Sayah M, Fortenberry Y, Cameron A, Lindberg I
Department of Biochemistry and Molecular Biology, LSU Health Science Center, New Orleans, LA 70112, USA.
J Neurochem. 2001 Mar;76(6):1833-41. doi: 10.1046/j.1471-4159.2001.00165.x.
The conversion of inactive precursor proteins into bioactive neuropeptides and peptide hormones involves regulated secretory proteins such as prohormone convertases PC1 and PC2. The neuroendocrine protein 7B2 represents a specific binding protein for PC2, and the protein proSAAS, which interacts with PC1, exhibits certain structural and functional homologies with 7B2. With the intention of better understanding the physiological role of proSAAS and its derived peptides, we investigated its tissue localization using a new radioimmunoassay (RIA) to a C-terminal proSAAS-derived peptide. Immunoreactivity corresponding to this SAAS-derived peptide is mostly localized to the brain and gut. Analysis of the brain distribution of the proSAAS-derived peptides indicates that the hypothalamus and pituitary are the two richest areas, consistent with the previously described high expression of PC1 in these two areas. In order to investigate the cleavage of proSAAS by prohormone convertases, we incubated recombinant His-tagged proSAAS with recombinant mouse proPC2 or furin, separated the cleavage products using high-pressure gel permeation chromatography and analyzed the products by RIA. Our results indicate that either PC2 or furin can accomplish in vitro rapid removal and efficient internal processing of the C-terminal peptide, exposing the inhibitory hexapeptide to possible further digestion by carboxypeptidases. Finally, we also studied proSAAS processing in the brains of wild-type and PC2 null mice and found that proSAAS is efficiently processed in vivo. Whereas the C-terminal peptide is mostly internally cleaved in wild-type mouse brain, it is not processed as efficiently in the brain of PC2 null mice, suggesting that PC2 is partially responsible for this cleavage in vivo.
无活性前体蛋白转化为生物活性神经肽和肽类激素涉及到一些受调控的分泌蛋白，如激素原转化酶PC1和PC2。神经内分泌蛋白7B2是PC2的一种特异性结合蛋白，与PC1相互作用的蛋白proSAAS与7B2在结构和功能上具有一定同源性。为了更好地理解proSAAS及其衍生肽的生理作用，我们使用一种针对C端proSAAS衍生肽的新型放射免疫分析法（RIA）研究了其组织定位。与这种SAAS衍生肽相对应的免疫反应性主要定位于脑和肠道。对proSAAS衍生肽的脑部分布分析表明，下丘脑和垂体是含量最丰富的两个区域，这与之前描述的这两个区域中PC1的高表达一致。为了研究激素原转化酶对proSAAS的切割作用，我们将重组His标签的proSAAS与重组小鼠proPC2或弗林蛋白酶一起孵育，使用高压凝胶渗透色谱法分离切割产物，并通过RIA分析产物。我们的结果表明，PC2或弗林蛋白酶都可以在体外快速去除并高效地对C端肽进行内部加工，使抑制性六肽暴露于可能被羧肽酶进一步消化的环境中。最后，我们还研究了野生型和PC2基因敲除小鼠脑中proSAAS的加工过程，发现proSAAS在体内能被有效加工。在野生型小鼠脑中，C端肽大多在内部被切割，而在PC2基因敲除小鼠脑中其加工效率不高，这表明PC2在体内对这种切割起部分作用。
