Collagen folding and Interactions: from basic principles to bone disorders

I型胶原蛋白是一种长的三螺旋蛋白质，形成骨、皮肤和其他组织的基质。在过去的一年中，我们继续表征I型三螺旋中成骨不全（OI）突变的结构后果。我们构建了由螺旋沿着34个不同位点的36个点突变引起的三螺旋稳定性（解链温度）变化的图谱。为了将这张图与基于肽的稳定性预测联系起来，我们提出了一个模型，用于从报告的肽数据中提取局部螺旋展开的活化能。我们测试了模型，并确定其参数，通过测量的H-D交换率的甘氨酸NH残基参与链间氢键的胶原蛋白。通过比较基于肽和基于突变的稳定性图，我们改进了三螺旋结构中区域变化的模型。精细区域与胶原纤维组装和配体结合的重要区域对齐，并且它们似乎有助于至少一些观察到的OI表型的区域变化。 胶原蛋白与配体，特别是其他细胞外基质蛋白和蛋白聚糖的相互作用的破坏，是一个可能的机制，在OI的功能异常的胶原蛋白三螺旋结构缺陷。因此，我们正在开发一种新的共聚焦显微镜检测，这将允许一个可视化和定量测量结合的各种基质蛋白质的基础上差异荧光标记的胶原蛋白和配体的个别胶原纤维。通过该测定，例如，我们发现哺乳动物胶原酶通过优先结合微纤维攻击受损和组装不良的胶原纤维，微纤维在纤维缺陷处变得更加暴露。在过去的一年中，我们专注于荧光标记和未标记的核心蛋白聚糖之间的竞争性结合实验，这揭示了依赖于连接到核心蛋白聚糖的荧光标记的数量的结合增强。基于这些测量，我们开发了一个协议的表征和校正这样的荧光团效应。目前，我们正在进行系统的测量之间的相互作用的胶原纤维和不同的基质蛋白质潜在的重要性，在OI和其他结缔组织废用性疾病。 绝大多数严重的OI病例是由单个氨基酸取代引起的。然而，几个隐性OI和EDS的情况下，其中所有的I型胶原蛋白合成的α-1同源三聚体的形式，而不是两个α-1和一个α-2链的正常异源三聚体。在乳腺癌细胞培养物和易患常见的年龄相关性骨质疏松症的个体的正常骨细胞培养物中也观察到I型同源三聚体的形成。我们先前的研究揭示了同源三聚体三螺旋的区域稳定性改变和整个分子的变性温度的小幅增加，但它们没有提供潜在病理机制的线索。在过去的一年里，我们取得了一个潜在的突破。我们发现主要组织胶原酶MMP-1和MMP-13的同源三聚体裂解率显著降低。更详细的检查表明，最初的MMP与胶原蛋白的结合不受缺乏α-2链的影响，但该链对于裂解前三螺旋解旋和打开的下一步是必不可少的。在具有混合胶原成分的组织中，例如，含有I型同源三聚体和异源三聚体或I型同源三聚体和III型胶原蛋白，一种组分的异常裂解率将改变正常的重塑过程。 我们认为，由此产生的异常重塑可能在与同源三聚体合成相关的各种病理中发挥重要作用，并希望更好地了解潜在的分子机制可能有助于开发新的治疗策略。 大多数但不是所有具有OI临床症状的病例是由I型胶原突变引起的。与NICHD骨和细胞外基质分支的临床研究人员合作，我们刚刚报道了软骨相关蛋白（CRTAP）或脯氨酰-3-羟化酶（P3 H1）的隐性无效突变也会引起严重/致死性骨骼病理学，使人联想到OI。CRTAP和P3 H1在内质网（ER）中与亲环素B形成紧密的三蛋白复合物。我们的测量结果表明，这种复合物的破坏显着延迟I型前胶原折叠，导致过度羟基化和过度糖基化的赖氨酸残基，这也是在许多OI的情况下观察到的。我们认为，CRTAP和P3 H1可能是必不可少的保留亲环素B在ER和亲环素B脯氨酰异构酶活性是必不可少的前胶原折叠。这一假设目前正在几个研究小组中进行调查。

Type I collagen is a long, triple helical protein, which forms the matrix of bone, skin and other tissues. During the last year, we continued characterization of structural consequences of Osteogenesis Imperfecta (OI) mutations in the type I triple helix. We constructed the map of changes in the triple helix stability (melting temperature) caused by 36 point mutations at 34 different sites along the helix. To relate this map to peptide-based stability predictions, we proposed a model for extracting the activation energy of local helix unfolding from the reported peptide data. We tested the model and determined its parameters by measuring the H-D exchange rate for glycine NH residues involved in inter-chain hydrogen bonds in collagen. From comparison of the peptide-based and mutation-based stability maps, we refined our model of regional variations in the triple helix structure. The refined regions align with regions important for collagen fibril assembly and ligand binding, and they appear to contribute to at least some of the observed regional variations in OI phenotype. Disruption of collagen interactions with ligands, particularly other extracellular matrix proteins and proteoglycans, is one of possible mechanisms relating structural defects in collagen triple helix to functional abnormalities in OI. Therefore, we are developing a novel confocal microscopy assay, which will allow one to visualize and quantitatively measure binding of various matrix proteins to individual collagen fibrils based on differential fluorescent labeling of collagen and the ligand. With this assay, e.g., we found that mammalian collagenases attack damaged and poorly assembled collagen fibers via preferential binding to microfibrils, which become more exposed at fiber defects. In the last year, we focused on competitive binding experiments between fluorescently labeled and unlabeled decorin, which revealed binding enhancement dependent on the number of fluorescent labels attached to decorin. Based on these measurement, we developed a protocol for characterization and correction for such fluorophore effects. Presently, we are conducting systematic measurements of interactions between collagen fibers and different matrix proteins potentially important in OI and other connective disuse disorders. The overwhelming majority of severe OI cases are caused by single amino acid substitutions. However, several recessive OI and EDS cases were described, in which all type I collagen was synthesized in the form of alpha-1 homotrimer rather than the normal heterotrimer of two alpha-1 and one alpha-2 chains. Formation of type I homotrimers was also observed in cultures of breast cancer cells and in cultures of normal bone cells from individuals predisposed to common, age-related osteoporosis. Our previous studies revealed altered regional stability of the homotrimer triple helix and a small increase in the denaturation temperature of the whole molecule, but they did not offer clues to potential mechanisms of pathology. During the last year, we made a potential breakthrough. We discovered a significant reduction in the homotrimer cleavage rate by major tissue collagenases, MMP-1 and MMP-13. More detailed examination suggested that the initial MMP binding to collagen is not affected by the lack of the alpha-2 chain, but that this chain is essential for the next step of triple helix unwinding and opening, preceding the cleavage. In tissues with mixed collagen composition, e.g., containing type I homo- and heterotrimers or type I homotrimers and type III collagen, the abnormal cleavage rate of one component will alter the normal remodeling process. We believe that the resulting abnormal remodeling may play an important role in various pathologies associated with homotrimer synthesis and hope that better understanding of the underlying molecular mechanism may help to develop new treatment strategies. Most but not all cases with clinical symptoms of OI are caused by mutations in type I collagen. In collaboration with clinical researchers from Bone and Extracellular Matrix Branch of NICHD, we just reported that severe/lethal skeletal pathology reminiscent of OI is also caused by recessive null mutations in the cartilage associated protein (CRTAP) or prolyl-3-hydroxylase (P3H1). CRTAP and P3H1 form a tight three-protein complex with cyclophilin B in the Endoplasmic Reticulum (ER). Our measurements revealed that the disruption of this complex significantly delays type I procollagen folding, resulting in overhydroxylation and overglycosylation of Lys residues, which is also observed in many OI cases. We believe that the CRTAP and P3H1 may be essential for retaining cyclophilin B within ER and that the prolyl isomerase activity of cyclophilin B is essential for procollagen folding. This hypothesis is currently under investigation in several research groups.