Gingras D, White D, Garin J, Cosson J, Huitorel P, Zingg H, Cibert C, Gagnon C
Urology Research Laboratory, Royal Victoria Hospital, McGill University, Montreal H3A 1A1, Quebec, Canada.
Mol Biol Cell. 1998 Feb;9(2):513-22. doi: 10.1091/mbc.9.2.513.
Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40 degrees C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form alpha-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.
针对海胆精子轴丝蛋白产生的单克隆抗体已被用于研究鞭毛运动所涉及的调控机制。在此，我们报告其中一种抗体，单克隆抗体D - 316，对海胆精子模型的运动具有不寻常的干扰作用；它不影响摆动频率、摆动幅度或活动精子模型的百分比，而是促进鞭毛摆动模式从二维运动显著转变为三维运动。在通过SDS - PAGE分离的轴丝蛋白免疫印迹中，D - 316识别出一条90 kDa的单一多肽。通过将轴丝在40摄氏度短暂热处理后提取该蛋白，随后对其进行了纯化。该蛋白与43 kDa和34 kDa的蛋白共纯化且共免疫沉淀，表明它以天然形式作为复合物存在。使用D - 316作为探针，从海胆cDNA文库中获得了编码90 kDa蛋白的全长cDNA克隆。该序列预测这是一种由552个氨基酸组成的高度酸性蛋白（pI = 4.0），质量为62,720 Da（p63）。与数据库中的蛋白质序列比较表明，该蛋白与莱茵衣藻的径向辐条蛋白4和6（RSP4和RSP6）相关，分别与p63有37%和25%的相似性。然而，海胆蛋白具有与RSP4和RSP6不同的结构特征，例如存在三个主要的酸性区域，分别包含25、17和12个天冬氨酸和谷氨酸残基，这些区域分别由34、22和14个氨基酸长的片段组成，预计会形成α - 螺旋卷曲螺旋二级结构。这些结果表明p63在维持精子鞭毛摆动的平面形式中起主要作用，并为研究更进化物种中径向辐条头部的功能提供了新工具。
