Beers M F, Wali A, Eckenhoff M F, Feinstein S I, Fisher J H, Fisher A B
Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6068.
Am J Respir Cell Mol Biol. 1992 Oct;7(4):368-78. doi: 10.1165/ajrcmb/7.4.368.
Surfactant protein C (SP-C) is a lung-specific, hydrophobic peptide found in organic extracts of pulmonary surfactant. Alveolar SP-C (3.5 kD) is produced from proteolytic cleavage of a larger precursor molecule (pro-SP-C; 21 kD). While SP-C is synthesized by type II cells, the pathways for processing and secretion have remained elusive due, in part, to the lack of monospecific antibodies against SP-C or its precursors. This report describes production and characterization of a new antibody directed against pro-SP-C epitopes. Polyclonal antisera (anti-CPRO-SP-C) was prepared using a synthetic peptide corresponding to a portion of rat SP-C cDNA sequence (Ile26-Ser72). This contained amino acids 3-35 of mature SP-C plus additional C-terminal residues (His59-Ser72). On Western blots, anti-CPRO-SP-C competitively reacted to CPRO-SP-C but not to mature SP-C. Immunoblots of in vitro synthesized pro-SP-C confirmed that the antisera also recognized native protein. Immunocytochemistry with anti-CPRO-SP-C demonstrated staining for pro-SP-C peptides in isolated type II cells as well as in alveolar epithelial cells of rat lung sections. Pro-SP-C preferentially co-localized to cells that stained positive for Maclura pomifera antigen. Anti-CPRO-SP-C staining was not observed in lung interstitium, pulmonary vasculature, or several control tissues (brain, heart, and liver were negative). Western blotting of subcellular fractions demonstrated pro-SP-C peptides in plasma membrane (20 kD) and microsomal (20 and 21 kD) fractions with a 16 kD peptide present in lamellar bodies. No pro-SP-C peptides were detected in purified surfactant. These results demonstrate the use of a synthetic peptide to generate specific antiserum against more hydrophilic domains of pro-SP-C sequences and confirm that SP-C propeptides are unique to the lung.
表面活性蛋白C（SP-C）是一种在肺表面活性剂有机提取物中发现的肺特异性疏水肽。肺泡SP-C（3.5 kD）由较大前体分子（前SP-C；21 kD）的蛋白水解切割产生。虽然SP-C由II型细胞合成，但由于部分缺乏针对SP-C或其前体的单特异性抗体，其加工和分泌途径仍不清楚。本报告描述了一种针对前SP-C表位的新抗体的产生和特性。使用对应于大鼠SP-C cDNA序列一部分（Ile26-Ser72）的合成肽制备多克隆抗血清（抗CPRO-SP-C）。这包含成熟SP-C的第3至35位氨基酸以及额外的C末端残基（His59-Ser72）。在蛋白质印迹上，抗CPRO-SP-C与CPRO-SP-C发生竞争性反应，但不与成熟SP-C反应。体外合成的前SP-C的免疫印迹证实该抗血清也识别天然蛋白。用抗CPRO-SP-C进行免疫细胞化学显示，在分离的II型细胞以及大鼠肺切片的肺泡上皮细胞中，前SP-C肽呈阳性染色。前SP-C优先共定位于对桑橙抗原呈阳性染色的细胞。在肺间质、肺血管或几个对照组织（脑、心脏和肝脏均为阴性）中未观察到抗CPRO-SP-C染色。亚细胞组分的蛋白质印迹显示，质膜（20 kD）和微粒体（20和21 kD）组分中有前SP-C肽，板层小体中有16 kD肽。在纯化的表面活性剂中未检测到前SP-C肽。这些结果证明了使用合成肽产生针对前SP-C序列更亲水结构域的特异性抗血清，并证实SP-C前肽是肺所特有的。
