Hu G, Gura T, Sabsay B, Sauk J, Dixit S N, Veis A
Division of Oral Biology, Northwestern University, Chicago, Illinois 60611, USA.
J Cell Biochem. 1995 Nov;59(3):350-67. doi: 10.1002/jcb.240590307.
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 with 35[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 alpha 1(I)-N-propeptide at 25 micrograms/ml completely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86-90 of the pro alpha 1(I)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro alpha 1(I)-N-propeptide domain were confirmed by examination of polysome-associated pro alpha chains. The nascent pro alpha 1(I)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro alpha 1 (I)-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 alpha 1 (I)- 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 alpha 1(I)- and pro alpha 2(I)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro alpha 2(I)-chains and hsp47, whereas there was strong interaction between pro alpha 1(I)-chains and nascent hsp47. SP1.D8 could not precipitate pro alpha 1(I)-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 pro alpha 1 (I) chain, and the pro alpha 1(I) amino-propeptide region in particular.
热休克蛋白47（Hsp47）是成纤维细胞中一种内质网驻留热休克蛋白，具有明胶结合特性。曾有假说认为它作为伴侣蛋白调节前胶原链的折叠和/或组装，但Hsp47与I型前胶原相互作用的机制尚不清楚。Hsp47能与变性和天然的I型前胶原结合。进行了一系列竞争研究，在将各种胶原蛋白和胶原结构域肽与[³⁵S] - 甲硫氨酸标记的小鼠3T6细胞裂解物孵育后，再与明胶 - 琼脂糖4B珠混合。收集结合在明胶上的蛋白质，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I型前胶原具有相同的效果，表明前肽是主要的相互作用位点。以25微克/毫升添加完整的α1（I）-N-前肽完全抑制Hsp47与明胶 - 琼脂糖的结合。甚至α1（I）-N-前肽86 - 90位残基的五肽VPTDE也能抑制Hsp47与明胶的结合。通过检查多聚核糖体相关的α链证实了这些涉及α1（I）-N-前肽结构域的数据。具有完整N-前肽区域的新生α1（I）链可被单克隆Hsp47抗体11D10沉淀，但除非从Hsp47解离，否则不能被单克隆抗α1（I）-N-前肽抗体SP1.D8沉淀。α1（I）-N-前肽内23 - 108（NP1）、23 - 151（NP2）和23 - 178（NP3）位残基的谷胱甘肽S-转移酶（GST）融合蛋白构建体与琼脂糖4B偶联，并用作从3T6细胞裂解物中收集Hsp47的亲和珠。NP1和NP2均显示出与裂解物Hsp47的强特异性结合。最后，在无膜体外共翻译系统中研究了这种相互作用，其中单独以及相互混合并与Hsp47 RNA一起翻译完整的α1（I）链和α2（I）链RNA。α2（I）链与Hsp47之间未观察到明显相互作用，而α1（I）链与新生Hsp47之间存在强相互作用。如果存在新生Hsp47，SP1.D8不能从翻译混合物中沉淀α1（I）链。这些数据均表明，如果Hsp47在I型前胶原链加工和折叠过程中具有“伴侣”作用，那么它通过与α1（I）链，特别是α1（I）氨基前肽区域的优先相互作用来发挥这一特定作用。
