蜜蜂级型分化相关蛋白MRJP1的结构生物学研究

Royal jelly is the primary food that is secreted from the hypopharyngeal and mandibular glands of nurse bees, a kind of special milky substance that fed to larvaes in 3-day-old and adult queens in the colony. MRJP1 was the most abundant protein in MRJPs, the proportion reached 31%, and it represents 48% of the water-soluble proteins of Royal jelly. MRJP1 determined caste differentiation of female bees. As MRJP1 was a kind of glycoprotein that was difficult to crystallize, so its structure was not being reported yet. Based on this background, this project will screen and optimize the crystal growth conditions of MRJP1 to obtain crystals which are suitable for X-ray diffraction, collect MRJP1 crystal diffraction data by synchrotron radiation X-ray diffraction,and then resolve the space structure of MRJP1 in the molecular level and speculate its function. Our research focused on the structural biology of MRJP1 from the view point of structural biology. This work are significant to discover and resolve the mechanism of physiological functions in honeybees and healthcare in other species.

蜂王浆是由工蜂哺育蜂咽下腺和上颚腺共同分泌，用于饲喂蜂王及3日龄内幼虫的一种浆状物质，与蜜蜂的级型分化密切相关。MRJP1在蜂王浆主要蛋白中含量最丰富，所占比例达到了31%，占蜂王浆中水溶性蛋白的48%，参与决定蜂群中雌性蜂的级型分化。由于MRJP1是一种糖蛋白，难于结晶，至今其结构还未被解析。在目前已有的工作基础上，本项目将对MRJP1的晶体生长条件进行优化研究，获得适合X射线衍射的MRJP1晶体。通过同步辐射X—射线衍射收集MRJP1晶体的衍射数据，从分子水平解析出MRJP1的空间结构并推测其功能。对MRJP1结构生物学的研究有助于深入认识MRJP1在蜜蜂中的重要生理功能及探明对其他生物的医疗保健中所起作用和机理，具有重要的生物学意义。

蜂王浆是由工蜂哺育蜂咽下腺和上颚腺共同分泌，用于饲喂蜂王及3日龄内幼虫的一种浆状物质。蜂王浆与蜜蜂的级型分化密切相关。MRJP1在蜂王浆主要蛋白中含量最丰富，在蜜蜂营养和幼虫发育中起着关键作用，对于人体具有广泛的药理活性。Kamakura研究发现MRJP1是蜂王浆中决定蜜蜂级型分化的关键因子。蛋白质的结构决定其功能，由于MRJP1是一种糖蛋白，难于结晶，其晶体结构还未见报道。. 本项目通过从天然蜂王浆中分离纯化得到纯度很高的MRJP1蛋白。MRJP1蛋白经PNGase F切除糖链以及一系列的纯化步骤得到高纯度的MRJP1聚体，采用坐滴和悬滴蒸气扩散法获得其单晶，X射线衍射分辨率达到2.65 Å。通过弱同源解结构的方法解析出MRJP1异质三元复合物结构。MRJP1四聚体由4个MRJP1单体、4个多肽apisimin和8个24-亚甲基胆甾醇小分子组成。MRJP1的结构是采用经典的六叶β-折叠组成螺旋桨式结构，主要以疏水相互作用和氢键结合，其中apisimin起关键的连接作用，另外还含有3个二硫键。两个MRJP1单体通过C末端与多肽apisimin和24-亚甲基胆甾醇相互作用组成MRJP1二聚体，两个MRJP1二聚体通过apisimin小肽形成四聚体。结构比对表明apisimin结构是一种新的模体。采用凝胶过滤层析、分析超速离心、小角散射、GC-MS和NMR等实验对MRJP1复合物的大小、形状、组成成分等进行了确证。通过MRJP1复合物离群饲喂蜜蜂幼虫研究，表明MRJP1复合物不能促进蜜蜂幼虫的生长发育，但经过蛋白酶处理后则出现显著的促进作用。. 本研究为深入理解MRJPs的药理作用及其在蜜蜂中独特的生理作用提供了结构基础，具有重要的生物学意义。