Human tryptases α and β/II are functionally distinct due, in part, to a single amino acid difference in one of the surface loops that forms the substrate-binding cleft

Abstract

Tryptases α andβ/II were expressed in insect cells to try to ascertain why human mast cells express these two nearly identical granule proteases. In contrast to that proposed by others, residue -3 in the propeptide did not appear to be essential for the three-dimensional folding, post-translational modification, and/or activation of this family of serine proteases. Both recombinant tryptases were functional and bound the active-site inhibitor diisopropyl fluorophosphate. However, they differed in their ability to cleave varied trypsin-susceptible chromogenic substrates. Structural modeling analyses revealed that tryptase α differs from tryptase β/II in that it possesses an Asp, rather than a Gly, in one of the loops that form its substrate-binding cleft. A site-directed mutagenesis approach was therefore carried out to determine the importance of this residue. Because the D215G derivative of tryptase α exhibited potent enzymatic activity against fibrinogen and other tryptase β/II-susceptible substrates, Asp215 dominantly restricts the substrate specificity of tryptase α. These data indicate for the first time that tryptases α and β/II are functionally different human proteases. Moreover, the variation of just a single amino acid in the substrate-binding cleft of a tryptase can have profound consequences in the regulation of its enzymatic activity and/or substrate preference.