Expression of extracellular dermal glycoprotein (EDGP) is induced by biotic or abiotic stress.The amino acid sequence alignment showed that EDGP shared significant homology with proteins from legumes,tomato,Arabidopsis,wheat,and cotton.These proteins are involved in signal transduction or stress response systems.Most of the Cys residues in these proteins are conserved,suggesting that they share similar tertiary structures.Surface plasmon resonance (SPR) analysis shows that EDGP binds a soybean 4-kDa hormone-like peptide (4-kDa peptide) in vitro and reduction of EDGP decreased significantly the binding activity,implying that posttranslational modifications are important for its function.Therefore,we investigated the posttranslational modifications in EDGP using mass spectrometry.As the result,six disulfide bonds in EDGP were identified:Cys~(70)-Cys~(158),Cys~(84)-Cys~(89),Cys~(97)-Cys~(113),Cys~(100)-Cys~(108),Cys~(201)-Cys~(426),and Cys~(332)-Cys~(378).In addition,the N-terminal glutamine was cyclized into pyroglutamic acid.All four putative glycosylation sites were occupied by N-linked glycans,which have similar masses of m/z 1171.Finally,measuring the mass of the native protein showed that the posttranslational modifications of EDGP (pI9.5) involved only disulfide bonds,N-terminal modification,and glycosylation.
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机译:生物或非生物胁迫诱导了细胞外真皮糖蛋白(EDGP)的表达。氨基酸序列比对表明EDGP与豆类,番茄,拟南芥,小麦和棉花中的蛋白质具有显着的同源性,这些蛋白质参与信号转导或胁迫。这些蛋白质中的大部分Cys残基均被保守,暗示它们具有相似的三级结构。表面等离振子共振(SPR)分析表明EDGP在体外与大豆4-kDa激素样肽(4-kDa肽)结合。 EDGP的还原和还原作用显着降低了结合活性,这说明翻译后修饰对其功能很重要。因此,我们使用质谱法研究了EDGP中的翻译后修饰。结果,鉴定了EDGP中的六个二硫键:Cys〜(70) -Cys〜(158),Cys〜(84)-Cys〜(89),Cys〜(97)-Cys〜(113),Cys〜(100)-Cys〜(108),Cys〜(201)-Cys 〜(426)和Cys〜(332)-Cys〜(378)。此外,N末端谷氨酸己内酯被环化成焦谷氨酸。所有四个假定的糖基化位点都被N-连接的聚糖占据,它们的m / z 1171的质量相似。最后,测量天然蛋白的质量表明EDGP的翻译后修饰(pI9.5 )仅涉及二硫键,N末端修饰和糖基化。
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