Site-directed Mutagenesis of Rabbit Proacrosin
Site-directed Mutagenesis of Rabbit Proacrosin
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兔顶大体素原的定点诱变
DOI：
--
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发表时间：
1996
影响因子：
4.8
通讯作者：
M. O'Rand
中科院分区：
文献类型：
--
作者：
R. Richardson;M. O'Rand
哺乳动物顶体精子蛋白酶原顶体蛋白通过卵母细胞外部投资的蛋白水解在受精中发挥作用。除了其丝氨酸蛋白酶活性外，已知来自多个物种的顶体蛋白对透明带具有结合活性，并且这种作用可能有助于在透明带穿透过程中锚定精子。在这项研究中，从兔精子的酸提取物中纯化了顶体素原，并通过体外测定证明其与同源透明带结合。这种结合活性的测量表明与 KD = 1.4 × 10−8 M 存在高亲和力饱和相互作用。使用从先前克隆和测序的兔原顶体素和编码更短顶体素形式的剪接变体获得的 cDNA（Richardson, R. T., and O'Rand, M. G. (1994) Biochim. Biophys. Acta 1219, 215-218），使用聚合酶链式反应产生不同大小的构建体并表达为重组蛋白。在相同的体外透明带结合测定中，发现代表兔顶体素原残基 1-279 的构建体以与天然顶体素原相似的高亲和力与透明带结合，KD = 2.1 × 10−8 M。通过制备更小的重组片段并测定它们的透明带结合活性，结合位点的位置被映射到残基 47-94。使用胰凝乳蛋白酶原 A 作为三维模型对兔顶体蛋白原进行蛋白质建模，表明胰凝乳蛋白酶原 A 中暴露的环 Asp35 至 His40 延伸有兔顶体蛋白原中从 Ile43 至 His53 的额外五个氨基酸残基，其中包含精氨酸残基 Arg47、Arg50 和 Arg51。将精氨酸残基 Arg50 和 Arg51 定点诱变为丙氨酸产生没有显着透明带结合活性的重组体。这些结果与兔顶体蛋白原含有特定透明带结合位点并且含有精氨酸残基 50 和 51 的环对于透明带结合活性至关重要的假设一致。
The mammalian acrosomal sperm protease proacrosin plays a role in fertilization by proteolysis of the oocyte's outer investments. In addition to its serine protease activity, acrosin from several species is known to have binding activity for the zona pellucida, and this action may serve to anchor sperm during zona penetration. In this study, proacrosin was purified from acid extracts of rabbit sperm and shown to bind to homologous zona pellucida using an in vitro assay. Measurement of this binding activity indicated a high affinity saturable interaction with a KD = 1.4 × 10−8 M. Using cDNAs obtained from previously cloned and sequenced rabbit proacrosin and a splice variant that encodes a shorter form of acrosin (Richardson, R. T., and O'Rand, M. G. (1994) Biochim. Biophys. Acta 1219, 215-218), constructs of various sizes were produced using polymerase chain reaction and expressed as recombinant proteins. In the same in vitro zona binding assay, a construct representing residues 1-279 of rabbit proacrosin was found to bind to zona with a high affinity similar to that of native proacrosin, KD = 2.1 × 10−8 M. By making smaller recombinant fragments and assaying them for zona binding activity, the location of the binding site was mapped to residues 47-94. Protein modeling of rabbit proacrosin using chymotrypsinogen A as a three-dimensional model indicated that an exposed loop Asp35 to His40 in chymotrypsinogen A is extended with an additional five amino acid residues in rabbit proacrosin from Ile43 to His53 containing arginine residues Arg47, Arg50 and Arg51. Site-directed mutagenesis of arginine residues Arg50 and Arg51 to alanine produced a recombinant without significant zona binding activity. These results are consistent with the hypothesis that rabbit proacrosin contains a specific zona pellucida binding site and that the loop containing arginine residues 50 and 51 is critical for zona binding activity.
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兔精子膜自身抗原 RSA 的表征，作为凝集素样透明带结合蛋白。
影响因子：
2.7
作者：
O'Rand,MG;Widgren,EE;Fisher,SJ
通讯作者：
Fisher,SJ
兔前顶体素原的 cDNA 和新型前顶体素原相关 cDNA 的克隆和测序。
DOI：
10.1016/0167-4781(94)90275-5
发表时间：
1994
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Richardson,RT;O'Rand,MG
通讯作者：
O'Rand,MG
受精过程中精子膜和透明带相互作用。
影响因子：
--
作者：
O'Rand,MG;Welch,JE;Fisher,SJ
通讯作者：
Fisher,SJ
影响因子：
2.7
作者：
RICHARDSON, RT;YAMASAKI, N;ORAND, MG
通讯作者：
ORAND, MG
固定化透明带基质诱导顶体反应期间兔精子顶体蛋白的定位。
影响因子：
3.6
作者：
Richardson,RT;Nikolajczyk,BS;Abdullah,LH;Beavers,JC;O'Rand,MG
通讯作者：
O'Rand,MG