Huq N Laila, Loganathan Ashwini, Cross Keith J, Chen Yu-Yen, Johnson Neil I, Willetts Matthew, Veith Paul D, Reynolds Eric C
Centre for Oral Health Science, School of Dental Science, The University of Melbourne, 711 Elizabeth Street, Melbourne, Victoria 3000, Australia.
Arch Oral Biol. 2005 Sep;50(9):807-19. doi: 10.1016/j.archoralbio.2005.02.002.
Bovine dentine phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser(P)) residues, is synthesized by odontoblasts and believed to be involved in matrix-mediated biomineralization of dentine. Phosphophoryn was purified from bovine dentine using EDTA extraction, Ca(2+) precipitation, anion exchange and size exclusion chromatography. The purified protein migrated on SDS-PAGGE as a single band. The protein was dephosphorylated using a chelex alkaline dialysis procedure, repurified using anion exchange and size exclusion chromatography and then subjected to cleavage with trypsin. The digest was subjected to reversed-phase HPLC and analysed by Q-TOF mass spectrometry. The only non-trypsin peptides that could be identified were two collagen Type I alpha2 peptides whose sequence was determined by fragmentation analysis. The association of collagen fragments with highly purified phosphophoryn suggests that the EDTA extraction method yields BDP that is strongly bound to collagen fragments. This association now helps explain discrepancies in molecular weight and amino acid composition data for various phosphophoryn preparations compared with the same data calculated from the C-terminal extension of mouse, rat and human dentine sialophosphoprotein (DSPP) gene products. Analysis of the mutation pattern of the clinical disorder Osteogenesis Imperfecta within the region enclosed by the identified collagen fragments reveals that phosphophoryn associates with a segment of collagen that is crucial for structure and/or function.
牛牙本质磷蛋白（BDP）是一种富含天冬氨酰（Asp）和O-磷酰丝氨酰（Ser(P)）残基的蛋白质，由成牙本质细胞合成，被认为参与牙本质的基质介导生物矿化过程。采用EDTA提取、Ca(2+)沉淀、阴离子交换和尺寸排阻色谱法从牛牙本质中纯化磷蛋白。纯化后的蛋白质在SDS-PAGGE上迁移为单一条带。使用螯合树脂碱性透析法使该蛋白质去磷酸化，再通过阴离子交换和尺寸排阻色谱法重新纯化，然后用胰蛋白酶进行切割。对消化产物进行反相高效液相色谱分析，并通过Q-TOF质谱进行分析。唯一能鉴定出的非胰蛋白酶肽段是两条I型胶原α2肽段，其序列通过片段分析确定。胶原片段与高度纯化的磷蛋白的结合表明，EDTA提取法得到的BDP与胶原片段紧密结合。这种结合现在有助于解释各种磷蛋白制剂的分子量和氨基酸组成数据与从小鼠、大鼠和人类牙本质涎磷蛋白（DSPP）基因产物的C末端延伸计算得到的相同数据之间的差异。对在已鉴定的胶原片段所包围区域内的临床疾病成骨不全症的突变模式进行分析表明，磷蛋白与对结构和/或功能至关重要的一段胶原相关联。
