Inhibition of tryptase TL2 from human T4+ lymphocytes and inhibition of HIV-1 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 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 HIV-1 gp120 showed a specific inhibition of tryptase TL2 (> 80%). However, the [Leu15, Phe17, Glu52] aprotinin variant with hydrophobic 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 chymotrypsin-like 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 micro M concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter-alpha-trypsin inhibitor. Only the single-headed variant [Arg94] delta 2 bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2 micro M 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.
Brinkmann
T
T
Institut für Laboratoriums- und Transfusionsmedizin, Herzund Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universitt Bochum, Bad Oeynhausen, Germany.
Schäfers
J
J
Gürtler
L
L
Kido
H
H
Niwa
Y
Y
Katunuma
N
N
Tschesche
H
H
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000031:analogs & derivatives / Q000494:pharmacology*; D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology*; D002460:Cell Line; D006023:Glycoproteins / Q000737:chemistry / Q000494:pharmacology*; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000187:drug effects* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014360:Trypsin Inhibitor, Kunitz Soybean; D053802:Tryptases; D014779:Virus Replication / Q000187:drug effects*
D006023:Glycoproteins; D015699:HIV Envelope Protein gp120; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C109148:SPINT2 protein, human; D015842:Serine Proteinase Inhibitors; D007611:Aprotinin; D014360:Trypsin Inhibitor, Kunitz Soybean; D012697:Serine Endopeptidases; D053802:Tryptases; C070201:tryptase TL(2)