Histidines are critical for heparin-dependent activation of mast cell tryptase.
Mast cell tryptase is a tetrameric serine protease that is stored in complex with negatively charged heparin proteoglycans in the secretory granule. Tryptase has potent proinflammatory properties and has been implicated in diverse pathological conditions such as asthma and fibrosis. Previous studies have shown that tryptase binds tightly to heparin, and that heparin is required in the assembly of the tryptase tetramer as well as for stabilization of the active tetramer. Because the interaction of tryptase with heparin is optimal at acidic pH, we investigated in this study whether His residues are of importance for the heparin binding, tetramerization, and activation of the tryptase mouse mast cell protease 6. Molecular modeling of mouse mast cell protease 6 identified four His residues, H35, H106, H108, and H238, that are conserved among pH-dependent tryptases and are exposed on the molecular surface, and these four His residues were mutated to Ala. In addition, combinations of different mutations were prepared. Generally, the single His-Ala mutations did not cause any major defects in heparin binding, activation, or tetramerization, although some effect of the H106A mutation was observed. However, when several mutations were combined, large defects in all of these parameters were observed. Of the mutants, the triple mutant H106A/H108A/H238A was the most affected with an almost complete inability to bind to heparin and to form active tryptase tetramers. Taken together, this study shows that surface-exposed histidines mediate the interaction of mast cell tryptase with heparin and are of critical importance in the formation of active tryptase tetramers.
Hallgren
Jenny
J
Department of Molecular Biosciences, The Biomedical Centre, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Bäckström
Stefan
S
Estrada
Sergio
S
Thuveson
Maria
M
Pejler
Gunnar
G
D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers; D002460:Cell Line; D016384:Consensus Sequence; D004789:Enzyme Activation; D006493:Heparin / Q000378:metabolism*; D006639:Histidine / Q000502:physiology*; D006801:Humans; D007231:Infant, Newborn; D007668:Kidney; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013045:Species Specificity; D053802:Tryptases
D001704:Biopolymers; D011993:Recombinant Fusion Proteins; C509190:Tpsb2 protein, mouse; D006639:Histidine; D006493:Heparin; D012697:Serine Endopeptidases; C509222:Tpsab1 protein, mouse; D053802:Tryptases