Cawley N X, Pu L P, Loh Y P
Section on Cellular Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Endocrinology. 1996 Nov;137(11):5135-43. doi: 10.1210/endo.137.11.8895388.
The novel aspartic proteases, yeast aspartic protease 3 and the mammalian POMC-converting enzyme (PCE), can process prohormones at specific basic residue cleavage sites. We show that an antibody against yeast aspartic protease 3 (YAP3p) cross-reacted with purified bovine PCE on Western blot, indicating structural homology between these two enzymes, but not with other aspartic proteases, such as renin or cathepsin D. A PCE-sized anti-YAP3p-immunoreactive band was detected on Western blots of bovine intermediate lobe where PCE activity has been found. YAP3p antiserum also cross-reacted with a protein of approximately 90 kDa from mouse hypothalamus and anterior pituitary, and bovine anterior pituitary secretory granules. Distribution studies showed the presence of anti-YAP3p-immunopositive cells in bovine pituitary and peptide-rich brain regions, including the mouse arcuate nucleus and hippocampus and the rat supraoptic nucleus, paraventricular nucleus, cortex, striatum, and reticular nucleus. In the bovine intermediate pituitary, a subpopulation of cells was intensely stained with the YAP3p antiserum, and in combination with in situ hybridization, these cells were shown to contain POMC messenger RNA (mRNA). Only a subpopulation of cells was immunopositive for anti-YAP3p in bovine anterior pituitary, and most of these cells were identified by double immunostaining with ACTH antiserum as corticotrophs. In situ hybridization in combination with immunocytochemistry provided evidence for the localization of arginine vasopressin mRNA in YAP3p-immunopositive neurons in the rat supraoptic nucleus, whereas cholecystokinin mRNA was detected in YAP3p-immunopositive cells in the rat cortex and hippocampus. These results support the hypothesis that YAP3p-like aspartic proteases, including PCE, play a role in prohormone processing in endocrine/neuroendocrine cells in vivo.
新型天冬氨酸蛋白酶，即酵母天冬氨酸蛋白酶3和哺乳动物的阿黑皮素原转化酶（PCE），能够在特定的碱性残基切割位点加工激素原。我们发现，一种抗酵母天冬氨酸蛋白酶3（YAP3p）的抗体在蛋白质免疫印迹法中能与纯化的牛PCE发生交叉反应，这表明这两种酶在结构上具有同源性，但与其他天冬氨酸蛋白酶，如肾素或组织蛋白酶D无交叉反应。在已发现有PCE活性的牛垂体中间叶的蛋白质免疫印迹中，检测到了一条PCE大小的抗YAP3p免疫反应带。YAP3p抗血清还与来自小鼠下丘脑、垂体前叶以及牛垂体前叶分泌颗粒中一种约90 kDa的蛋白质发生交叉反应。分布研究表明，在牛垂体以及富含肽的脑区，包括小鼠弓状核和海马以及大鼠视上核、室旁核、皮质、纹状体和网状核中，存在抗YAP3p免疫阳性细胞。在牛垂体中间叶，有一群细胞被YAP3p抗血清强烈染色，结合原位杂交显示，这些细胞含有阿黑皮素原信使核糖核酸（mRNA）。在牛垂体前叶，只有一小部分细胞对抗YAP3p呈免疫阳性，通过与促肾上腺皮质激素抗血清进行双重免疫染色，其中大多数细胞被鉴定为促肾上腺皮质激素细胞。原位杂交与免疫细胞化学相结合的方法证明，大鼠视上核中抗YAP3p免疫阳性神经元中有精氨酸加压素mRNA定位，而在大鼠皮质和海马的抗YAP3p免疫阳性细胞中检测到胆囊收缩素mRNA。这些结果支持这样一种假说，即包括PCE在内的YAP3p样天冬氨酸蛋白酶在体内内分泌/神经内分泌细胞的激素原加工过程中发挥作用。
