Endoplasmic reticulum protein Hsp47 binds specifically to the N-terminal globular domain of the amino-propeptide of the procollagen I alpha 1 (I)-chain.
Endoplasmic reticulum protein Hsp47 binds specifically to the N-terminal globular domain of the amino-propeptide of the procollagen I alpha 1 (I)-chain.
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内质网蛋白 Hsp47 特异性结合 I 型前胶原 α1 (I) 链氨基前肽的 N 末端球状结构域。
DOI：
10.1002/jcb.240590307
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Veis,A
中科院分区：
文献类型：
--
作者：
Hu,G;Gura,T;Sabsay,B;Sauk,J;Dixit,SN;Veis,A
Hsp 47是一种存在于成纤维细胞中的内质网热休克蛋白，具有明胶结合特性。有人假设它作为一个伴侣调节前胶原链折叠和/或组装，但热休克蛋白47-前胶原I相互作用的机制尚不清楚。热休克蛋白47可与变性和天然Ⅰ型前胶原结合。进行了一系列竞争研究，其中将各种胶原和胶原结构域肽与35 [S]-甲硫氨酸标记的鼠3 T6细胞裂解物一起孵育，然后与明胶-琼脂糖4 B珠混合。收集明胶结合蛋白，并通过凝胶电泳和放射自显影进行分析。胶原酶消化的I型前胶原与变性的完整前胶原具有相同的作用，表明前肽是主要的相互作用位点。添加25 μg/ml的完整pro α1（l）-N-前肽完全抑制hsp 47与明胶-琼脂糖凝胶的结合。即使是五肽VPTDE，前α1（1）-N-前肽的残基86-90，也抑制hsp 47-明胶结合。通过检查多核糖体相关的pro α链，证实了这些涉及pro α1（l）-N-前肽结构域的数据。具有完整N-前肽区域的新生pro α1（l）-链可被单克隆hsp 47抗体11 D10沉淀，但不能被单克隆抗pro α1（l）-N-前肽抗体SP1.D8沉淀，除非与hsp 47解离。将原α1（1）-N-前肽内残基23-108（NP 1）、23-151（NP 2）和23-178（NP 3）的GST-融合蛋白构建体偶联至琼脂糖4 B，并用作亲和珠，用于从3 T6细胞裂解物中收集hsp 47。NP 1和NP 2均显示出对裂解物hsp 47的强特异性结合。最后，在无膜的体外共翻译系统中研究了相互作用，在该系统中，完整的pro α1（l）-和pro α2（l）-链RNA单独翻译，以及与hsp 47 RNA混合翻译。pro α2（l）-链与hsp 47之间没有明显的相互作用，而pro α1（l）-链与新生hsp 47之间有很强的相互作用。如果存在新生hsp 47，SP1.D8不能从翻译混合物中沉淀pro α1（l）-链。这些数据都表明，如果hsp 47在前胶原链加工和折叠过程中具有“伴侣”作用，则它通过与proα1（l）链，特别是pro α1（l）氨基前肽区域的优先相互作用来发挥这种特定作用。© 1995 Wiley利斯公司
Hsp47, an endoplasmic reticulum‐resident heat shock protein in fibroblasts has gelatin‐binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47‐procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with35[S]‐methionine‐labeled murine 3T6 cell lysates prior to mixing with gelatin‐Sepharose 4B beads. The gelatin‐bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro α1 (l)‐N‐propeptide at 25 μg/ml compeletely inhibited hsp47 binding to the gelatin‐Sepharose. Even the pentapeptide VPTDE, residues 86–90 of the pro α1 (l)‐N‐propeptide, inhibits hsp47‐gelatin binding. These data implicating the pro α1 (l)‐N‐propeptide domain were confirmed by examination of polysome‐associated pro α chains. The nascent pro α1(l)‐chains with intact N‐propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti‐pro α1 (l)‐N‐propeptide antibody SP1.D8 unless dissociated from the hsp47. GST‐fusion protein constructs of residues 23–108 (NP1), 23–151 (NP2), and 23–178 (NP3) within the pro α1 (l)‐N‐propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane‐free in vitro cotranslation systems in which the complete pro α1(l)‐ and pro α2(l)‐chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro α2(l)‐chains and hsp47, whereas there was strong interaction between pro α1 (l)‐chains and nascent hsp47. SP1.D8 could not precipitate pro α1 (l)‐chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a “chaperone” role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the proα1 (l) chain, and the pro α1 (l) amino‐propeptide region in particular. © 1995 Wiley‐Liss, Inc.