人类精浆中明胶结合蛋白的分子多样性

Molecular heterogeneity of gelatin-binding proteins from human seminal plasma

研究人类精浆中各种蛋白质的分子特征是了解这些蛋白质的生理和病理功能的必要步骤。据预测，人类精浆中的明胶结合蛋白（由纤维粘连蛋白[FN]和其相关分子组成）对男性不育十分重要，所以本文研究了该蛋白的免疫糖生化特征。将精液参数正常的研究对象的精浆在明胶琼脂糖凝胶上分离，用SDS-PAGE和免疫印迹（不同FN的抗体）分析。用蛋白质芯片技术结合表面增强激光解吸电离飞行时间质谱（SELDI—TOF—MS）技术（亲水性、疏水性和金属亲和芯片）来鉴定分离到的分子的多样性。用甘露醇、海藻糖和硅铝酸特异的植物血凝素和半乳糖凝集素-1检测碳水化合物组成。结果表明分离到的蛋白质组成与已知的FN一致，免疫反应试验也验证了这一结果。这些蛋白质中具有肝磷脂结合力的蛋白质优先集中于小分子成分中。实验发现了一些特别片段的磷酸化和糖基化现象。植物血凝素与带有明胶结合位点的片段（尤其是Ricinus communis凝集素-1）的结合力强于带有FN细胞结合位点的片段。实验还发现了少量能与唾液酸糖苷化和特有伴刀豆球蛋白A以及Lensculinaris凝集素结合的蛋白质成分。半乳糖凝集素-1与分离到的物质无任何反应。深入了解正常人类精浆中FN的分子多样性和它们与已知FN分子可能的相似及不同之处，是将这些特征应用于临床之前首当其冲的一步。

Defining the molecular characteristics of seminal plasma proteins is essential for understanding their function in physiological and pathological conditions. Starting from the predicted importance of human seminal plasma gelatin-binding proteins, comprising fibronectin （FN） and FN-related molecules, for male fertility, this study aims at gaining insight into their immuno-glycobiochemical properties. Human seminal plasma from subjects with normal semen parameters were separated on a gelatin-Sepharose column and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting using antibodies against distinct FN forms. Heterogeneity of the isolated molecular species was examined by protein chip arrays combined with surface-enhanced laser desorption/ionization time of flight mass spectrometry, on normal, metal and hydrophobic surfaces. Carbohydrate composition was investigated using mannose-, fucose- and sialic acid-specific plant lectins and galectin-1. The results obtained indicated a pattern of isolated proteins corresponding to that of known FN fragments, as confirmed by immunoreactivity. Among them heparin-binding ability was preferentially associated with low molecular mass species. As for posttranslational modifications, phosphorylation and glycosylation of distinct fragments were revealed. Lectin binding to fragments containing the gelatin-binding domain, particularly with Ricinus communis agglutinin I, was stronger than to fragments containing the cell-binding site of FN. A low level of sialylation and distinctive concanavalin A- and Lens culinaris agglutinin-reactive species were also observed. Galectin-1 did not interact with the isolated preparation. Resolving the molecular heterogeneity of normal human seminal plasma FN and gaining initial insight into possible similarities/differences with known FN molecular species may be considered a prerequisite step preceding challenging the clinical usefulness of these molecular properties.