Blocking of proteolytic processing and deletion of glycosaminoglycan side chain of mouse DMP1 by substituting critical amino acid residues.
Blocking of proteolytic processing and deletion of glycosaminoglycan side chain of mouse DMP1 by substituting critical amino acid residues.
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DOI：
10.1159/000151373
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Qin C
中科院分区：
文献类型：
--
作者：
Peng T;Huang B;Sun Y;Lu Y;Bonewald L;Chen S;Butler WT;Feng JQ;D'Souza RN;Qin C
牙本质基质蛋白1（Dentin matrix protein 1，DMP 1）是牙本质和骨的细胞外基质（extracellular matrix，ECM）中的一种蛋白质，在大鼠DMP 1的加工过程中，由于天冬氨酸残基的NH 2端发生蛋白水解而形成的NH 2-和COOH-末端片段。一个切割位点残基，Asp 181（对应于小鼠DMP 1的Asp 197），及其侧翼区在物种间高度保守。我们推测，在天冬氨酸197的小鼠DMP 1的NH 2末端的裂解代表了初始的，在整个级联的蛋白水解加工的第一步切断。为了测试Asp 197是否对启动小鼠DMP 1的蛋白水解加工至关重要，我们通过突变编码该氨基酸残基的小鼠cDNA的相应核苷酸来用Ala 197取代Asp 197。将突变体DMP 1cDNA克隆到pcDNA3.1载体中。来自转染实验的数据表明，该单一取代阻断了小鼠DMP 1在HEK-293细胞中的蛋白水解加工，表明Asp 197的NH 2末端的切割对于暴露用于将DMP 1转化为其片段的其他切割位点至关重要。DMP 1的NH 2-末端片段以含有糖胺聚糖（GAG）链的蛋白聚糖形式（DMP 1-PG）存在。以前，我们表明，GAG链连接到大鼠DMP 1中的Ser 74（小鼠DMP 1中的Ser 89）。为了证实小鼠DMP 1-PG具有与Ser 89连接的单个GAG链，我们用Gly 89取代Ser 89。来自转染分析的数据表明，该取代完全阻止了含GAG形式的形成，证实了DMP 1-PG含有与小鼠DMP 1中的Ser 89连接的单个GAG链。
Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH2- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH2 termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp 181 (corresponding to Asp 197 of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH2 terminus of Asp 197 of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp 197 is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp 197 with Ala197 by mutating the corresponding nucleotides of mouse cDNA that encode this amino acid residue. This mutant DMP1 cDNA was cloned into a pcDNA3.1 vector. Data from transfection experiments indicated that this single substitution blocked the proteolytic processing of mouse DMP1 in HEK-293 cells, indicating that cleavage at the NH2 terminus of Asp 197 is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH2-terminal fragment of DMP1 occurs as a proteoglycan form (DMP1-PG) that contains a glycosaminoglycan (GAG) chain. Previously, we showed that a GAG chain is linked to Ser 74 in rat DMP1 (Ser 89 in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser 89, we substituted Ser 89 by Gly89. Data from transfection analysis indicated that this substitution completely prevented formation of the GAG-containing form, confirming that DMP1-PG contains a single GAG chain attached to Ser 89 in mouse DMP1.
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影响因子：
3.5
作者：
Toyosawa, S;O'hUigin, C;Klein, J
通讯作者：
Klein, J
影响因子：
30.8
作者：
Lorenz-Depiereux, Bettina;Bastepe, Murat;Strom, Tim M.
通讯作者：
Strom, Tim M.
影响因子：
6.2
作者：
MacDougall, M;Gu, TT;Chen, JK
通讯作者：
Chen, JK
影响因子：
4.8
作者：
Ye, L;Mishina, Y;Feng, JQ
通讯作者：
Feng, JQ
影响因子：
4.8
作者：
Qin, CL;Huang, BZ;Butler, WT
通讯作者：
Butler, WT