Glasser S W, Korfhagen T R, Weaver T, Pilot-Matias T, Fox J L, Whitsett J A
Proc Natl Acad Sci U S A. 1987 Jun;84(12):4007-11. doi: 10.1073/pnas.84.12.4007.
Hydrophobic surfactant-associated protein of Mr 6000-14,000 was isolated from ether/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant. Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Tyr-Cys-Trp-Leu-Cys-Arg-Ala-Leu-. Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe). Antiserum generated against hydrophobic surfactant protein(s) from bovine pulmonary surfactant recognized protein of Mr 6000-14,000 in immunoblot analysis and was used to screen a lambda gt11 expression library constructed from adult human lung poly(A)+ RNA. This resulted in identification of a 1.4-kilobase cDNA clone that was shown to encode the N-terminus of the surfactant polypeptide SPL(Phe) (Phe-Pro-Ile-Pro-Leu-Pro-) within an open reading frame for a larger protein. Expression of a fused beta-galactosidase-SPL(Phe) gene in Escherichia coli yielded an immunoreactive Mr 34,000 fusion peptide. Hybrid-arrested translation with this cDNA and immunoprecipitation of [35S]methionine-labeled in vitro translation products of human poly(A)+ RNA with a surfactant polyclonal antibody resulted in identification of a Mr 40,000 precursor protein. Blot hybridization analysis of electrophoretically fractionated RNA from human lung detected a 2.0-kilobase RNA that was more abundant in adult lung than in fetal lung. The larger RNA and translation product indicates that SPL(Phe) is derived by proteolysis of a large polypeptide precursor. The amino acid sequence of the predicted protein, beginning Phe-Pro-Ile-Pro-Leu-Pro-Try-, comprises a hydrophobic peptide that is a major protein component of surfactant lipid extracts used successfully to treat hyaline membrane disease in newborn infants. These proteins, and specifically SPL(Phe), may therefore be useful for synthesis of replacement surfactants for treatment of hyaline membrane disease in newborn infants or of other surfactant-deficient states.
从哺乳动物肺表面活性剂的乙醚/乙醇或氯仿/甲醇提取物中分离出分子量为6000 - 14,000的疏水表面活性剂相关蛋白。在气相测序仪中进行的自动埃德曼降解显示，人低分子量蛋白的主要N端为苯丙氨酸-脯氨酸-异亮氨酸-脯氨酸-亮氨酸-脯氨酸-酪氨酸-半胱氨酸-色氨酸-亮氨酸-半胱氨酸-精氨酸-丙氨酸-亮氨酸-。由于N端为苯丙氨酸，该表面活性剂蛋白被命名为SPL（苯丙氨酸）。针对牛肺表面活性剂中疏水表面活性剂蛋白产生的抗血清在免疫印迹分析中识别出分子量为6000 - 14,000的蛋白，并用于筛选由成人肺多聚腺苷酸加尾RNA构建的λgt11表达文库。这导致鉴定出一个1.4千碱基的cDNA克隆，该克隆在一个更大蛋白质的开放阅读框内编码表面活性剂多肽SPL（苯丙氨酸）（苯丙氨酸-脯氨酸-异亮氨酸-脯氨酸-亮氨酸-脯氨酸-）的N端。在大肠杆菌中表达融合的β-半乳糖苷酶-SPL（苯丙氨酸）基因产生了一种免疫反应性的分子量为34,000的融合肽。用该cDNA进行杂交捕获翻译，并用表面活性剂多克隆抗体对人多聚腺苷酸加尾RNA的[35S]甲硫氨酸标记的体外翻译产物进行免疫沉淀，从而鉴定出一种分子量为40,000的前体蛋白。对人肺电泳分离的RNA进行印迹杂交分析，检测到一种2.0千碱基的RNA，其在成人肺中比在胎儿肺中更丰富。较大的RNA和翻译产物表明SPL（苯丙氨酸）是由一种大的多肽前体经蛋白水解产生的。预测蛋白的氨基酸序列从苯丙氨酸-脯氨酸-异亮氨酸-脯氨酸-亮氨酸-脯氨酸-色氨酸-开始，包含一个疏水肽，它是成功用于治疗新生儿透明膜病的表面活性剂脂质提取物的主要蛋白质成分。因此，这些蛋白质，特别是SPL（苯丙氨酸），可能有助于合成用于治疗新生儿透明膜病或其他表面活性剂缺乏状态的替代表面活性剂。
