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Mouse mast cell protease 7 (mMCP-7) is a tryptase stored in the secretory granules of mast cells. At the granule pH of 5.5, mMCP-7 is fully active and is bound to heparin-containing serglycin proteoglycans. to understand the interaction of mMCP-7 with heparin inside and outside the mast cell, this trytase was first studied by comparative protein modeling. The "pro" form of mMCP-7 was then expressed in insect cells and studied by site-directed mutagenesis. Although mMCP-7 lacks known linear sequences of amino acis that interact with heparin, the three-dimensional model of mMCP-7 revealed an area on the surface of the folded protein away from the substrate-binding site that exhibits a strong positive electrostatic potential at the acidic pH of the granule. In agreement with this calculation, recombinant pro-mMCP-7 bound to a heparin-affinity column at pH 5.5 and readily dissociated from the column at pH > 6.5. Site-directed mutagenesis confirmed the prediction that the conversion of His residues 8,68, and 70 in the positively charged region into Glu prevents the binding of pro-mMCP-7 to heparin. Because the binding requires positively charged His residues, native mMCP-7 is able to dissociate from the protease/proteoglycan macromolecular complex when the complex is exocytosed from bone marrow-derived mast cells into a neutral pH environment. Many hematopoietic effector cells store positively charged proteins in granules that contain serglycin proteoglycans. The heparin/mMCP-7 interaction, which depends on the tertiary structure of the tryptase, may be representative of a general control mechanism by which hematopoietic cells maximize storage of properly folded, enzymatically active proteins in their granules.
Matsumoto et al. (Tue,) studied this question.