Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
J Biol Chem. 2012 Nov 23;287(48):40598-610. doi: 10.1074/jbc.M112.406850. Epub 2012 Oct 11.
α1(V) is an extensively modified collagen chain important in disease.
Comprehensive mapping of α1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets.
The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties.
Positions, numbers, and occupancy of modified sites can provide insights into α1(V) biological properties. Aberrant expression of the type V collagen α1(V) chain can underlie the connective tissue disorder classic Ehlers-Danlos syndrome, and autoimmune responses against the α1(V) chain are linked to lung transplant rejection and atherosclerosis. The α1(V) collagenous COL1 domain is thought to contain greater numbers of post-translational modifications (PTMs) than do similar domains of other fibrillar collagen chains, PTMs consisting of hydroxylated prolines and lysines, the latter of which can be glycosylated. These types of PTMs can contribute to epitopes that underlie immune responses against collagens, and the high level of PTMs may contribute to the unique biological properties of the α1(V) chain. Here we use high resolution mass spectrometry to map such PTMs in bovine placental α1(V) and human recombinant pro-α1(V) procollagen chains. Findings include the locations of those PTMs that vary and those PTMs that are invariant between these α1(V) chains from widely divergent sources. Notably, an unexpectedly large number of hydroxyproline residues were mapped to the X-positions of Gly-X-Y triplets, contrary to expectations based on previous amino acid analyses of hydrolyzed α1(V) chains from various tissues. We attribute this difference to the ability of tandem mass spectrometry coupled to nanoflow chromatographic separations to detect lower-level PTM combinations with superior sensitivity and specificity. The data are consistent with the presence of a relatively large number of 3-hydroxyproline sites with less than 100% occupancy, suggesting a previously unknown mechanism for the differential modification of α1(V) chain and type V collagen properties.
α1(V) 是一种广泛修饰的胶原蛋白链，在疾病中很重要。
对 α1(V) 翻译后修饰的综合作图揭示了 Gly-X-Y 氨基酸三肽中出乎意料数量的 X 位羟脯氨酸。
X 位羟脯氨酸的丰富程度表明了胶原蛋白性质差异修饰的机制。
修饰位点的位置、数量和占有率可以为了解 α1(V) 的生物学特性提供依据。经典的埃勒斯-当洛斯综合征的结缔组织疾病的基础是 V 型胶原 α1(V) 链的异常表达，而针对 α1(V) 链的自身免疫反应与肺移植排斥和动脉粥样硬化有关。α1(V) 胶原蛋白 COL1 结构域被认为比其他纤维状胶原蛋白链的类似结构域包含更多的翻译后修饰（PTM），PTM 由羟脯氨酸和赖氨酸组成，后者可以糖基化。这些类型的 PTM 可以有助于针对胶原蛋白的免疫反应的表位，并且高水平的 PTM 可能有助于 α1(V) 链的独特生物学特性。在这里，我们使用高分辨率质谱法来绘制牛胎盘 α1(V) 和人重组 pro-α1(V) 前胶原链中的这些 PTM。研究结果包括在广泛不同来源的这些 α1(V) 链之间变化的 PTM 位置和不变的 PTM 位置。值得注意的是，出乎意料的是，大量的羟脯氨酸残基被映射到 Gly-X-Y 三肽的 X 位，这与基于来自各种组织的水解 α1(V) 链的先前氨基酸分析的预期相反。我们将这种差异归因于串联质谱法与纳流色谱分离相结合的能力，以具有更高灵敏度和特异性的方式检测较低水平的 PTM 组合。这些数据与存在相对大量羟脯氨酸位点但占有率低于 100%的情况一致，这表明了一种以前未知的 α1(V) 链和 V 型胶原蛋白性质差异修饰的机制。
