Identification and characterization of the major chicken bone phosphoprotein. Analysis of its synthesis by cultured embryonic chick osteoblasts.
Identification and characterization of the major chicken bone phosphoprotein. Analysis of its synthesis by cultured embryonic chick osteoblasts.
复制标题
主要鸡骨磷蛋白的鉴定和表征。
DOI：
10.1111/j.1432-1033.1990.tb15276.x
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Glimcher,MJ
中科院分区：
文献类型：
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作者：
Gotoh,Y;Gerstenfeld,LC;Glimcher,MJ
培养的鸡胚成骨细胞合成的主要磷蛋白的分子量约为 66 kDa。酸性磷酸酶处理裂解蛋白质上的32P标记，部分酸水解后可鉴定O-[32P]磷酸丝氨酸和O-[32P]磷酸苏氨酸。磷蛋白约占成骨细胞合成总蛋白的 2.0%，并且如其在培养基中的存在所示，显示出被分泌。糖基化通过它可以用[3H]半乳糖胺标记的事实得到证明。主要的 ~ 66-kDa 磷蛋白通过等电聚焦分解为三个主要变体，pI 值范围超过 3.7-3.9；所有三种形式似乎都是蛋白质磷酸化程度变化的结果。可以从鸡骨中提取相同的 ~ 66-kDa 磷蛋白，其在 DEAE-Sephacel 色谱后具有相同的 pI 值范围和相同的洗脱位置。对从骨中分离的蛋白质的分析表明存在唾液酸，虽然约 25% 的蛋白质的氨基末端序列分析和内部胰蛋白酶片段序列分析显示与大鼠磷蛋白骨桥蛋白几乎没有相似性，但在 ~ 66-kDa 鸡蛋白中鉴定出跨越大鼠蛋白骨桥蛋白的 Arg-Gly-Asp 细胞结合位点的保守九残基序列。与体外合成的蛋白质相比，用金黄色葡萄球菌V8蛋白酶对体内蛋白质进行的肽图分析显示出相同的肽指纹。这两种蛋白质也具有相似的氨基酸组成。在骨的 HC1 提取物中还观察到了几种分子量较小的磷蛋白，其大小范围在约 55-29 kDa 之间。这些物种的肽图谱表明~ 66-kDa、~ 55-kDa 和~ 45-kDa 物种具有共同的肽片段核心。培养物中的脉冲/追踪实验表明，没有证据表明与 ∼ 66-kDa 物种相关的明确的细胞内蛋白水解途径，因为这种磷蛋白在 24 小时追踪后仍然是普遍的物种。由于所有较小分子质量形式与细胞层的主要关联，并且在 24 小时的追踪中没有定量转化为任何较小形式，这些结果表明较低分子质量种类不是合成或分泌过程中蛋白水解加工的结果，而是代表细胞外基质中 ~ 66-kDa 成分的蛋白水解。总之，这些结果表明，培养的成骨细胞合成一种主要的 ~ 66-kDa 磷蛋白，该磷蛋白与出生后鸡骨中存在的主要 HCl 可提取磷蛋白相同。
The major phosphoprotein synthesized by cultured chicken embryo osteoblasts had a molecular mass of approximately 66 kDa. The32P label on the protein was cleaved by acid phosphatase treatment andO‐[32P]phosphoserine andO‐[32P]phosphothreonine could be identified after partial acid hydrolysis. The phosphoprotein contributed approximately 2.0% of the total protein synthesized by osteoblasts and was shown to be secreted, as shown by its presence in the culture media. Glycosylation was demonstrated by the fact that it could be labelled with [3H]galactosamine. The major ∼ 66‐kDa phosphoprotein was resolved by isoelectric focusing into three major variants with pI values ranging over 3.7–3.9; all three forms appear to be the result of variation in the extent of protein phosphorylation. An identical ∼ 66‐kDa phosphoprotein could be extracted from chicken bones which had both the same range of pI values and an identical elution position following DEAE‐Sephacel chromatography. Analysis of the protein isolated from bone demonstrated the presence of sialic acid and, while amino‐terminal sequence analysis and internal tryptic fragment sequence analysis of about 25% of the protein revealed little similarity to the rat phosphoprotein osteopontin, a conserved nine‐residue sequence spanning the Arg‐Gly‐Asp cell‐binding site of the rat protein osteopontin, was identified in the ∼ 66‐kDa chicken protein. Peptide mapping withStaphylococcus aureusV8 protease of thein vivoprotein compared to thein vitrosynthesized protein demonstrated identical peptide fingerprints. The two proteins also had comparable amino acid compositions.Several smaller‐molecular‐mass phosphoproteins ranging in size over about 55–29 kDa were also observed in the HC1 extracts of bone. Peptide mapping of these species demonstrated that the ∼ 66‐kDa, ∼ 55‐kDa, and ∼ 45‐kDa species had a common core of peptide fragments. Pulse/chase experiments in culture revealed no evidence for a defined pathway of intracellular proteolysis associated with the ∼ 66‐kDa species since this phosphoprotein remained the prevalent species after a 24‐h chase. Because of the predominant association of all the smaller‐molecular‐mass forms with the cell layer and an absence of a quantitative conversion to any of the smaller forms over a 24‐h chase, these results suggested that the lower‐molecular‐mass species were not the result of proteolytic processing during synthesis or secretion, but rather represent proteolysis of the ∼ 66‐kDa component in the extracellular matrix. In summary, these results demonstrate that cultured osteoblasts synthesize a major ∼ 66‐kDa phosphoprotein that is identical to the major HCl‐extractable phosphoprotein present in postnatal chick bone.