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Active sites of human MEPE-ASARM regulating bone matrix mineralization.
Molecular and Cellular Endocrinology ( IF 3.8 ) Pub Date : 2020-07-24 , DOI: 10.1016/j.mce.2020.110931
Tomoko Minamizaki 1 , Kaoru Sakurai 2 , Ikue Hayashi 3 , Masaaki Toshishige 1 , Hirotaka Yoshioka 1 , Katsuyuki Kozai 4 , Yuji Yoshiko 1
Affiliation  

The proteolytic fragment ASARM (acidic serine- and aspartate-rich motif) of MEPE (matrix extracellular phosphoglycoprotein) (MEPE-ASARM) may act as an endogenous anti-mineralization factor involved in X-linked hypophosphatemic rickets/osteomalacia (XLH). We synthesized MEPE-ASARM peptides and relevant peptide fragments with or without phosphorylated Ser residues (pSer) to determine the active site(s) of MEPE-ASARM in a rat calvaria cell culture model. None of the synthetic peptides elicited changes in cell death, proliferation or differentiation, but the peptide (pASARM) with three pSer residues inhibited mineralization without causing changes in gene expression of osteoblast markers tested. The anti-mineralization effect was maintained in peptides in which any one of three pSer residues was deleted. Polyclonal antibodies recognizing pASARM but not ASARM abolished the pASARM effect. Deletion of six N-terminal residues but leaving the recognition sites for PHEX (phosphate regulating endopeptidase homolog, X-linked), a membrane endopeptidase responsible for XLH, intact and two C-terminal amino acid residues did not alter the anti-mineralization activity of pASARM. Our results strengthen understanding of the active sites of MEPE-pASARM and allowed us to identify a shorter more stable sequence with fewer pSer residues still exhibiting hypomineralization activity, reducing peptide synthesis cost and increasing reliability for exploring biological and potential therapeutic effects.



中文翻译:

人类MEPE-ASARM的活性部位调节骨基质矿化。

MEPE(基质细胞外磷酸糖蛋白)(MEPE-ASARM)的蛋白水解片段ASARM(酸性富含丝氨酸和天冬氨酸的基序)可能充当X连锁低磷病/骨软化症(XLH)的内源性抗矿化因子。我们合成了具有或不具有磷酸化Ser残基(pSer)的MEPE-ASARM肽和相关肽片段,以确定在大鼠颅盖细胞培养模型中MEPE-ASARM的活性位点。没有一种合成的肽引起细胞死亡,增殖或分化的改变,但是具有三个pSer残基的肽(pASARM)抑制了矿化作用,而没有引起测试的成骨细胞标记基因表达的变化。在其中三个pSer残基中的任何一个被缺失的肽中保持了抗矿化作用。识别pASARM但不能识别ASARM的多克隆抗体消除了pASARM的作用。删除6个N末端残基,但保留PHEX的识别位点(磷酸调节内肽酶同源物,X连锁),负责XLH的膜内肽酶,完整和2个C末端氨基酸残基,不会改变其抗矿化活性pASARM。我们的结果加强了对MEPE-pASARM活性位点的理解,使我们能够鉴定出较短的更稳定的序列,同时具有更少的pSer残基仍具有矿化作用,降低肽合成成本,并提高了探索生物学和潜在治疗作用的可靠性。负责XLH,完整和两个C末端氨基酸残基的膜内肽酶不会改变pASARM的抗矿化活性。我们的结果加强了对MEPE-pASARM活性位点的理解,使我们能够鉴定出较短的更稳定的序列,同时具有更少的pSer残基仍具有矿化作用,降低肽合成成本,并提高了探索生物学和潜在治疗作用的可靠性。负责XLH,完整和两个C末端氨基酸残基的膜内肽酶不会改变pASARM的抗矿化活性。我们的结果加强了对MEPE-pASARM活性位点的理解,使我们能够鉴定出较短的更稳定的序列,同时具有更少的pSer残基仍具有矿化作用,降低肽合成成本,并提高了探索生物学和潜在治疗作用的可靠性。

更新日期:2020-08-06
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