Comparison of prostaglandin H synthase isoform structures using limited proteolytic digestion.
Comparison of prostaglandin H synthase isoform structures using limited proteolytic digestion.
复制标题
使用有限蛋白水解消化比较前列腺素 H 合酶异构体结构。
DOI：
10.1006/abbi.1997.0192
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Kulmacz,RJ
中科院分区：
文献类型：
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作者：
Guo,Q;Chang,S;Diekman,L;Xiao,G;Kulmacz,RJ
前列腺素H合酶（PGHS）催化多种生物活性脂质介质生物合成的关键步骤。两种已知的异构体（PGHS-1和-2）共享约60%的氨基酸同一性，但表现出与底物、激活剂和抑制剂的不同相互作用。绵羊PGHS-1先前已被证明在Arg 277附近具有独特的蛋白酶敏感位点;胰蛋白酶、胰凝乳蛋白酶或蛋白酶K的切割产生33和38 kDa的片段并丧失活性。绵羊PGHS-1晶体结构显示Arg 277位于暴露的环结构中;同源性建模预测两种人亚型（hPGHS-1和-2）的环结构相似。我们已经使用重组hPGHS-1和hPGHS-2的有限蛋白水解消化来探测它们的结构。孵育的hPGHS-1与胰蛋白酶或蛋白酶K产生33-和38-kDa的片段和活性损失。与此相反，孵育的hPGHS-2与相同的蛋白酶导致切割只有2至3 kDa的片段，没有降低活性。用位点特异性抗体的免疫印迹证明切割的片段来自hPGHS-2的C-末端。类似的免疫印迹实验表明，胰蛋白酶不攻击绵羊PGHS-1的C-末端。用精氨酸取代hPGHS-2的Pro 263（对应于绵羊PGHS-1的Arg 277），插入潜在的胰蛋白酶位点。该P263 R hPGHS-2突变体与胰蛋白酶或蛋白酶K孵育导致C-末端附近的切割和活性保留，就像野生型hPGHS-2一样。含有P263 R突变体的残基259-268的肽被胰蛋白酶以与对应于hPGHS-1残基272-281的肽相同的速率切割，证明序列差异不是hPGHS-2突变体中残基263处缺乏胰蛋白酶切割的原因。在与蛋白酶K孵育之前，用分级水平的盐酸胍预孵育hPGHS-2不会产生大的蛋白水解片段，这表明hPGHS-2环区域没有选择性地展开。结果指出PGHS-1和PGHS-2之间存在两个显著结构差异的区域：Arg 277环，其在PGHS-1中是蛋白酶敏感的，但在PGHS-2中是蛋白酶抗性的，以及C末端，其在PGHS-2中是蛋白酶敏感的，但在PGHS-1中不是。
Prostaglandin H synthase (PGHS) catalyzes a key step in the biosynthesis of a variety of bioactive lipid mediators. The two known isoforms (PGHS-1 and -2) share about 60% amino acid identity, but exhibit distinct interactions with substrates, activators, and inhibitors. Ovine PGHS-1 has previously been shown to have a distinctive protease-sensitive site near Arg277; cleavage by trypsin, chymotrypsin, or proteinase K produces fragments of 33 and 38 kDa and loss of activity. The ovine PGHS-1 crystal structure shows Arg277 located in an exposed loop structure; homology modeling predicts similar loop structures for both human isoforms (hPGHS-1 and -2). We have used limited proteolytic digestion of recombinant hPGHS-1 and hPGHS-2 to probe their structures. Incubation of hPGHS-1 with either trypsin or proteinase K produced 33- and 38-kDa fragments and loss of activity. In contrast, incubation of hPGHS-2 with the same proteases led to cleavage of only a 2- to 3-kDa fragment, with no decrease in activity. Immunoblotting with site-specific antibodies demonstrated that the cleaved fragment originated from the hPGHS-2 C-terminus. Similar immunoblotting experiments indicated that trypsin did not attack the ovine PGHS-1 C-terminus. Mutagenesis was used to replace Pro263 of hPGHS-2 (corresponds to Arg277 of ovine PGHS-1) with arginine, inserting a potential trypsin site. Incubation of this P263R hPGHS-2 mutant with either trypsin or proteinase K resulted in cleavage near the C-terminus and retention of activity, just as with wild-type hPGHS-2. A peptide containing residues 259–268 of the P263R mutant was cleaved by trypsin at the same rate as a peptide corresponding to hPGHS-1 residues 272–281, demonstrating that the sequence differences were not responsible for the lack of tryptic cleavage at residue 263 in the hPGHS-2 mutant. Preincubation of hPGHS-2 with graded levels of guanidinium HCl before incubation with proteinase K did not produce large proteolytic fragments, indicating that the hPGHS-2 loop region was not selectively unfolding. The results point to two regions of significant structural difference between PGHS-1 and -2: the Arg277 loop, which is protease-sensitive in PGHS-1 but protease-resistant in PGHS-2, and the C-terminus, which is protease-sensitive in PGHS-2 but not in PGHS-1.