鹅颈藤壶多糖提取、理化性质及N-糖链表达谱分析研究

Gooseneck Barnacle Polysaccharide Extraction,Physicochemical Properties and N-Glycans Expression Spectrum Analysis

本文以南海海域鹅颈藤壶(Lepas anatifera)为原料,提取鹅颈藤壶背甲与肌肉质柄中的多糖,基于其基本理化性质的分析,比较不同生长时期鹅颈藤壶N-糖链表达谱的差异性。经过脱脂、碱水解/蛋白酶解、乙醇沉淀方法提取得到粗多糖;利用高效液相色谱法、高效凝胶渗透色谱法、苯酚-硫酸法、考马斯亮蓝法对其单糖组成、分子量分布、总糖含量及蛋白质含量进行分析;采用液质分析(PGC-ESI-MS/MS)方法对鹅颈藤壶成体(A-GB)与幼体(L-GB)肌肉质柄中N-糖链进行分析并比较。表明肌肉质柄中多糖分子量为18.40 kDa;总糖含量为11.99%,蛋白质含量60.14%,富含甘露糖、葡萄糖、氨基半乳糖,其中甘露糖的含量高达80%以上。在鹅颈藤壶成体与幼体中均鉴定得到26条N-糖链,成体中高甘露糖型N-糖链占总糖链的20.81%,且相对丰度较高的糖型组成为F_(1)H_(3)N_(2)(39.06%)、F_(1)H_(3)N_(3)(24.44%)、H_(3)N_(3)(9.35%);幼体中高甘露糖型N-糖链占总糖链的52.17%,相对丰度较高的糖型组成为F_(1)H_(3)N_(2)(21.65%)、H_(6)N_(2)(15.93%)、H_(5)N_(2)(14.75%)。鹅颈藤壶多糖是一类N-糖基化的蛋白复合物,推测N-糖链在鹅颈藤壶长发育过程中发挥着至关重要的作用,可能高甘露糖型N-糖链与其附着黏附能力密切相关。

To extract polysaccharide from Lepas anatifera,a gooseneck barnacle,in the South China Sea.Based on the analysis of its basic physical and chemical properties,the expression profile of N-glycans in Gooseneck barnacle at different growth stages was compared.Polysaccharide was extracted by degreasing,alkaline hydrolysis and ethanol precipitation.The monosaccharide composition,molecular distribution,total polysaccharide content and protein content were analyzed by high performance liquid chromatography,high performance gel permeation chromatography,pheno-lsulfuric acid method and Coomassie brilliant blue method.Liquid mass analysis(PGC-ESI-MS/MS)was used to analyze and compare the N-glycans in the musculature stalk of adult(A-GB)and larvae(L-GB)barnacles.The molecular weight of polysaccharide in muscle stalk was 18.40 kDa.The total polysaccharide content is 11.99%,the protein content is 60.14%,rich in mannose,glucose,galactose amino,among which the content of mannose is up to 80%.Twenty-six N-glycans were identified in both adult and larvae of barnacles.The high mannose-type N-glycans accounted for 20.81%of the total glycans in adults,and the glycotypes with high relative abundance were F_(1)H_(3)N_(2)(39.06%)、F_(1)H_(3)N_(3)(24.44%)、H_(3)N_(3)(9.35%).The high mannose-type N-glycans accounted for 52.17%of the total N-glycans in the larvae,and the glycotypes with high relative abundance were F_(1)H_(3)N_(2)(21.65%)、H_(6)N_(2)(15.93%)、H_(5)N_(2)(14.75%).Barnacle polysaccharide is a kind of N-glycosylated protein complex.It is speculated that N-glucans plays a crucial role in the growth and development of barnacle,and high mannose-type N-glycosyl chain may be closely related to its adhesion ability.