Harris N L, Rattray K R, Tye C E, Underhill T M, Somerman M J, D'Errico J A, Chambers A F, Hunter G K, Goldberg H A
Department of Biochemistry, Faculty of Medicine & Dentistry, University of Western Ontario, London, ON, Canada.
Bone. 2000 Dec;27(6):795-802. doi: 10.1016/s8756-3282(00)00392-6.
Mammalian bone sialoprotein (BSP) is a mineralized tissue-specific protein containing an RGD (arginine-glycine-aspartic acid) cell-attachment sequence and two distinct glutamic acid (glu)-rich regions, with each containing one contiguous glu sequence. These regions have been proposed to contribute to the attachment of bone cells to the extracellular matrix and to the nucleation of hydroxyapatite (HA), respectively. To further delineate the domains responsible for these activities, porcine BSP cDNA was used to construct expression vectors coding for two partial-length recombinant BSP peptides: P2S (residues 42-87), containing the first glutamic acid-rich domain; and P1L (residues 69-300), containing the second glutamic acid-rich region and the RGD sequence. These peptides were expressed in Escherichia coli as his-tag fusion proteins and purified by nickel affinity columns and FPLC chromatography. Digestion with trypsin released the his-tag fusion peptide, which generated P2S-TY (residues 42-87) and P1L-TY (residues 132-239). Using a steady-state agarose gel system, P2S-TY promoted HA nucleation, whereas P2S, P1L, and P1L-TY did not. This implies that the minimum requirement for nucleation of HA resides within the amino acid sequence of the first glutamic acid-rich domain, whereas the second glutamic acid-rich domain may require posttranslational modifications for activity. P1L, but not P2S, promoted RGD-mediated attachment of human gingival fibroblasts in a manner similar to that of native BSP. Deletion of the RGD domain or conversion of it to RGE (arginine-glycine-glutamic acid) abolished the cell-attachment activity of P1L. This suggests that, at least for human gingival fibroblasts, the major cell-attachment activity in the recombinant BSP peptides studied (residues 42-87 and 69-300) requires the RGD sequence located at the C-terminal domain.
哺乳动物骨唾液蛋白（BSP）是一种矿化组织特异性蛋白，含有RGD（精氨酸 - 甘氨酸 - 天冬氨酸）细胞附着序列和两个不同的富含谷氨酸（Glu）的区域，每个区域包含一个连续的Glu序列。这些区域分别被认为有助于骨细胞附着于细胞外基质以及羟基磷灰石（HA）的成核。为了进一步确定负责这些活性的结构域，使用猪BSP cDNA构建编码两种部分长度重组BSP肽的表达载体：P2S（第42 - 87位氨基酸残基），包含第一个富含谷氨酸的结构域；以及P1L（第69 - 300位氨基酸残基），包含第二个富含谷氨酸的区域和RGD序列。这些肽在大肠杆菌中作为His标签融合蛋白表达，并通过镍亲和柱和快速蛋白质液相色谱法纯化。用胰蛋白酶消化释放出His标签融合肽，产生P2S - TY（第42 - 87位氨基酸残基）和P1L - TY（第132 - 239位氨基酸残基）。使用稳态琼脂糖凝胶系统，P2S - TY促进HA成核，而P2S、P1L和P1L - TY则没有。这意味着HA成核的最低要求存在于第一个富含谷氨酸结构域的氨基酸序列中，而第二个富含谷氨酸的结构域可能需要翻译后修饰才能具有活性。P1L促进人牙龈成纤维细胞的RGD介导的附着，而P2S则不能，其方式与天然BSP相似。RGD结构域的缺失或将其转化为RGE（精氨酸 - 甘氨酸 - 谷氨酸）消除了P1L的细胞附着活性。这表明，至少对于人牙龈成纤维细胞来说，所研究的重组BSP肽（第42 - 87位氨基酸残基和第69 - 300位氨基酸残基）中的主要细胞附着活性需要位于C末端结构域的RGD序列。
