Proteolytic fragments of anti-HIV and anti-tumor proteins MAP30 and GAP31 are biologically active

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 digestio n 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 t o proteolysis, whereas the N- and C-termini are accessible. Peptide fragmen ts were purified by FPLC on Superdex 75 columns, characterized by gel elect rophoresis, 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. T hese 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, l ittle cytotoxicity to normal cells was observed. In addition, these fragmen ts 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 portio ns of the N- and C-termini are not essential for antiviral and anti-tumor a ctivities, but do appear to be required for ribosome inactivation. These re sults may provide novel strategies for rational design and targeted develop ment of mimetic antiviral and anti-tumor therapeutics, (C) 1999 Academic Pr ess.