Vacuolar system-associated protein-60:: A protein characterized from bovine granulosa and luteal cells that is associated with intracellular vesicles and related to human 80K-H and murine β-glucosidase II
Vacuolar system-associated protein-60:: A protein characterized from bovine granulosa and luteal cells that is associated with intracellular vesicles and related to human 80K-H and murine β-glucosidase II
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DOI：
10.1095/biolreprod62.3.642
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发表时间：
2000-03-01
影响因子：
3.6
通讯作者：
Lussier, JG
中科院分区：
文献类型：
--
作者：
Brûlé, S;Rabahi, F;Lussier, JG
分子量为56-58 kDa的蛋白质在牛卵巢卵泡发育和卵母细胞成熟中起重要作用。一个多克隆抗体提出了对56- 58-kDa的蛋白带纯化的牛颗粒细胞，并用于筛选颗粒或黄体细胞的cDNA表达文库。这项工作导致在鉴定的cDNA编码的蛋白质的60.1 kDa的13个残基的信号肽。牛60.1-kDa蛋白分别与人80 K-H蛋白和小鼠葡萄糖苷酶II推定β亚基共有86.7%和81.8%的同一性（beta-GII），并命名为空泡系统相关蛋白-60（VASAP-60），序列同一性的显著差异在含有串联D和E氨基酸段的推定分子衔接子结构域中被注意到，富含脯氨酸的序列呈现最小的PXXP SH 3基序。VASAP-60被证明是非糖基化使用糖苷内切酶H处理，并发现主要在牛黄体的细胞膜部分。VASAP-60被定位在大鼠肝高尔基体/内体部分和麦胚凝集素（WGA）亲和色谱洗脱液，从而表明存在与膜糖蛋白的相互作用。针对重组VASAP-60的推定接头结构域产生多克隆抗体;这显示出识别主要的88-kDa和两个次要的58-kDa和50-kDa蛋白，表明主要的88-kDa蛋白条带代表完整的VASAP-60蛋白，而58-kDa和50-kDa条带代表其蛋白水解片段。北方印迹分析表明，在所有分析的牛组织中存在单一的2.3-腺苷酸酶转录物，组织之间的稳态水平存在差异。免疫组化结果显示VASAP-60在牛组织中广泛分布，主要定位于细胞质周膜和核周膜。在上皮细胞中，染色呈现与细胞内空泡相关的基底侧或顶端极性。总之，我们的特点是一种新的酸性膜蛋白，与细胞器的液泡系统，这是广泛和组织特异性表达在牛组织。VASAP-60代表牛直系同源物或先前表征的人80 K-H和鼠β-GII蛋白的新家族成员。我们的研究结果表明，VASAP-60提出了一个分子衔接蛋白的特点，在膜运输事件的功能。
It has been suggested that proteins of molecular size 56-58 kDa play an important role in bovine ovarian follicular development and oocyte maturation. A polyclonal antibody was raised against a 56- to 58-kDa protein band purified from bovine granulosa cells and was used to screen granulosa or luteal cell cDNA expression libraries. This work resulted in the identification of a cDNA encoding for a protein of 60.1 kDa with a signal peptide of 13 residues. The bovine 60.1-kDa protein shared an overall 86.7% and 81.8% identity with, respectively, the human 80K-H protein and the mouse putative beta subunit of glucosidase II (beta-GII), and was named vacuolar system-associated protein-60 (VASAP-60), Marked differences in sequence identity were noted in a putative molecular adapter domain containing a tandem D and E amino acid stretch flanked by proline-rich sequences presenting the minimal PXXP SH3 motif. VASAP-60 was shown to be unglycosylated using endoglycosidase H treatment and was found mainly in a cellular membrane fraction of bovine corpus luteum. VASAP-60 was localized in a rat hepatic Golgi/endosome fraction and in wheat germ agglutinin (WGA) affinity chromatographic eluates, thereby suggesting the presence of interactions with membrane glycoproteins. A polyclonal antibody was raised against the putative adapter domain of the recombinant VASAP-60; this was shown to recognize a major 88-kDa and two minor 58-kDa and 50-kDa proteins, suggesting that the major 88-kDa protein band represents the complete VASAP-60 protein whereas the 58-kDa and the 50-kDa bands represent its proteolytic fragments. Northern blot analysis demonstrated the presence of a single 2.3-kilobase transcript in all the bovine tissues analyzed with variation in the steady state level between tissues. Immunohistochemical observations showed that VASAP-60 was widely distributed in bovine tissues and was localized in pericytoplasmic and perinuclear membranes. In epithelial cells, the staining presented a basolateral or apical polarity associated with intracellular vacuoles. In conclusion, we have characterized a novel acidic membrane protein, associated with organelles of the vacuolar system, that is widely and histospecifically expressed in bovine tissues. VASAP-60 represents either the bovine ortholog or a new family member of the previously characterized human 80K-H and murine beta-GII proteins. Our results suggest that VASAP-60 presents characteristics of a molecular adaptor protein with functions in membrane-trafficking events.