Rivkin E, Tres L L, Kaplan-Kraicer R, Shalgi R, Kierszenbaum A L
Department of Cell Biology and Anatomical Sciences, The City University of New York Medical School, New York, New York.
Mol Reprod Dev. 2000 Jul;56(3):401-11. doi: 10.1002/1098-2795(200007)56:3<401::AID-MRD11>3.0.CO;2-7.
Rat sperm galactosyl receptor is a member of the C-type animal lectin family showing preferential binding to N-acetylgalactosamine compared to galactose. Binding is mediated by a Ca(2+)-dependent carbohydrate-recognition domain (CRD) identical to that of the minor variant of rat hepatic lectin receptor 2/3 (RHL-2/3). The molecular organization of the genomic DNA, cDNA, and derived amino acid sequence of rat testis galactosyl receptor have been determined and in vitro fertilization studies were conducted to ascertain its role. We have determined that the rat testis galactosyl receptor gene generates two mRNA species: one species, designated liver-type, is identical to RHL-2/3; the other, designated testis-type, contains one unspliced intron (86 nt) which alters the reading frame and changes the amino acid sequence of the carboxyl terminus. As a result, the CRD (glutamine-proline-aspartic acid/QPD) and flanked Ca(2+)-binding amino acid sequences were not present in the testis-type protein. Northern and Southern blots demonstrated presence of transcripts with unspliced intron in rat sperm but not liver. Similarly, antibody, raised against a synthetic 12-amino acid peptide (p12) encoded by the unspliced intron, recognized in immunoblots a 54 kDa receptor protein in protein extracts from testis but not from liver. Immunofluorescence and immunogold electron microscopy studies demonstrated that both protein species localized on the plasma membrane surface of the head and tail of rat sperm. Furthermore, capacitated rat sperm preincubated with polyclonal antisera to RHL-2/3 or to the CRD of the liver-type galactosyl receptor showed a statistically significant decrease in the in vitro fertilization rate. We conclude that rat sperm galactosyl receptor may play a role in egg binding and that an undetermined molecular mechanism operates to generate two proteins with identical intracellular amino terminal domain but only one of them displays a CRD and associated Ca(2+)-binding sites at the carboxyl terminal extracellular domain.
大鼠精子半乳糖基受体是C型动物凝集素家族的成员，与半乳糖相比，它对N-乙酰半乳糖胺具有优先结合能力。这种结合由一个与大鼠肝凝集素受体2/3（RHL-2/3）的次要变体相同的Ca(2+)依赖性碳水化合物识别结构域（CRD）介导。已经确定了大鼠睾丸半乳糖基受体的基因组DNA、cDNA和推导的氨基酸序列的分子结构，并进行了体外受精研究以确定其作用。我们已经确定大鼠睾丸半乳糖基受体基因产生两种mRNA：一种称为肝型，与RHL-2/3相同；另一种称为睾丸型，包含一个未剪接的内含子（86 nt），该内含子改变了阅读框并改变了羧基末端的氨基酸序列。结果，睾丸型蛋白中不存在CRD（谷氨酰胺-脯氨酸-天冬氨酸/QPD）和侧翼的Ca(2+)结合氨基酸序列。Northern和Southern印迹显示大鼠精子中存在带有未剪接内含子的转录本，而肝脏中则没有。同样，针对由未剪接内含子编码的合成12氨基酸肽（p12）产生的抗体，在免疫印迹中识别出睾丸而非肝脏的蛋白质提取物中的一种54 kDa受体蛋白。免疫荧光和免疫金电子显微镜研究表明，这两种蛋白质都定位于大鼠精子头部和尾部的质膜表面。此外，用针对RHL-2/3或肝型半乳糖基受体CRD的多克隆抗血清预孵育的获能大鼠精子在体外受精率上显示出统计学上的显著下降。我们得出结论，大鼠精子半乳糖基受体可能在卵子结合中起作用，并且一种未确定的分子机制起作用以产生两种具有相同细胞内氨基末端结构域的蛋白质，但其中只有一种在羧基末端细胞外结构域显示CRD和相关的Ca(2+)结合位点。
