Chen H, Pyluck A L, Janik M, Sampson N S
Department of Chemistry, State University of New York, Stony Brook 11794-3400, USA.
Biopolymers. 1998;47(4):299-307. doi: 10.1002/(SICI)1097-0282(1998)47:4<299::AID-BIP5>3.0.CO;2-X.
A key step leading to fertilization is the binding of sperm to the egg plasma membrane. When a mammalian sperm reaches the egg plasma membrane, fertilin, an extracellular sperm membrane protein, is believed to bind to an egg plasma membrane receptor mediating fusion. Fertilin is composed of two subunits, and each subunit contains several domains, i.e., metalloprotease, disintegrin, epidermal growth factor (EGF)-like and fusion domains. This investigation examined the role of the EGF-like domains of mouse fertilin alpha and fertilin beta. Peptides corresponding to the N-terminal subdomain, containing four cysteines, and the C-terminal subdomain, containing two cysteines, were synthesized by solid-phase synthesis methods. Disulfide bonds were formed regioselectively according to the canonical EGF-like disulfide pattern. The activity of these peptides and their linear counterparts were tested for activity in a mouse in vitro fertilization assay. One peptide, 4a, corresponding to the cystine-constrained N-terminal subdomain of fertilin beta, had an activating effect on fertilization. The fertilization rate (number of eggs fertilized), fertilization index (number of sperm fused per egg), and level of polyspermy (three or more sperm fused per egg) increased in the presence of 500 microM 4a (12, 56, and 190%, respectively). Its linear counterpart, 4b, had no effect on in vitro fertilization. These data suggest that the EGF-like domain of fertilin beta has a function in sperm-egg binding and fusion. Previously, it has been shown that the fertilin beta disintegrin domain has a role in sperm-egg binding. Considered together, these studies suggest that fertilin is a modular, multidomain protein with more than one mechanism of action. This modularity may be used to design inhibitors of fertilin-receptor interactions that have high specificities for the fertilization process.
受精过程中的一个关键步骤是精子与卵质膜的结合。当哺乳动物的精子到达卵质膜时，受精素（一种细胞外精子膜蛋白）被认为会与介导融合的卵质膜受体结合。受精素由两个亚基组成，每个亚基包含几个结构域，即金属蛋白酶结构域、解整合素结构域、表皮生长因子（EGF）样结构域和融合结构域。本研究探讨了小鼠受精素α和受精素β的EGF样结构域的作用。通过固相合成方法合成了与含有四个半胱氨酸的N端亚结构域和含有两个半胱氨酸的C端亚结构域相对应的肽段。根据典型的EGF样二硫键模式区域选择性地形成二硫键。在小鼠体外受精试验中测试了这些肽段及其线性对应物的活性。一种肽段4a，对应于受精素β的胱氨酸限制型N端亚结构域，对受精有激活作用。在500微摩尔4a存在的情况下，受精率（受精的卵数）、受精指数（每个卵融合的精子数）和多精受精水平（每个卵有三个或更多精子融合）分别提高了12%、56%和190%。其线性对应物4b对体外受精没有影响。这些数据表明受精素β的EGF样结构域在精卵结合和融合中具有功能。此前已表明受精素β解整合素结构域在精卵结合中起作用。综合考虑，这些研究表明受精素是一种具有多种作用机制的模块化、多结构域蛋白。这种模块化可用于设计对受精过程具有高特异性的受精素-受体相互作用抑制剂。
