Craniofacial and Skeletal Diseases Branch, NIDCR, NIH, DHHS, Bethesda, MD, USA.
Matrix Biol. 2010 May;29(4):295-303. doi: 10.1016/j.matbio.2010.01.002. Epub 2010 Jan 15.
The protease that cleaves the most abundant non-collagenous protein of dentin matrix, dentin sialophosphoprotein (DSPP), into its two final dentin matrix products, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), has not been directly identified. In this study, full-length recombinant mouse DSPP was made for the first time in furin-deficient mammalian LoVo cells and used to test the ability of three different isoforms of one candidate protease, bone morphogenetic protein-1 (BMP1) to cleave DSPP at the appropriate site. Furthermore, two reported enhancers of BMP1/mTLD activity (procollagen C-endopeptidase enhancer-1, PCPE-1, and secreted frizzled-related protein-2, sFRP2) were tested for their abilities to modulate BMP1-mediated processing of both DSPP and another SIBLING family member with a similar cleavage motif, dentin matrix protein-1 (DMP1). Three splice variants of BMP1 (classic BMP1, the full-length mTolloid (mTLD), and the shorter isoform lacking the CUB3 domain, BMP1-5) were all shown to cleave the recombinant DSPP in vitro although mTLD was relatively inefficient at processing both DSPP and DMP1. Mutation of the MQGDD peptide motif to IEGDD completely eliminated the ability of all three recombinant isoforms to process full-length recombinant DSPP in vitro thereby verifying the single predicted cleavage site. Furthermore when human bone marrow stromal cells (which naturally express furin-activated BMP1) were transduced with the adenovirus-encoding either wild-type or mutant DSPP, they were observed to fully cleave wild-type DSPP but failed to process the mutant DSPP(MQDeltaIE) during biogenesis. All three BMP1 isoforms were shown to process type I procollagen as well as DSPP and DMP1 much more efficiently in low-salt buffer (< or = 50 mM NaCl) compared to commonly used normal saline buffers (150 mM NaCl). Neither PCPE-1 nor sFRP2 were able to enhance any of the three BMP1 isoforms in cleaving either DSPP or DMP1 under either low or normal saline conditions. Interestingly, we were unable to reproduce sFRP2's reported ability to enhance the processing of type I procollagen by BMP1/mTLD. In summary, three isoforms of BMP1 process both DSPP and DMP1 at the MQX/DDP motif, but the identity of a protein that can enhance the cleavage of the two SIBLING proteins remains elusive.
目前尚未直接鉴定出能将牙本质基质中含量最丰富的非胶原蛋白——牙本质涎磷蛋白（DSPP）切割成最终的牙本质基质产物牙本质涎蛋白（DSP）和牙本质磷蛋白（DPP）的蛋白酶。在这项研究中，首次在缺乏furin 的哺乳动物 LoVo 细胞中制备全长重组鼠 DSPP，并用于测试三种不同候选蛋白酶之一的骨形态发生蛋白 1（BMP1）同工型切割 DSPP 适当位置的能力。此外，还测试了两种报道的增强 BMP1/mTLD 活性的增强子（原胶原 C-端肽酶增强子 1[PCPE-1]和分泌卷曲相关蛋白 2[sFRP2]）调节 BMP1 介导的另一种具有相似切割模体的 SIBLING 家族成员牙本质基质蛋白 1（DMP1）加工的能力。三种 BMP1 剪接变体（经典 BMP1、全长 mTolloid[mTLD]和缺乏 CUB3 结构域的较短同工型 BMP1-5）均能体外切割重组 DSPP，尽管 mTLD 对切割 DSPP 和 DMP1 的效率相对较低。将 MQGDD 肽基序突变为 IEGDD 可完全消除所有三种重组同工型体外切割全长重组 DSPP 的能力，从而验证了单一预测的切割位点。此外，当用编码野生型或突变型 DSPP 的腺病毒转导人骨髓基质细胞（其天然表达 furin 激活的 BMP1）时，观察到它们能完全切割野生型 DSPP，但在生物发生过程中不能加工突变型 DSPP（MQDeltaIE）。与常用的生理盐水缓冲液（150 mM NaCl）相比，三种 BMP1 同工型在低盐缓冲液（<或=50 mM NaCl）中更有效地处理 I 型原胶原以及 DSPP 和 DMP1。PCPE-1 和 sFRP2 均不能增强三种 BMP1 同工型在低盐或生理盐水条件下切割任何一种 DSPP 或 DMP1。有趣的是，我们无法重现 sFRP2 报道的增强 BMP1/mTLD 切割 I 型原胶原的能力。总之，三种 BMP1 同工型在 MQX/DDP 基序切割 DSPP 和 DMP1，但能增强两种 SIBLING 蛋白切割的蛋白质的身份仍难以捉摸。
