Mapping of the tryptase locus on chromosome 17 revealed a novel gene 2.3 kilobase 3' of the mouse mast cell protease (mMCP) 6 gene. This 3.7-kilobase gene encodes the first example of a protease in the tryptase family that contains a membrane-spanning segment located at its COOH terminus. Comparative structural studies indicated that the putative transmembrane tryptase (TMT) possesses a unique substrate-binding cleft. As assessed by RNA blot analyses, mTMT is expressed in mice in both strain- and tissue-dependent manners. Thus, different transcriptional and/or post-transcriptional mechanisms are used to control the expression of mTMT in vivo. Analysis of the corresponding tryptase locus in the human genome resulted in the isolation and characterization of the hTMT gene. The hTMT transcript is expressed in numerous tissues and is also translated. Analysis of the tryptase family of genes in mice and humans now indicates that a primordial serine protease gene duplicated early and often during the evolution of mammals to generate a panel of homologous tryptases in each species that differ in their tissue expression, substrate specificities, and physical properties.

Tryptases ␣ and ␤/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 ␣ differs from tryptase ␤/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 ␣ exhibited potent enzymatic activity against fibrinogen and other tryptase ␤/II-susceptible substrates, Asp 215 dominantly restricts the substrate specificity of tryptase ␣. These data indicate for the first time that tryptases ␣ and ␤/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. Mast cells (MCs)1 reside in connective tissue matrices and epithelial surfaces and are important effector cells in acquired and innate immunity. Human MCs express at least four closely related tryptases (designated human tryptases I, ␤/II, III, and ␣) 2 (1-4), and this family of serine proteases has been implicated in asthma and other allergy-related disorders. Although the amino acid sequences of the varied tryptases are Ն93% identical, there are at least four genes on human chromosome 16 that encode related but distinct tryptases (6, 7). It has been shown recently that the two related tryptases designated mouse MC protease (mMCP)-6 (8, 9) and mMCP-7 (10, 11) are metabolized differently in vivo (12) and have dissimilar substrate specificities (13,14). Nevertheless, it is presently unclear why human MCs express so many homologous tryptases. Native (15) and recombinant (16) human tryptase ␤/II can degrade fibrinogen but whether or not human tryptase ␣ is a functional enzyme is controversial. While normal human basophils contain a small amount of tryptase ␣ protein (17) and mRNA (18), substantial numbers of tryptase ␣ ϩ cells have been found in the blood of patients with asthma, chronic allergies, or adverse drug reactions (19). The level of tryptase ␣ is also elevated in the sera of patients with systemic mastocytosis (20). Thus, whether or not tryptase ␣ is a functional neutral protease in humans is of critical importance.Using an expression/site-directed mutagenesis approach, we now show that tryptases ␣ and ␤/II are functional enzymes but that tryptase ␣ exhibits a more restricted substrate specificity due to an alteration in one of the ...

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Background: Three functionally distinct tryptases have been identified in the mouse, one of which encodes an unusual protease that possesses a membrane–spanning domain located in its C terminus. Methods and Results: Using the deduced nucleotide sequence of this mouse transmembrane tryptase (mTMT) gene in a polymerase chain reaction approach, cDNAs were isolated from a number of tissues which encode its human homolog. The amino acid sequences of hTMT and mTMT are 74% identical, and the human tryptase also has the novel membrane–spanning domain. Conclusion: The discovery that the human genome contains a large number of homologous, but distinct, tryptase genes suggests that the individual members of this family of proteases evolved to carry out discrete functions in mast cell–mediated allergic reactions.

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