Beers M F, Kim C Y, Dodia C, Fisher A B
Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
J Biol Chem. 1994 Aug 12;269(32):20318-28.
Surfactant protein C (SP-C), a 3.7-kDa hydrophobic lung-specific protein, is synthesized and secreted by pulmonary type II cells through proteolytic processing of a 21-kDa propeptide (SP-C21) by currently undefined pathways. Previously, we reported the production of a polyclonal antibody against rat SP-C21 (anti-CPROSP-C) using a synthetic peptide as the immunizing antigen (Beers, M. F., Wali, A., Eckenhoff, M. E. F., Feinstein, S., Fisher, J. H., and Fisher, A. B. (1992) Am. J. Respir. Cell. Mol. Biol. 7, 368-378). In this study, two additional epitope-specific proSP-C antibodies produced using synthetic peptide sequences were utilized to examine synthetic processing of SP-C. Anti-NPROSP-C (Met10-Gln23) and anti-CTERMSP-C (Ser149-Ser166) recognized native proSP-C21 produced from in vitro translation of SP-C cDNA. Immunocytochemistry using anti-NPROSP-C confirmed the localization of proSP-C peptides exclusively in type II cells. Western analysis of subcellular fractions identified a single 21-kDa band in microsomes and a 16-kDa form in lamellar bodies each recognized by all three antisera, while anti-NPROSP-C also uniquely identified a prominent 5-6-kDa form in lamellar bodies. In a perfused rat lung model labeled with [35S]cysteine/methionine, immunoprecipitation of lung homogenate and lamellar body fractions identified early appearances of 35S-labeled 21-, 18-, and 16-kDa SP-C forms in homogenate and a 16-kDa intermediate form in lamellar bodies. Anti-NPROSP-C also exclusively detected time-dependent appearances of 5-10-kDa proSP-C forms in lamellar bodies and homogenates. Processing of proSP-C21 was completely blocked by inclusion of brefeldin A (15 micrograms/ml) in the perfusate. These results demonstrate that synthetic peptides can be used to produce epitope-specific antisera which recognize more hydrophilic domains of proSP-C and show that proSP-C processing occurs intracellularly in subcellular compartments of type II cells which are distal to the trans-Golgi network.
表面活性蛋白C（SP-C）是一种3.7 kDa的疏水性肺特异性蛋白，由肺II型细胞通过目前尚不清楚的途径对21 kDa前体肽（SP-C21）进行蛋白水解加工后合成并分泌。此前，我们报道了使用合成肽作为免疫抗原制备的针对大鼠SP-C21的多克隆抗体（抗CPROSP-C）（Beers，M.F.，Wali，A.，Eckenhoff，M.E.F.，Feinstein，S.，Fisher，J.H.，以及Fisher，A.B.（1992年）《美国呼吸细胞与分子生物学杂志》7卷，368 - 378页）。在本研究中，利用另外两种使用合成肽序列制备的表位特异性前SP-C抗体来研究SP-C的合成加工过程。抗NPROSP-C（Met10 - Gln23）和抗CTERMSP-C（Ser149 - Ser166）识别从SP-C cDNA体外翻译产生的天然前SP-C21。使用抗NPROSP-C进行免疫细胞化学证实前SP-C肽仅定位于II型细胞中。对亚细胞组分的蛋白质印迹分析在微粒体中鉴定出一条单一的21 kDa条带，在板层小体中鉴定出一条16 kDa条带，这两条带均被所有三种抗血清识别，而抗NPROSP-C还在板层小体中独特地鉴定出一条突出的5 - 6 kDa条带。在以[35S]半胱氨酸/甲硫氨酸标记的灌注大鼠肺模型中，对肺匀浆和板层小体组分进行免疫沉淀，在匀浆中鉴定出35S标记的21 kDa、18 kDa和16 kDa SP-C形式的早期出现，在板层小体中鉴定出一种16 kDa的中间形式。抗NPROSP-C还仅在板层小体和匀浆中检测到5 - 10 kDa前SP-C形式随时间的出现。在前灌流液中加入布雷菲德菌素A（15微克/毫升）可完全阻断前SP-C21的加工过程。这些结果表明合成肽可用于制备识别前SP-C更亲水结构域的表位特异性抗血清，并表明前SP-C的加工过程发生在II型细胞中位于反式高尔基体网络远端的亚细胞区室的细胞内。
