Inhibition of tryptase TL₂ from human T4⁺ lymphocytes and inhibition of HIV1 replication in h9 cells by recombinant aprotinin and bikunin homologues

The serine esterase TL₂ from human T4⁺ lymphocytes is a binding component to HIV-1 glycoprotein gp120 and seems to play a role in the HIV-1 infection mechanism. Recombinant variants of the Kunitz-type serine proteinase inhibitor aprotinin were investigated for their ability to inhibit tryptase TL₂ and the binding of gp120 to this enzyme. Furthermore, the viral replication of HIV-1 was investigated in H9 cell cultures under the influence of recombinant aprotinin and bikunin variants. In contrast to native aprotinin, the recombinant variant [Arg¹⁵, Phe¹⁷, Glu⁵²]aprotinin with a reactive-site sequence homologous to the V3 loop of HIV4 gp120 showed a specific inhibition of tryptase TL₂ (>80%). However, the [Leu¹⁵ Phe¹⁷, Glu⁵²]aprotinin variant with hydrophohie subsites was the most potent inhibitor of the binding of gp120 to tryptase TL₂ (68%). Our results show that the enzyme activity of purified tryptase TL₂ is inhibited not only by variants with basic amino acids, but also those with hydrophobic residues in the reactive-site region. Therefore, tryptase TL₂ is not a typical trypsin- like or chymotrypsinlike protease. Investigations on inhibition of HIV-1 replication in H9 cell cultures showed that tryptase TL₂ is involved in the mechanism of virus internalization into human lymphocytes. The [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin showed a significant retardation of syncytium formation over a period of 5 days in a 1 μM concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter-α-trypsin inhibitor. Only the single-headed variant [Arg⁹⁴](δ²) bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2μM concentration. Wild-type bikunin and all full- length variants showed no effect, possibly due to steric hindrance by the second domain of the double-headed inhibitor.