Bhattacharyya S N, Sahu S, Lynn W S
Biochim Biophys Acta. 1976 Mar 18;427(1):91-106. doi: 10.1016/0005-2795(76)90288-9.
A major glycoprotein 36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 mol of mannose, 4.0 mol of galactose, 6.0 mol of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NH2-terminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4% hydroxyproline, 14% proline, 28% glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of -Fly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of asparitic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.
从肺泡蛋白沉着症患者的肺灌洗液中分离出一种主要的糖蛋白（分子量36000），发现它含有5个羟脯氨酸残基、50个甘氨酸残基、3个甲硫氨酸残基、3摩尔唾液酸、4.4摩尔甘露糖、4.0摩尔半乳糖、6.0摩尔葡糖胺和1摩尔岩藻糖。正如预期的那样，用溴化氰（CNBr）处理该糖蛋白后，得到了4种表观分子量分别为18000、12000、5000和1000的肽段。CNBr肽段的化学组成表明，除了一个肽段外，其他所有肽段都含有羟脯氨酸和大量的甘氨酸；4个CNBr肽段中有2个含有碳水化合物。对天然糖蛋白及其CNBr肽段进行凝胶过滤、丙烯酰胺凝胶电泳和端基分析表明，这些肽段是均一的。对CNBr裂解产物的端基分析将18000分子量的肽段归属于天然糖蛋白分子的NH2末端部分，而1000分子量的肽段归属于COOH末端部分。用链霉蛋白酶消化36000分子量的糖蛋白，然后进行凝胶过滤和阳离子交换色谱，得到两个组分。一个组分呈酸性，含有所有的碳水化合物、高含量的天冬氨酸且不含羟脯氨酸。另一个组分呈碱性，含有8.4%的羟脯氨酸、14%的脯氨酸、28%的甘氨酸且不含碳水化合物，这表明肽链中存在类似胶原蛋白的序列。对碱性组分进行纸电泳显示有两个组分，其氨基酸组成与胶原蛋白的相同。对其中一个CNBr肽段（18000分子量）进行部分氨基末端序列分析表明，肽链中存在-Fly-Pro-HyP-Gly-序列，这证实了我们的推测，即天然糖蛋白分子中存在类似胶原蛋白的区域。对酸性组分进行有限酸水解，随后用Dowex柱对酸水解产物进行分级分离，得到一个与茚三酮试剂反应产生棕色的组分。对该组分进行纸色谱分析显示有一个大量的组分，它与茚三酮试剂反应也呈棕色。酸水解后，发现该组分由等量的天冬氨酸和葡糖胺组成，这表明寡糖的N-乙酰葡糖胺与肽段的天冬酰胺残基相连。不存在丝氨酸或苏氨酸连接。
