Kokoza V A, Snigirevskaya E S, Raikhel A S
Department of Entomology, Michigan State University, East Lansing 48824-1115, USA.
Insect Mol Biol. 1997 Nov;6(4):357-68. doi: 10.1046/j.1365-2583.1997.00191.x.
We have deduced the amino acid sequences of clathrin heavy chain (CHC) polypeptides based on cDNA and genomic clones from the mosquito, Aedes aegypti. Two isoforms which differ in the very beginning of the N-terminal domain, ovary-specific AaCHCa and somatic-specific AaCHCb, were identified, characterized and compared to one another as well as to CHC polypeptides from different species. The 1682 amino acid sequence of the AaCHCa isoform predicts a molecular mass (M[r]) of 191,743 daltons and an isoelectric point of 5.80, whereas the 1674 amino acid sequence of the AaCHCb isoform predicts a M(r) of 191,033 daltons and an isoelectric point of 5.71. Both mosquito AaCHC isoforms are highly conserved, with full-sequence identities of 88% to Drosophila melanogaster, 81% to mammal (rat, cow and human), 71% to C. elegans, 58% to Dictyostelium discoideum, and 49% to yeast CHC polypeptides. The highest degree of conservation is in the middle portion of the mosquito CHC molecule which includes the linker region and extended triskelion arm, with decreasing conservation through the N-terminal domain, trimerization domain, and the relatively diverged C-terminal region. The protein domains do not directly correspond to specific exons of the mosquito AaCHC gene, with the exception of exon 6 which encodes the C-terminal domain of the CHC polypeptide. Polyclonal antibodies raised against a bacteria-expressed AaCHC fusion protein recognized one major band of about 180 kDa in vitellogenic ovary whole-lysate. Immunogold labelling of the AaCHC polypeptide localized it to the coat of coated pits and coated vesicles in oocytes from vitellogenic follicles. Northern blot and in situ hybridization analyses suggest that regulation of AaCHC gene expression in the ovary is complex, and it likely involves both developmental and hormonal signals.
我们基于来自埃及伊蚊的cDNA和基因组克隆推导了网格蛋白重链（CHC）多肽的氨基酸序列。鉴定并表征了两种在N端结构域起始处不同的异构体，即卵巢特异性的AaCHCa和体细胞特异性的AaCHCb，并将它们相互比较，同时也与来自不同物种的CHC多肽进行了比较。AaCHCa异构体的1682个氨基酸序列预测分子量（M[r]）为191,743道尔顿，等电点为5.80，而AaCHCb异构体的1674个氨基酸序列预测M[r]为191,033道尔顿，等电点为5.71。两种埃及伊蚊AaCHC异构体都高度保守，与黑腹果蝇的全序列同一性为88%，与哺乳动物（大鼠、牛和人类）为81%，与秀丽隐杆线虫为71%，与盘基网柄菌为58%，与酵母CHC多肽为49%。最高程度的保守性存在于埃及伊蚊CHC分子的中部，包括连接区和延伸的三脚蛋白臂，从N端结构域、三聚化结构域到相对分化的C端区域，保守性逐渐降低。蛋白质结构域并不直接对应于埃及伊蚊AaCHC基因的特定外显子，除了编码CHC多肽C端结构域的外显子6。针对细菌表达的AaCHC融合蛋白产生的多克隆抗体在卵黄发生期卵巢全裂解物中识别出一条约180 kDa的主要条带。AaCHC多肽的免疫金标记将其定位在卵黄发生期卵泡卵母细胞中被膜小窝和被膜小泡的包被上。Northern印迹和原位杂交分析表明，卵巢中AaCHC基因表达的调控很复杂，可能涉及发育和激素信号。
