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。我们检查了二硫键形成的反应，这不会导致Tyr（SO3H）居住地上硫酸盐的损失。通过两种方法制备了包含两个二硫键的α-抗毒素（PNIA和PNIB），它们含有两个二硫键。简单的氧化方法和两步选择性氧化方法。在整个合成过程中，两种方法均未产生脱硫肽。还通过侧链锚定策略合成了另一种硫化的A-抗毒素EPI，其中ASP住宅的β-羧基与聚合物支持。与非硫酸盐的同类产品相比，这三种硫酸化的α-核毒素几乎是对生物活性的几乎有计划的。该结果意味着这些宠物上的硫酸酯不控制其生物学活性。2。含有Tyr（SO3H）保留的肽硫醇的制备对于化学合成大分子硫酸化肽的化学合成至关重要。通过FMOC-SPPS组装Tyr（SO3H）肽的肽后，对受保护的肽进行了硫代疗法，然后去除具有TFA的保护组（4℃）。 Tyr（SO3H）含有含肽的肽用于Ag+介导的片段冷凝，以制备客观硫化肽3。我们制备的各种硫酸化肽经过MALDI-TOF-MS分析，以获取有关硫酸化肽和蛋白质MS的信息。我们还研究了新的方法，以确定硫化物和蛋白质的硫化位点，结合甲状腺胆素消化和MS分析4。将大鼠胆囊化蛋白（CCK） - 变化分子大小的肽合成并用作HPLC上的真实标记，以确定前体蛋白质的蛋白水解产物。从这些研究中，提出各种蛋白酶和激素转化酶参与CCK加工，其作用是细胞和组织特异性的。