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 ␣ 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 ...

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

“…They have been implicated in asthma and other allergy disorders. Secreted as catalytically active, noncovalently bound tetramers, there are at least four closely related tryptases: ␣, ␤I, II, and III, sharing at least 93% identical amino acid sequences (40). Human lung tryptase (␣) showed a preference for the polar amino acids Asn, Ser, and Thr in the P 2 position for both the P 1 ϭ Arg and Lys sublibraries (Fig.…”

Evaluation of the Substrate Specificity of Human Mast Cell Tryptase βI and Demonstration of Its Importance in Bacterial Infections of the Lung

“…Thus, it is highly unlikely that these two human tryptases are functionally identical in vivo. Using an expression/site-directed mutagenesis approach, we demonstrated that the ability of recombinant tryptase ␣ to cleave fibrinogen and various colorimetric substrates in vitro differs from that of other mouse and human tryptases due, in part, to a single Asp/Gly difference in loop 2 (33). Neutrophils express PAR2 and can be activated by trypsin and PAR2 receptor agonists (57), and the constituents of this cell's secretory granules contribute to airway hyperreactivity in various in vivo model systems of inflammation (58,59).…”

“…Expression constructs were then prepared that encode pseudo-zymogen forms of these two tryptases that both have a 5-residue enterokinase-susceptible peptide between the natural propeptide and the mature portion of the enzyme and an 8-residue FLAG peptide attached to the C terminus. The resulting cDNAs were subcloned into the expression vector pVL1393, and the recombinant pseudo-zymogens were expressed in baculovirus-infected High Five ® insect cells as previously described for human tryptase ␣, mMCP-6, and mMCP-7 (25,26,33). After their purification, the mature forms of the two human tryptases were obtained by proteolytic removal of the bioengineered propeptides.…”

“…After their purification, the mature forms of the two human tryptases were obtained by proteolytic removal of the bioengineered propeptides. The enzymatic activities of the resulting recombinant tryptases were monitored in preliminary studies with the chromogenic substrate tosyl-Gly-Pro-Lys-p-nitroanilide and by measuring the incorporation of [ 3 H]diisopropyl fluorophosphate (Amersham Pharmacia Biotech) into each activated enzyme (33).…”

Definition of the Extended Substrate Specificity Determinants for β-Tryptases I and II

“…One possibility is that they each perform different proteolytic functions that may be reflected in their substrate specificity preferences. Indeed, it has recently been shown that a single amino acid substitution between tryptase ␣ and tryptase ␤II accounts for discrimination in substrate preference for the two enzymes (9).…”