (HBV-binding protein; HBV-BP), which was found to closely correspond to the human squamous cell carcinoma antigen 1 (SCCA1), a member of the ovalbumin family of serine protease inhibitors. Comparison of SCCA1 sequence with the sequence of the corresponding HBV-BP cDNA, cloned by polymerase chain reaction starting from RNA poly(A) + fractions extracted from HepG2 cells, indicated the presence of only four nucleotide substitutions in the coding region, leading to three amino acid changes. Intact recombinant HBV-BP lacked inhibitory activity for serine proteases such as α-chymotrypsin and trypsin but inhibited with high potency cysteine proteases such as papain and cathepsin L. Direct binding experiments confirmed the interaction of recombinant HBV-BP with the HBV preS1 domain. HepG2 cells overexpressing HBV-BP after transfection of corresponding cDNA showed a virus binding capacity increased by 2 orders of magnitude compared with untransfected cells, while Chinese hamster ovary cells, which normally do not bind to HBV, acquired susceptibility to HBV binding after transfection. Native HBV particle entry was enhanced in transfected cells. Both recombinant HBV-BP and antibodies to recombinant HBV-BP blocked virus binding and internalization in transfected cells as well as in primary human hepatocytes in a dose-dependent manner. Our findings suggest that this protein plays a major role in HBV infection.
Cloning and Expression of a Novel Hepatitis B Virus-binding Protein from HepG2 Cells
Kállay C, Dávid A, Timári S, Nagy EM, Sanna D, Garribba E, Micera G, De Bona P, Pappalardo G, Rizzarelli E, Sóvágó I * Copper(II) complexes of rat amylin fragments(554 views) Dalton T (ISSN: 1477-9234, 1477-9226, 1477-9234electronic), 2011 Oct 14; 40(38): 9711-9721. Impact Factor:3.838 ViewExport to BibTeXExport to EndNote