Ustriyana Putu, Schulte Fabian, Gombedza Farai, Gil-Bona Ana, Paruchuri Sailaja, Bidlack Felicitas B, Hardt Markus, Landis William J, Sahai Nita
School of Polymer Science and Polymer Engineering, The University of Akron, Akron, OH 44325, USA.
The Forsyth Institute, Cambridge, MA 02142, USA.
Bone Rep. 2021 Feb 10;14:100754. doi: 10.1016/j.bonr.2021.100754. eCollection 2021 Jun.
Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca(PO)(OH)) within the organic matrix of a tissue. The ability of certain vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing) and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work generated a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then yielded generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicated that osteocalcin was closely involved in mineralization but did not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues and defines the proteome of turkey leg tendons as a model for vertebrate mineralization.
骨生物矿化是一个复杂的过程，其中I型胶原蛋白和相关的非胶原蛋白（NCPs），包括糖蛋白和蛋白聚糖，与无机钙和磷酸根离子密切相互作用，以控制纳米级、非化学计量的羟基磷灰石（HAP，理想化学计量为Ca(PO)(OH)）在组织有机基质中的沉淀。某些脊椎动物组织的矿化能力与其功能的几个方面密切相关。本研究的目的是在大鼠和火鸡这两种动物模型的矿化和非矿化组织中鉴定特定的NCPs，并确定某些NCPs是否为每种组织所特有。所研究的组织是大鼠股骨（矿化组织）和尾腱（非矿化组织）以及火鸡腿腱（在同一个体标本中有矿化和非矿化区域）。通过将顺序蛋白质提取与使用蛋白质组学和蛋白质印迹的全面蛋白质图谱分析相结合，设计了一种实验方法用于该研究。提取方法能够根据各种NCPs与组织的细胞外有机胶原基质相或无机矿质相的关联来分离它们。蛋白质组学工作生成了不同组织和动物物种中NCPs的完整图谱。随后，蛋白质印迹为一些蛋白质组学发现提供了验证。该调查随后得出了与各种蛋白质家族相关的一般性结果，而不仅仅是单个NCPs。本研究主要关注属于富含亮氨酸小分子蛋白聚糖（SLRP）家族和小整合素结合配体N-连接糖蛋白（SIBLINGs）的NCPs。发现SLRPs仅与胶原基质相关，这一结果表明它们主要参与结构基质组织而非矿化过程。SIBLINGs以及基质Gla（γ-羧基谷氨酸）蛋白严格定位于矿化组织的无机矿质相中，这一发现表明它们的作用仅限于矿化。本研究结果表明骨钙素密切参与矿化过程，但并不排除其作为一种激素可能具有的其他作用。本报告首次提供了来自矿化和非矿化组织的NCPs的空间调查和比较，并将火鸡腿腱的蛋白质组定义为脊椎动物矿化的模型。
