title: Proteolytic fragments of anti-HIV and anti-tumor proteins MAP30 and GAP31 are biologically active.
abstract: We analyzed the structural and functional organization of anti-HIV and anti-tumor proteins MAP30 and GAP31 by limited proteolysis with endopeptidases Lys-C and Glu-C (V8). MAP30 and GAP31 are resistant to proteolytic digestion under conditions of as much as 5% (w/w) proteases. In the presence of 10% (w/w) protease, the central regions of the proteins are still resistant to proteolysis, whereas the N- and C-termini are accessible. Peptide fragments were purified by FPLC on Superdex 75 columns, characterized by gel electrophoresis, identified by amino acid sequencing, and analyzed for anti-HIV, anti-tumor, and other biochemical activities. We report here that limited proteolysis yields biologically active fragments of both MAP30 and GAP31. These fragments are active against HIV-1 and tumor cells with EC(50)s in the sub-nanomolar ranges, 0.2-0.4 nM. At the dose levels used in the assays, little cytotoxicity to normal cells was observed. In addition, these fragments remain fully active in HIV-integrase inhibition and HIV-LTR topological inactivation, but not ribosome inactivation. These results demonstrate that the antiviral and anti-tumor activities of MAP30 and GAP31 are independent of ribosome inactivation activity. In addition, we demonstrate that portions of the N- and C-termini are not essential for antiviral and anti-tumor activities, but do appear to be required for ribosome inactivation. These results may provide novel strategies for rational design and targeted development of mimetic antiviral and anti-tumor therapeutics.
authors.affiliation: American Biosciences, New York, New York, 10021, USA.
authors.forename: P L
authors.initials: PL
authors.lastname: Huang
authors.forename: Y
authors.initials: Y
authors.lastname: Sun
authors.forename: H C
authors.initials: HC
authors.lastname: Chen
authors.forename: H F
authors.initials: HF
authors.lastname: Kung
authors.forename: S
authors.initials: S
authors.lastname: Lee-Huang
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000737:chemistry / Q000633:toxicity*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000633:toxicity*; D001943:Breast Neoplasms; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D019427:HIV Integrase / Q000187:drug effects; D016325:HIV Long Terminal Repeat / Q000187:drug effects*; D015497:HIV-1 / Q000187:drug effects*; D006801:Humans; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000633:toxicity*; D010940:Plant Proteins / Q000737:chemistry / Q000633:toxicity*; D054789:Ribosome Inactivating Proteins, Type 1; D054790:Ribosome Inactivating Proteins, Type 2; D012697:Serine Endopeptidases; D014407:Tumor Cells, Cultured
chemical_list: D019380:Anti-HIV Agents; D000970:Antineoplastic Agents; C071380:GAP31 protein, Gelonium multiflorum; C066347:MAP30 protein, Momordica charantia; D010446:Peptide Fragments; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D054790:Ribosome Inactivating Proteins, Type 2; D019427:HIV Integrase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase