Rat sperm galactosyl receptor: purification and identification by polyclonal antibodies raised against multiple antigen peptides.
Rat sperm galactosyl receptor: purification and identification by polyclonal antibodies raised against multiple antigen peptides.
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大鼠精子半乳糖基受体：针对多种抗原肽的多克隆抗体的纯化和鉴定。
DOI：
10.1002/mrd.1080410314
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Kierszenbaum,AL
中科院分区：
文献类型：
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作者：
Mertz,JR;Banda,PW;Kierszenbaum,AL
我们先前已经报道了大鼠睾丸半乳糖受体的纯化，其相当于大鼠肝凝集素-2/3（RHL-2/3）的Ca 2+依赖性（C-型）微小变体。本文报道了从大鼠精子中纯化半乳糖受体，并以多抗原肽（MAP）为免疫原制备多克隆抗体，对半乳糖受体在大鼠睾丸和精子中的免疫定位。使用两种MAP抗原（指定为27-mer和28-mer）进行免疫，其对应于RHL-2/3的碳水化合物识别结构域（半乳糖）和相邻Ca 2+结合位点的氨基酸序列。抗RHL-2/3、抗p27和抗p28血清与大鼠肝细胞RHL-2/3及其大鼠睾丸和精子等效物半乳糖受体发生交叉反应，半乳糖受体通过色谱聚焦，然后通过半乳糖-Hydropore-EP亲和色谱纯化。抗p27和抗p28血清均不与主要肝细胞变体RHL-1发生交叉反应。在大鼠睾丸和精子中未检测到RHL-1-等效物。免疫荧光研究表明，抗p27和抗p28血清识别支持细胞-生精细胞界面和精子头背表面上覆盖顶体的半乳糖受体位点。诱导顶体反应后，精子中特征性的新月形免疫反应模式消失。进一步的研究应确定MAP抗原27-mer和28-mer（对应于特定的蛋白基序）的抗血清是否可以作为免疫学探针，用于检查精子发生和受精过程中的细胞-细胞相互作用事件。© 1995 Wiley利斯公司
We have previously reported the purification of rat testis galactosyl receptor, an equivalent to the Ca2+‐dependent (C‐type) minor variant of rat hepatic lectin‐2/3 (RHL‐2/3). We now report the purification of galactosyl receptor from rat sperm and its immunolocalization in the intact rat testis and sperm by polyclonal antibodies prepared using multiple antigen peptides (MAP) as immunogens. Two MAP antigens (designated 27‐mer and 28‐mer), corresponding to amino acid sequences of the carbohydrate‐recognition domain (galactose) and adjacent Ca2+‐binding sites of RHL‐2/3, were used for immunization. Anti‐RHL‐2/3, anti‐p27, and anti‐p28 sera crossreacted with rat hepatocyte RHL‐2/3 and its rat testis and sperm equivalent, galactosyl receptor, purified by chromatofocusing followed by galactose‐Hydropore‐EP affinity chromatography. Neither anti‐p27 nor anti‐p28 sera crossreacted with the major hepatocyte variant, RHL‐1. A RHL‐1‐equivalent was not detected in rat testis and sperm. Immunofluorescence studies demonstrated that anti‐p27 and anti‐p28 sera recognize galactosyl receptor sites at the Sertoli cell‐spermatogenic cell interface and on the dorsal surfacae of the sperm head, overlying the acrosome. The characteristic crescent‐shaped immunoreactive pattern in sperm was lost after induction of the acrosome reaction. Further studies should determine whether antisera to MAP antigens 27‐mer and 28‐mer, corresponding to specific protein motifs, can serve as immunological probes for examining cell‐cell interaction events during spermatogenesis and at fertilization. © 1995 Wiley‐Liss, Inc.