Peptide synthesis and its application to elucidate the functional aspects of sulfated peptides and proteins

肽合成及其在阐明硫酸化肽和蛋白质功能方面的应用

基本信息

批准号：
16590021
负责人：
KITAGAWA Kouki
金额：
$ 2.24万
依托单位：
Nigata University of Phermacy and Applied Life Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

1. We examined the disulfide-bond forming reaction which does not cause the loss of sulfate ester on Tyr(SO3H) residue. α-Conotoxins (PnIA and PnIB), which contain two disulfide-bonds, were prepared by two approaches; a simultaneous oxidation approach and a two-step selective oxidation approach. Both approaches did not produce the desulfated peptide throughout the synthesis. Another sulfated a-conotoxin, EpI, was also synthesized by the side-chain anchoring strategy in which β-carboxyl group of Asp residue was anchored with polymer-support. These three sulfated α-conotoxins were proved to be almost equipotent on biological activity compared with their non-sulfate counterparts. This result implies that the sulfate ester on these peptides does not govern their biological activities.2. Preparation of peptide thioester which contains the Tyr(SO3H) residue(s) is critical for the chemical synthesis of the large-molecular-form of sulfated peptide. After assembly of the Tyr(SO3H)-containing peptide by Fmoc-SPPS, the protected peptide was subjected to the thioesterification followed by the removal of protecting groups with TFA (4℃). The Tyr(SO3H)-containing peptide thioester was used for the Ag+-mediated segment condensation to prepare the objective sulfated peptide.3. Various sulfated peptides prepared by us were subjected to MALDI-TOF-MS analyses to obtain information on MS of sulfated peptides and proteins. Also we examined the novel approach to determine the sulfated site(s) of peptides and proteins using a combination of chymotrypsin digestion and MS analysis.4. Rat cholecystokinin (CCK)-peptides varying the molecular size, were synthesized and used as authentic markers on HPLC to determine the proteolytic products from the precursor protein. From these studies, it is suggested that various proteases and prohormone convertases are involved in CCK processing and their actions are cell and tissue specific.

1. 我们检查了二硫键反应的形成，该反应不会导致 Tyr(SO3H) 残基上硫酸酯的损失。α-芋螺毒素（PnIA 和 PnIB）是通过两种方法同时制备的。氧化方法和两步选择性氧化方法在整个合成过程中都没有产生另一种硫酸化α-芋螺毒素。 EpI 也是通过侧链锚定策略合成的，其中 Asp 残基的 β-羧基与聚合物载体锚定，这三种硫酸化 α-芋螺毒素与它们的非硫酸盐对相比，生物活性几乎相同。该结果表明这些肽上的硫酸酯不控制其生物活性。2．含有Tyr(SO3H)残基的肽硫酯的制备。对于大分子形式硫酸化肽的化学合成至关重要。用 Fmoc-SPPS 组装含 Tyr(SO3H) 的肽后，对受保护的肽进行硫酯化，然后用 TFA 去除保护基团。 4℃)。用含Tyr(SO3H)的肽硫酯进行Ag+介导的片段缩合，制备目标硫酸化肽。我们制备的硫酸化肽进行了 MALDI-TOF-MS 分析，以获得硫酸化肽和蛋白质的 MS 信息。我们还研究了结合胰凝乳蛋白酶消化和蛋白质硫酸化位点的新方法。 MS 分析。4. 合成不同分子大小的大鼠胆囊收缩素 (CCK) 肽，并用作 HPLC 的真实标记物以确定蛋白水解产物。这些研究表明，多种蛋白酶和激素原转化酶参与 CCK 加工，并且它们的作用具有细胞和组织特异性。