Cottrell G A, Lutz E M, Price D A, Sommerville J
School of Biological and Medical Sciences, University of St. Andrews, Fife, Scotland.
Mol Cell Neurosci. 1994 Dec;5(6):632-41. doi: 10.1006/mcne.1994.1077.
Sequencing of cDNA clones reveals a precursor protein that can be processed into 10 different hepta-FaRPs. Two of the peptides are previously undescribed and are N-terminally extended forms of-YMRFamide, making them the only methionine-containing peptides in the precursor. They are separated from the main cluster of hepta-FaRPs by a recognition site (RQKR) for the Golgi-resident proteolytic enzyme furin. Antisera raised against the synthetic peptide KQDPFLRFGK specifically stain the clusters of neurons in the parietal ganglia that have been shown to contain hepta-FaRP mRNA. These antisera recognize two major protein bands of 35 and 23 kDa on immunoblots. Evidence is presented to identify the larger band as the precursor protein and the smaller band as the fragment containing the main cluster of hepta-FaRPs produced after furin cleavage. A series of immunostaining bands of 22-13 kDa suggests sequential and non-preferential N- or C-terminal cleavage at the mainly monobasic (K and R) sites that link all of the peptide sequences throughout the 23-kDa fragment, to yield the preamidated hepta-FaRPs. Immunostaining of sections shows punctate staining in the perikarya of the parietal cluster neurons commensurate with label within the endoplasmic reticulum and Golgi apparatus. Staining is followed through the axons to many fibers in the nerve trunks and is picked up as fine processes within the skin. These observations indicate that the antiserum used here recognizes one or more of the processed hepta-FaRPs, a view confirmed by radioimmunoassay. The abundance of immunoreactive fibers within the skin suggests a major role for the peptides in this tissue.
对cDNA克隆进行测序发现一种前体蛋白，它可被加工成10种不同的七肽FaRP。其中两种肽此前未被描述，是YMRFamide的N端延伸形式，使其成为前体中仅有的含甲硫氨酸的肽。它们通过高尔基体驻留蛋白水解酶弗林蛋白酶的识别位点（RQKR）与七肽FaRP的主要簇分开。针对合成肽KQDPFLRFGK产生的抗血清特异性地标记了壁神经节中已被证明含有七肽FaRP mRNA的神经元簇。这些抗血清在免疫印迹上识别出两条主要的蛋白带，分子量分别为35 kDa和23 kDa。有证据表明较大的条带是前体蛋白，较小的条带是弗林蛋白酶切割后产生的包含七肽FaRP主要簇的片段。一系列22 - 13 kDa的免疫染色条带表明，在连接23 kDa片段中所有肽序列的主要单碱性（K和R）位点上，N端或C端进行了连续且非优先的切割，从而产生酰胺化前的七肽FaRP。切片的免疫染色显示，壁神经节簇状神经元的胞体中有点状染色，这与内质网和高尔基体中的标记一致。染色沿着轴突延伸到神经干中的许多纤维，并在皮肤中以精细的突起形式呈现。这些观察结果表明，此处使用的抗血清识别一种或多种加工后的七肽FaRP，放射免疫测定证实了这一观点。皮肤中免疫反应性纤维的丰富表明这些肽在该组织中起主要作用。
