Collagen folding and Interactions: from basic principles to bone disorders

I型胶原是一种长的三螺旋蛋白质，形成骨骼、皮肤和其他组织的基质。在过去的一年里，我们继续表征I型三螺旋中成骨不全(OI)突变的结构后果。我们构建了由螺旋上34个不同位置的36个点突变引起的三螺旋稳定性(熔化温度)的变化图谱。为了将这一图谱与基于肽的稳定性预测联系起来，我们提出了一个模型，用于从已报道的肽数据中提取局部螺旋展开的激活能。我们测试了该模型，并通过测量参与胶原链间氢键的甘氨酸NH残基的H-D交换率来确定它的参数。从基于多肽和基于突变的稳定性图的比较中，我们改进了我们的三螺旋结构区域变异模型。精细化区域与胶原纤维组装和配体结合的重要区域对齐，它们似乎至少对观察到的OI表型的部分区域差异有贡献。 胶原与配体，特别是其他细胞外基质蛋白和蛋白多糖相互作用的中断，是胶原三螺旋结构缺陷与OI功能异常的可能机制之一。因此，我们正在开发一种新的共聚焦显微镜分析方法，它将使人们能够基于胶原和配体的差异荧光标记来可视化和定量测量不同基质蛋白与单个胶原纤维的结合。例如，通过这种测试，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，微纤维更容易暴露在纤维缺陷处。在过去的一年里，我们重点研究了荧光标记的核心蛋白聚糖和未标记的核心蛋白聚糖之间的竞争结合实验，发现结合的增强依赖于连接到核心蛋白蛋白的荧光标记的数量。在这些测量的基础上，我们开发了一种用于表征和校正这种荧光团效应的协议。目前，我们正在对胶原纤维和不同基质蛋白之间的相互作用进行系统的测量，这些蛋白在OI和其他连接性废用障碍中可能具有重要作用。 绝大多数严重的OI病例是由单一氨基酸替代引起的。然而，一些隐性OI和EDS病例被描述，在这些病例中，所有的I型胶原都是以α-1同源三聚体的形式合成的，而不是两个α-1和一个α-2链的正常杂三聚体。在乳腺癌细胞的培养中也观察到了I型同源三聚体的形成，在来自普通的年龄相关性骨质疏松症患者的正常骨细胞的培养中也观察到了I型同源三聚体的形成。我们以前的研究显示，高三聚体三螺旋的区域稳定性发生了变化，整个分子的变性温度略有上升，但它们没有为潜在的病理机制提供线索。在过去一年里，我们取得了潜在的突破。我们发现，主要组织胶原酶-1和-13显著降低了同源三聚体的裂解率。更详细的研究表明，最初的基质金属蛋白酶与胶原结合不受缺乏α-2链的影响，但这条链对于切割前的下一步三螺旋解离和打开是必不可少的。在含有混合胶原成分的组织中，例如含有I型同源和异源三聚体或I型同源三聚体和III型胶原，一种成分的异常裂解速度将改变正常的重塑过程。我们认为，由此导致的异常重构可能在与高三聚体合成相关的各种病理过程中发挥重要作用，并希望更好地了解其潜在的分子机制，可能有助于开发新的治疗策略。 大多数但不是所有OI的临床症状都是由I型胶原突变引起的。我们与NICHD骨和细胞外基质分会的临床研究人员合作，刚刚报道了与OI相关的严重/致死性骨骼病理也是由软骨相关蛋白(CRTAP)或Pro-3-羟基酶(P3H1)的隐性零突变引起的。CRTAP和P3H1在内质网(ER)中与亲环素B形成紧密的三蛋白复合体。我们的测量显示，该复合体的破坏显著延迟了I型前胶原的折叠，导致Lys残基的过度羟化和过度糖基化，这在许多OI病例中也可以观察到。我们认为CRTAP和P3H1可能是在内质网中保留亲环素B所必需的，并且亲环素B的脯氨基异构酶活性对于前胶原折叠是必不可少的。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.