整合素结合序列N端Gly错义突变对重组胶原蛋白结构与功能的影响

Effects of Gly mutations N-terminal to the integrin-binding sequence on the structure and function of recombinant collagen

摘要:

胶原蛋白作为动物体内多种组织和功能的重要基质蛋白，在生物材料领域应用广泛。而在I型胶原蛋白的三螺旋结构中，Gly-Xaa-Yaa三联体中Gly的错义突变是引发成骨不全症(osteogenesis imperfecta,OI)的重要原因。其临床表现具有高度异质性，涵盖从轻度骨骼脆弱(I型)到严重肢体畸形(III型)，乃至致死性围产期型(II型)的广泛疾病谱。本研究以重组胶原蛋白作为研究模型，旨在进一步明确发生在整合素结合序列GFPGER N端的Gly→Ala/Val突变是否会影响胶原蛋白的结构和功能，探讨错义突变对胶原蛋白生物学功能的影响机制。通过在GFPGER序列N端的7个Gly位点分别引入Ala和Val突变，系统评估了氨基酸替换对重组胶原蛋白的三螺旋结构、热稳定性、整合素结合能力和细胞黏附能力的影响。研究发现，所有Gly错义突变体均能形成稳定的三螺旋结构，但热稳定性略有下降。通过胰蛋白酶酶解实验，发现Gly→Val替换导致重组胶原蛋白对胰蛋白酶的敏感性增加，表明突变位点周围可能存在局部的构象扰动。此外，Gly→Val突变体的整合素结合能力和HT1080细胞黏附能力均显著降低，且这种效应比Gly→Ala突变体更为显著。这表明当Gly被更大体积的Val替代时，对胶原蛋白的结构和功能影响更为负面。本研究为理解成骨不全症的分子机制提供了新见解，同时为胶原蛋白的序列设计和生物材料开发提供了重要的理论参考和实验基础。

Abstract:

Collagen, a vital matrix protein for various tissue and functions in animals, is widely applied in biomaterials. In type I collagen, missense mutations of glycine (Gly) in the Gly-Xaa-Yaa triplet of the triple helix are a major cause of osteogenesis imperfecta (OI). Clinical manifestations exhibit marked heterogeneity, spanning a broad disease spectrum from mild skeletal fragility (Type I) to severe limb deformities (Type III) and perinatal lethal forms (Type II). This study utilized recombinant collagen as a model to further elucidate whether Gly→Ala/Val mutations at the N-terminus of the integrin-binding sequence GFPGER affect collagen structure and function, and to explore the underlying mechanisms by which missense mutations impact the biological function of collagen. By introducing Ala and Val substitutions at seven Gly positions N-terminal to the GFPGER sequence, we systematically assessed the effects of these amino acid replacements on the triple-helical structure, thermal stability, integrin-binding ability, and cell adhesion of recombinant collagen. All constructs formed a stable triple-helix structure, with slightly compromised thermal stability. Gly→Val substitutions increased the susceptibility of recombinant collagen to trypsin, which suggested local conformational perturbations in the triple helix. In addition, Gly→Val substitutions significantly reduced the integrin-binding affinity and decreased HT1080 cell adhesion, with the effects stronger than Gly→Ala substitutions. Compared with Gly→Ala substitutions, substitution of Gly with the larger residue Val had enhanced negative effects on the structure and function of recombinant collagen. These findings provide new insights into the molecular mechanisms of osteogenesis imperfecta and offer theoretical references and experimental foundations for the design of collagen sequences and the development of collagen-based biomaterials.