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
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
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DOI：
10.1074/jbc.270.33.19524
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发表时间：
1995-08-18
影响因子：
4.8
通讯作者：
STEVENS, RL
中科院分区：
文献类型：
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作者：
MATSUMOTO, R;SALI, A;STEVENS, RL
小鼠肥大细胞蛋白酶7（mMCP-7）是一种储存在肥大细胞分泌颗粒中的类胰蛋白酶。在颗粒pH值为5.5时，mMCP-7是完全活性的，并与含肝素的丝甘肽蛋白聚糖结合。为了了解mMCP-7与肥大细胞内外肝素的相互作用，首先通过比较蛋白质建模研究了这种类胰蛋白酶。然后在昆虫细胞中表达mMCP-7的“原"形式，并通过定点突变进行研究。尽管mMCP-7缺乏已知的与肝素相互作用的氨基酸线性序列，但mMCP-7的三维模型揭示了折叠蛋白表面上远离底物结合位点的区域，该区域在颗粒的酸性pH下表现出强的正静电势。与该计算一致，重组pro-mMCP-7在pH 5.5下结合至肝素亲和柱，并且在pH > 6.5下容易地从柱解离。定点诱变证实了正电荷区中His残基8、68和70转化为Glu阻止pro-mMCP-7与肝素结合的预测。因为结合需要带正电荷的His残基，所以当复合物从骨髓来源的肥大细胞胞吐到中性pH环境中时，天然mMCP-7能够从蛋白酶/蛋白聚糖大分子复合物解离。许多造血效应细胞将带正电荷的蛋白质储存在含有丝甘肽蛋白聚糖的颗粒中。肝素/mMCP-7的相互作用，这取决于类胰蛋白酶的三级结构，可能是一个一般的控制机制，造血细胞最大限度地存储正确折叠，酶活性蛋白在其颗粒的代表。
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 tryptase 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 acids 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.