Rubio S, Lacaze-Masmonteil T, Chailley-Heu B, Kahn A, Bourbon J R, Ducroc R
INSERM U.319, Université Paris 7, France.
J Biol Chem. 1995 May 19;270(20):12162-9. doi: 10.1074/jbc.270.20.12162.
Surfactant protein A (SP-A) is the most abundant protein associated with phospholipids in pulmonary surfactant. There are several lines of evidence that pulmonary and gastrointestinal epithelium produce closely related surface-active materials, although the presence of SP-A in gastrointestinal tract has so far not been reported. Indirect immunofluorescence experiments using different antibodies raised against rat pulmonary SP-A showed that some jejunal and colonic but not gastric epithelial cells positively stained for SP-A. Analysis of the proteins in cell lysates from rat small intestine and colon studied by Western blot revealed several immuno-reactive bands, including the characteristic triplet of 26-, 32-, and 38-kDa monomeric proteins, less strongly labeled than in lung cells, and higher molecular mass forms of 66 and 120 kDa also present in lung cells. The 66- and 120-kDa bands displayed the expected isoelectric pH of SP-A after two-dimensional electrophoresis. Alkylation induced conversion of the 120-kDa form (almost completely) and the 66-kDa form (partly) into the 26-38-kDa monomeric species. The presence of SP-A mRNA in rat stomach, small intestine, and colon was then searched for by conventional cDNA/reverse transcriptase-polymerase chain reaction. Products of appropriate size (372 base pairs) identical to that of pulmonary tissue were amplified in small intestine and colon but not in stomach or in other tissues used as controls. Cloning and sequencing of rat colon SP-A cDNA revealed the same sequence as the one reported for rat lung SP-A. Furthermore, analysis of the transcriptional initiation site of SP-A gene in colon by anchored-polymerase chain reaction showed that transcription was initiated at the same site in both colon and lung. These data, which demonstrate that small intestine and colon express SP-A constitutively and that this protein is present in some epithelial cells, extend the concept of intestinal surfactant and underline its close relationships to pulmonary surfactant.
表面活性蛋白A（SP-A）是肺表面活性剂中与磷脂相关的最丰富的蛋白质。有几条证据表明，肺和胃肠道上皮会产生密切相关的表面活性物质，尽管迄今为止尚未报道胃肠道中存在SP-A。使用针对大鼠肺SP-A产生的不同抗体进行的间接免疫荧光实验表明，一些空肠和结肠上皮细胞而非胃上皮细胞对SP-A呈阳性染色。通过蛋白质印迹法对大鼠小肠和结肠细胞裂解物中的蛋白质进行分析，发现了几条免疫反应带，包括特征性的26 kDa、32 kDa和38 kDa单体蛋白三联体，其标记强度低于肺细胞，还有肺细胞中也存在的66 kDa和120 kDa的高分子量形式。在二维电泳后，66 kDa和120 kDa的条带显示出预期的SP-A等电点pH值。烷基化诱导120 kDa形式（几乎完全）和66 kDa形式（部分）转化为26 - 38 kDa的单体形式。然后通过常规的cDNA/逆转录酶-聚合酶链反应在大鼠胃、小肠和结肠中寻找SP-A mRNA。在小肠和结肠中扩增出了与肺组织大小合适（372个碱基对）相同的产物，但在胃或用作对照的其他组织中未扩增出。大鼠结肠SP-A cDNA的克隆和测序显示其序列与报道的大鼠肺SP-A序列相同。此外，通过锚定聚合酶链反应分析结肠中SP-A基因的转录起始位点表明，结肠和肺中的转录起始于同一位点。这些数据表明小肠和结肠组成性表达SP-A且该蛋白存在于一些上皮细胞中，扩展了肠表面活性剂的概念，并强调了其与肺表面活性剂的密切关系。
