Formation of Enzymatically Active, Homotypic, and Heterotypic Tetramers of Mouse Mast Cell Tryptases

Abstract

Mouse mast cell protease (mMCP) 6 and mMCP-7 are homologous tryptases stored in granules as macromolecular complexes with heparin and/or chondroitin sulfate E containing serglycin proteoglycans. When promMCP-7 and pseudozymogen forms of this tryptase and mMCP-6 were separately expressed in insect cells, all three recombinant proteins were secreted into the conditioned medium as properly folded, enzymatically inactive 33-kDa monomers. However, when their propeptides were removed, mMCP-6 and mMCP-7 became enzymatically active and spontaneously assumed an ϳ150-kDa tetramer structure. Heparin was not required for this structural change. When incubated at 37°C, recombinant mMCP-7 progressively lost its enzymatic activity in a time-dependent manner. Its N-linked glycans helped regulate the thermal stability of mMCP-7. However, the ability of this tryptase to form the enzymatically active tetramer was more dependent on a highly conserved Trp-rich domain on its surface. Although recombinant mMCP-6 and mMCP-7 preferred to form homotypic tetramers, these tryptases readily formed heterotypic tetramers in vitro. This latter finding indicates that the tetramer structural unit is a novel way the mast cell uses to assemble varied combinations of tryptases. Mouse mast cell (MC)1 protease (mMCP) 6 (1, 2) and mMCP-7 (3, 4) are homologous tryptases stored in abundance in their mature forms in the acidic secretory granules of numerous populations of MCs ionically bound to heparin and/or chondroitin sulfate E containing serglycin proteoglycans (1, 5, 6). Both tryptases are exocytosed when MCs are activated via their high affinity immunoglobulin (Ig) E receptors. mMCP-7 inhibits the formation of fibrin/platelet clots (7), whereas mMCP-6 induces the extravasation of neutrophils (8). Thus, the two tryptases often work in concert in the acute phases of MC-mediated inflammatory responses.A small amount of exocytosed mMCP-7 is retained in tissues for Ͼ1 h (5). However, most exocytosed mMCP-7 dissociates from its exocytosed protease/proteoglycan macromolecular complex in the extracellular matrix to allow the rapid diffusion of this tryptase away from the MC (5, 6). Because its proteoglycan-binding domain has more Lys and Arg residues than the corresponding domain in mMCP-7, exocytosed mMCP-6 cannot dissociate easily from its serglycin proteoglycan at neutral pH. Thus, due to its Ͼ10 million-dalton size, very little of the exocytosed mMCP-6/proteoglycan complex is able to enter the circulation.Pancreatic trypsin and most other serine proteases are enzymatically active in their ϳ30-kDa monomer states. Thus, it was a surprise when Schwartz and co-workers (9, 10) and then others (11-16) discovered that human MC tryptases purified from different tissues exist as tetramers. While it has been proposed that heparin is needed for human lung-and skinderived tryptases to form stable, enzymatically active tetramers (10, 14 -16), mMCP-7 is able to circulate in the blood of the V3 mastocytosis mouse for Ͼ1 h as an enzymatically active, homotypic tetrame...

Mast Cell-restricted Tryptases: Structure and Function in Inflammation and Pathogen Defense

“…Fifteen single nucleotide polymorphisms have been identified in the hPrss31 gene, which result in amino acid changes at residues 23, 123, 202, and 252 in the protease's mature domain. Residues 23 and 202 are predicted to reside in the protease's substrate-binding cleft (18) and residue 123 in the Trp-rich domain (7) that is required for activation of tryptase zymogens (25). It therefore is likely that some allelic variants of the hPrss31 gene encode defective or functionally distinct tryptases.…”

Mouse Chromosome 17A3.3 Contains 13 Genes That Encode Functional Tryptic-like Serine Proteases with Distinct Tissue and Cell Expression Patterns

“…Protease-enriched fractions were pooled and their protein contents estimated using the micro BCA protein assay reagent kit (Pierce). (37)(38)(39), and expression/site-directed mutagenesis analysis of mMCP-7 revealed that these glycans often are important in thermal stability (40). Analysis of their predicted primary amino acid sequences revealed potential N-linked glycosylation sites in mT5, mMCP-11, mBssp-4, mTessp1, and mPancreasin.…”

“…contain the residues that form the critical Trp-rich domain on the surface of other enzymatically active members of the family (40,47). Mouse and human TMT/tryptase ␥ remain anchored to the plasma membrane when MCs degranulate due to their C-terminal membrane-spanning domains (26).…”

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

“…Although the primary function(s) of each human tryptase remains to be determined, it is apparent that the individual mouse tryptases are metabolized differently during Fc⑀ receptor I-mediated inflammatory reactions (24) and are functionally distinct (25,26). The discovery that mMCP-6 and mMCP-7 can form homotypic and heterotypic tetramer complexes (27) alters the interpretation of earlier functional studies carried out with non-recombinant tryptases because heterotypic and homotypic tetramers probably degrade extracellular proteins quite differently.…”