Lopez A, Miraglia S J, Glabe C G
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Dev Biol. 1993 Mar;156(1):24-33. doi: 10.1006/dbio.1993.1056.
The functional domain structure of bindin, an adhesive protein component of the acrosomal granule of sea urchin sperm, has been analyzed using a series of carboxyl- and amino-terminal deletion analogs of recombinant bindin expressed in Escherichia coli. Bindin, which self-associates to form macromolecular aggregates, mediates the species-specific attachment of sperm to the egg surface as well as egg-to-egg agglutination in vitro by binding to egg surface-sulfated fucose-containing glycoconjugates. Cross-species egg agglutination activity and sperm adhesion were determined for two sea urchin species, Strongylocentrotus purpuratus and S. franciscanus. We found that S. franciscanus sperm bindin agglutinates both S. franciscanus and S. purpuratus eggs whereas S. purpuratus sperm bindin displays a much more restricted specificity for homologous S. purpuratus eggs. Consistent with this result, cross-species sperm adhesion experiments demonstrate that S. franciscanus sperm also bind more efficiently to S. purpuratus eggs in comparison to S. purpuratus sperm attachment to S. franciscanus eggs. Recombinant S. purpuratus bindin demonstrates the same species-specific egg agglutination properties observed for sperm bindin. Deletion mutants of S. purpuratus bindin were tested for their ability to species specifically agglutinate eggs. We found that residues 1-74 or residues 122-236 can be deleted without a loss of the species-specific agglutination properties of bindin. Comparison of the bindin amino acid sequences from the two species reveals that all of the active deletion analogs have a central segment in common corresponding to residues 75 through 121 of S. purpuratus bindin, where the sequences are identical except for a single amino acid, Arg 77, which is an Ala residue in S. franciscanus. However, substitution of Arg 77 for Ala does not alter the species specificity of recombinant bindin, suggesting that the more divergent amino- and carboxy-terminal sequences flanking the conserved central domain determine the species specificity of bindin and either end is sufficient to impart specificity. The amino- and carboxyl-terminal thirds of S. purpuratus bindin each contain two repeats of the sequence M G G P P/V. This sequence does not occur in S. franciscanus, which instead has five repeats of the sequence M G G A/V/Q F/V/P. It is conceivable that these repeats, which vary in sequence in bindins from different species, constitute an important part of the species recognition mechanism.
使用在大肠杆菌中表达的一系列重组结合蛋白的羧基末端和氨基末端缺失类似物，对海胆精子顶体颗粒的粘附蛋白成分——结合蛋白的功能结构域进行了分析。结合蛋白能自我缔合形成大分子聚集体，通过与卵表面含硫酸化岩藻糖的糖缀合物结合，介导精子与卵表面的物种特异性附着以及体外卵与卵的凝集。测定了两种海胆——紫球海胆和加州海胆的跨物种卵凝集活性和精子粘附情况。我们发现，加州海胆精子结合蛋白能凝集加州海胆和紫球海胆的卵，而紫球海胆精子结合蛋白对同源的紫球海胆卵表现出更为受限的特异性。与此结果一致，跨物种精子粘附实验表明，与紫球海胆精子附着于加州海胆卵相比，加州海胆精子与紫球海胆卵的结合效率也更高。重组紫球海胆结合蛋白表现出与精子结合蛋白相同的物种特异性卵凝集特性。对紫球海胆结合蛋白的缺失突变体进行了物种特异性凝集卵能力的测试。我们发现，删除1 - 74位残基或122 - 236位残基不会导致结合蛋白物种特异性凝集特性的丧失。比较这两个物种的结合蛋白氨基酸序列发现，所有活性缺失类似物都有一个共同的中央片段，对应于紫球海胆结合蛋白的75至121位残基，除了一个氨基酸（77位精氨酸，在加州海胆中为丙氨酸残基）外，序列相同。然而，将77位精氨酸替换为丙氨酸并不会改变重组结合蛋白的物种特异性，这表明保守中央结构域两侧差异较大的氨基末端和羧基末端序列决定了结合蛋白的物种特异性，且任一端都足以赋予特异性。紫球海胆结合蛋白的氨基末端和羧基末端三分之一区域各包含两个M G G P P/V序列重复。该序列在加州海胆中不出现，加州海胆中该序列有五个M G G A/V/Q F/V/P序列重复。可以想象，这些在不同物种结合蛋白中序列不同的重复序列构成了物种识别机制的重要部分。
