title: Packaging of proteases and proteoglycans in the granules of mast cells and other hematopoietic cells. A cluster of histidines on mouse mast cell protease 7 regulates its binding to heparin serglycin proteoglycans.
abstract: 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.
authors.affiliation: Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: R
authors.initials: R
authors.lastname: Matsumoto
authors.forename: A
authors.initials: A
authors.lastname: Sali
authors.forename: N
authors.initials: N
authors.lastname: Ghildyal
authors.forename: M
authors.initials: M
authors.lastname: Karplus
authors.forename: R L
authors.initials: RL
authors.lastname: Stevens
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells; D002460:Cell Line; D053818:Chymases; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D008407:Mast Cells / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011485:Protein Binding; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D018411:Spodoptera; D053802:Tryptases; D033921:Vesicular Transport Proteins
chemical_list: D011509:Proteoglycans; D011994:Recombinant Proteins; C509190:Tpsb2 protein, mouse; D033921:Vesicular Transport Proteins; C075430:serglycin; D006639:Histidine; D006493:Heparin; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; C509222:Tpsab1 protein, mouse; D053802:Tryptases