Egg yolk proteins in gray mullet (Mugil cephalus):: Purification and classification of multiple lipovitellins and other vitellogenin-derived yolk proteins and molecular cloning of the parent vitellogenin genes
Egg yolk proteins in gray mullet (Mugil cephalus):: Purification and classification of multiple lipovitellins and other vitellogenin-derived yolk proteins and molecular cloning of the parent vitellogenin genes
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DOI：
10.1002/jez.388
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发表时间：
2007-06-01
影响因子：
2.8
通讯作者：
Hara, Akihiko
中科院分区：
文献类型：
--
作者：
Amano, Haruna;Fujita, Toshiaki;Hara, Akihiko
采用羟基磷灰石、离子交换、免疫吸附和凝胶过滤等方法，从鲻鱼卵黄发生卵巢提取液中分离纯化了7种卵黄蛋白（YPs）、4种大脂蛋白（YPs 1 -4）和3种卵黄组分（YPs 5 -7），其中包括1种磷蛋白（YP 7）。天然Y-P1、YP 2、YP 3和YP 4的分子量估计分别为330、325、335和570 kDa。十二烷基硫酸钠聚丙烯酰胺凝胶电泳显示YP 1、YP 2和YP 3的三级结构是典型的硬骨鱼卵黄脂蛋白（Lvs），由一条重链组成（分别类似于110、类似于99和类似于97 kDa）和轻链5 kDa），而YP 4表现出一条重链（类似于110 kDa）和另外两条多肽带（类似于70和类似于54 kDa）。将纯化的YP的N-末端肽序列映射到从其各自的完整cDNA推导的多个鲻鱼卵黄蛋白原（Vgs）的一级结构，克隆并测序，最终确定YP 1、YP 2和YP 3分别为来自鲻鱼VgA、VgB和VgC的Lvs。第四个YP（YP 4）似乎是一个蛋白水解的变体，由Lv和VgA的Ekavitin组分组成。另外两种YP（YP 5和YP 6）基于它们的结构和对鲑鱼β '-组分的共同但不相同的抗原性而被鉴定为源自VgA和VgB的β'-组分，而纯化的YP 7（具有高含量丝氨酸残基的磷蛋白）被鉴定为源自VgB的卵黄蛋白。这是第一份报告，其中我们知道，纯化和分子分类的三种不同形式的吕从任何卵生脊椎动物。
Seven yolk proteins (YPs), four large lipoproteins (YPs1-4) and three minor yolk components (YPs5-7) including one phosphoprotein (YP7), were purified from extracts of vitellogenic ovaries of gray mullet (Mugil cephalus) by combinations of hydroxylapatite, ion exchange, immunoadsorbent, and gel filtration chromatography. The molecular masses of native Y-P1, YP2, YP3, and YP4 were estimated to be 330, 325, 335, and 570 kDa, respectively. The tertiary structures of YP1, YP2, and YP3 revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis were typical of teleost lipovitellins (Lvs), consisting of a heavy chain (similar to 110, similar to 99, and similar to 97 kDa, respectively) and a light chain (similar to 30, similar to 29, and similar to 21.5 kDa, respectively), while YP4 exhibited a heavy chain (similar to 110 kDa) and two more polypeptide bands (similar to 70 and similar to 54kDa). Mapping of N-terminal peptide sequences of the purified YPs to the primary structure of multiple mullet vitellogenins (Vgs) deduced from their respective complete cDNAs, which were cloned and sequenced, conclusively identified YP1, YP2, and YP3 as Lvs derived from mullet VgA, VgB, and VgC, respectively. The fourth YP (YP4) appeared to be a proteolytic variant consisting of Lv and phosvitin components of VgA. Two other YPs (YP5 and YP6) were identified as beta'-components derived from VgA and VgB based on their structures and common, but not identical, antigenicity to salmonid beta'-component, while purified YP7, a phosphoprotein with a high content of serine residues, was identified as a phosvitin derived from VgB. This is the first report, of which we are aware, on purification and molecular classification of three distinct forms of Lv from any oviparous vertebrate.