Identification of an O-glycosidic mannose-linked sialylated tetrasaccharide and keratan sulfate oligosaccharides in the chondroitin sulfate proteoglycan of brain.
Identification of an O-glycosidic mannose-linked sialylated tetrasaccharide and keratan sulfate oligosaccharides in the chondroitin sulfate proteoglycan of brain.
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发表时间：
1986-06
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通讯作者：
T. Krusius;J. Finne;R. Margolis;R. U. Margolis
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作者：
T. Krusius;J. Finne;R. Margolis;R. U. Margolis
用链霉蛋白酶消化大鼠脑硫酸软骨素蛋白聚糖，除去糖胺聚糖后，用碱性硼氢化物处理所得糖肽以释放O-糖苷连接的寡糖。这些被分馏的离子交换色谱法，凝胶过滤，和制备薄层色谱法，和它们的结构特性进行了研究，通过特定的酶降解，甲基化分析，和气-液色谱-质谱的二糖作为其三甲基硅烷化和全甲基化衍生物。除了先前表征的N-乙酰基-半乳糖胺连接的低聚糖和中性甘露醇含低聚糖[GlcNAc（β 1-3）Manol和Gal（beta 1-4）[Fuc（α 1-3）]GlcNAc（β 1-3）马诺]（其中Fuc是岩藻糖），我们现在已经鉴定了唾液酸化四糖NeuAc（α 2-3）Gal（β 1-4）GlcNAc（β 1-3）Manol，约占含甘露醇寡糖的20%。蛋白聚糖还含有甘露糖连接的硫酸角质素链（分子大小为3，000至10，000 Da），其由Gal（β 1-4）GlcNAc-6-O-SO 4（β 1-3）组成的二糖重复单元组成，在半乳糖残基的C-6处具有小比例的分支点。大约有一个硫酸角质素链每四个硫酸软骨素链的18，000 - 19，000 Da. After碱性硼氢化物处理的中性和单唾液酸糖肽馏分，甘露糖和N-乙酰半乳糖胺的组合减少是非常接近所观察到的破坏丝氨酸+苏氨酸，并伴随着等摩尔增加丙氨酸和α-氨基丁酸。一半的甘露糖被破坏的碱性硼氢化物处理的糖肽和化学计量转化为甘露醇，而其他糖和氨基酸的相对量只有很小的变化。数据表明，蛋白聚糖中超过一半的碳水化合物-肽键是甘露糖基-O-丝氨酸/苏氨酸类型。
The chondroitin sulfate proteoglycan of rat brain was digested with Pronase, and after removal of glycosaminoglycans, the resulting glycopeptides were treated with alkaline borohydride to release O-glycosidically linked oligosaccharides. These were fractionated by ion exchange chromatography, gel filtration, and preparative thin layer chromatography, and their structural properties were studied by specific enzymatic degradations, methylation analysis, and gas-liquid chromatography-mass spectrometry of disaccharides as their trimethylsilylated and permethylated derivatives. In addition to the previously characterized N-acetyl-galactosamine-linked oligosaccharides and neutral mannitol-containing oligosaccharides [GlcNAc(beta 1-3) Manol and Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Manol] (where Fuc is fucose), we have now identified the sialylated tetrasaccharide NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc (beta 1-3)Manol, which accounts for approximately 20% of the mannitol-containing oligosaccharides. The proteoglycan also contains mannose-linked keratan sulfate chains (with a molecular size of 3,000 to 10,000 Da) composed of disaccharide repeating units consisting of Gal(beta 1-4)GlcNAc-6-O-SO4(beta 1-3), with a small proportion of branch points at C-6 of galactose residues. There is approximately one keratan sulfate chain per four chondroitin sulfate chains of 18,000-19,000 Da. After alkaline borohydride treatment of the neutral and monosialyl glycopeptide fractions, the combined decrease in mannose and N-acetylgalactosamine was very close to the observed destruction of serine + threonine and was accompanied by an equimolar increase in alanine and alpha-aminobutyric acid. One half of the mannose was destroyed by alkaline borohydride treatment of the glycopeptides and stoichiometrically converted to mannitol, while there were only small changes in the relative amounts of the other sugars and amino acids. The data demonstrate that over half of the carbohydrate-peptide linkages in the proteoglycan are of the mannosyl-O-serine/threonine type.