Heparan Sulfate 6-O-Sulfotransferase Isoform-dependent Regulatory Effects of Heparin on the Activities of Various Proteases in Mast Cells and the Biosynthesis of 6-O-Sulfated Heparin
Heparan Sulfate 6-O-Sulfotransferase Isoform-dependent Regulatory Effects of Heparin on the Activities of Various Proteases in Mast Cells and the Biosynthesis of 6-O-Sulfated Heparin
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DOI：
10.1074/jbc.m112.416651
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发表时间：
2013-02-08
影响因子：
4.8
通讯作者：
Kimata, Koji
中科院分区：
文献类型：
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作者：
Anower-E-Khuda, Md. Ferdous;Habuchi, Hiroko;Kimata, Koji
硫酸乙酰肝素 6-O-磺基转移酶 (HS6ST) 是一种参与硫酸乙酰肝素 (HS) 生物合成的酶，可将硫酸残基转移至 HS 的 GlcNAc/GlcNSO(3) 残基的第 6 位，它由三种异构体组成。肝素是 HS 的高度硫酸化形式，存在于结缔组织肥大细胞中，并参与肥大细胞蛋白酶 (MCP) 的储存。然而，目前尚不清楚 HS6ST 的哪种异构体参与肝素的 6-O-硫酸化以及肝素中的 6-O-硫酸酯残基如何影响 MCP。为了研究这些问题，我们从野生型（WT）和HS6ST缺陷小鼠（HS6ST-1（-/-）、HS6ST-2（-/-）和HS6ST-1（-/-）/HS6ST-2（-/-））中制备了胎儿皮肤源性肥大细胞（FSMC），并测定了培养的FSMC中肝素的结构、蛋白酶活性和每个MCP的mRNA表达。 HS6ST-2(-/-)-FSMC 中类胰蛋白酶和羧肽酶-A 的活性降低，其中肝素 6-O-硫酸化在 WT-FSMC 中降低了 50%，而在几乎完全缺乏肝素 6-O-硫酸化的 HS6ST-1(-/-)/HS6ST-2(-/-)-FSMC 中几乎丧失。相比之下，甚至在 HS6ST-1(-/-)/HS6ST-2(-/-) FSMC 中也保留了食糜酶活性。在任何突变型 FSMC 中，每种 MCP mRNA 均未减少。蛋白质印迹分析显示，HS6ST-1(-/-)/HS6ST-2(-/-) FSMC 中几乎不存在类胰蛋白酶 (mMCP-6)，表明酶蛋白被降解/分泌。这些观察结果表明，HS6ST-1 和 HS6ST-2 均参与肝素的 6-O-硫酸化，并且类胰蛋白酶、羧肽酶-A 和食糜酶的正确包装和储存可能受到肝素中 6-O-硫酸酯残基的不同调节。因此，肝素的 6-O-硫酸化可能在调节 MCP 功能中发挥重要作用。
Heparan sulfate 6-O-sulfotransferase (HS6ST) is an enzyme involved in heparan sulfate (HS) biosynthesis that transfers a sulfate residue to position 6 of the GlcNAc/GlcNSO(3) residues of HS, and it consists of three isoforms. Heparin, the highly sulfated form of HS, resides in connective tissue mast cells and is involved in the storage of mast cell proteases (MCPs). However, it is not well understood which isoform(s) of HS6ST participates in 6-O-sulfation of heparin and how the 6-O-sulfate residues in heparin affect MCPs. To investigate these issues, we prepared fetal skin-derived mast cells (FSMCs) from wild type (WT) and HS6ST-deficient mice (HS6ST-1(-/-), HS6ST-2(-/-), and HS6ST-1(-/-)/HS6ST-2(-/-)) and determined the structure of heparin, the protease activity, and the mRNA expression of each MCP in cultured FSMCs. The activities of tryptase and carboxypeptidase-A were decreased in HS6ST-2(-/-)-FSMCs in which 6-O-sulfation of heparin was decreased at 50% of WT-FSMCs and almost lost in HS6ST-1(-/-)/HS6ST-2(-/-)-FSMCs, which lacked the 6-O-sulfation in heparin nearly completely. In contrast, chymase activity was retained even in HS6ST-1(-/-)/HS6ST-2(-/-) FSMCs. Each MCP mRNA was not decreased in any of the mutant FSMCs. Western blot analysis showed that tryptase (mMCP-6) was almost absent from HS6ST-1(-/-)/HS6ST-2(-/-) FSMCs indicating degradation/secretion of the enzyme protein. These observations suggest that both HS6ST-1 and HS6ST-2 are involved in 6-O-sulfation of heparin and that the proper packaging and storage of tryptase, carboxypeptidase-A, and chymase may be regulated differently by the 6-O-sulfate residues in heparin. It is thus likely that 6-O-sulfation of heparin plays important roles in regulating MCP functions.