Structural Studies on the Carbohydrate-Protein Linkage Region of Proteoglycans

鲸鱼软骨蛋白多糖主要含有4-硫酸软骨素，其糖蛋白连接区经放线酶E和软骨素酶ABC的彻底消化和b-去掉后，分离到一个非硫酸、两个单硫化和一个二硫化的六糖糖醛醇。通过软骨素酶ACII消化结合高效液相色谱和500 MHz~1H-核磁共振波谱对其结构进行了分析。非硫酸盐化合物(A)具有常规结构：DELTAGlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl-ol，，而其他化合物是化合物A的硫酸盐衍生物。两个单硫化化合物(B和C)分别在GalNAc残基的C-4或C-6上有一个酯硫酸酯，而二硫化化合物(D)有两个酯硫酸酯基团，一个在GalNAc残基的C-4上，另一个在Gal残基的C-4上。A：B：C：D的摩尔比为0.21：0.16：0.36：0.27。含半乳糖-…的化合物我们以前从大鼠软骨肉瘤软骨素4-硫酸酯蛋白多糖的糖-蛋白质连接区分离到更多的4-微米-硫酸盐结构，以硫酸糖丝氨酸[DELTAGlcAbeta1-3GAlNAc(4-omicron-sulfate)beta1-4GlcAbeta1-3Gal(4-omicron-sulfate)beta1-3Galbeta1-4Xylbeta1-omicron-Ser]的形式存在。在非肿瘤性软骨中4-硫酸软骨素蛋白多糖的碳水化合物-蛋白质连接区中发现了这种结构，这表明它不是肿瘤相关产物，而是一种生理生物合成产物，因为它代表着相当大的比例。猪14阶段肠肝素的制备方法是将丝氨酸作为主要氨基酸，用于分离肝素的碳水化合物-蛋白质连接区。经细菌肝素酶和肝素酶混合消化后，得到两种糖丝氨酸，摩尔比为96：4。其结构经组成分析、肝素酶消化、与正品糖丝氨酸共层析以及500 MHz 1D和2D 1H核磁共振波谱确证。大调的结构是DELTAGlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-omicron-Ser，小调的结构是DELTAGlcAbeta1-4GlcNAc(6-omicron-sulfate)alpha1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-omicron-Ser.新的6-奥微米-硫酸化GlcNAc残基被证明存在于碳水化合物-蛋白质连接区附近。与最近在硫酸软骨素蛋白多糖的碳水化合物-蛋白质连接区发现的半乳糖结构不同，半乳糖残基是非硫化的。较少

From the carbohydrate-protein linkage region of whale cartilage proteoglycans, which bear predominantly chondroitin 4-sulfate, one nonsulfated, two monosulfated and one disulfated hexasaccharide alditols were isolated after exhaustive digestions with Actinase E and chondroitinase ABC and subsequent b-elimnination. Their structures were analyzed by chondroitinase ACII digestion in conjunction with HPLC and by 500 MHz ^1H-NMR spectroscopy. The nonsulfated compound (A) had the conventional structure : DELTAGlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl-ol, while the other compounds were sulfated derivatives of the compound A. Two monosulfated compounds (B and C) had an ester sulfate on C-4 or C-6 of the GalNAc residue, respectively, and the disulfated compound (D) had two ester sulfate groups, namely, one on C-4 of the GalNAc and the other on C-4 of the Gal residue substituted by GlcA. The molar ratio of A : B : C : D was 0.21 : 0.16 : 0.36 : 0.27. The compound containing Gal- … More 4-omicron-sulfate structure was previously isolated by us in the form of sulfated glycoserine [DELTAGlcAbeta1-3GAlNAc(4-omicron-sulfate)beta1-4GlcAbeta1-3Gal(4-omicron-sulfate)beta1-3Galbeta1-4Xylbeta1-omicron-Ser] from the carbohydrate-protein linkage region of rat chondrosarcoma chondroitin 4-sulfate proteoglycans. The discovery of this structure in the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans from nontumorous cartilage indicates that it is not tumor-associated product but rather a physiological biosynthetic product since it represents a significant proportion.A preparation of porcine stage 14 intestinal heparin, which contains Ser as a predominant amino acid, was used for isolation of the carbohydrate-protein linkage region of heparin. Two glycoserines were isolated in a molar ratio of 96 : 4 after an exhaustive digestion with a mixture of bacterial heparinase and heparitinases. Their structures were determined by composition analysis, heparitinase digestion, co-chromatography with an authentic glycoserine on HPLC and by 500-MHz 1D and 2D ^1H-NMR spectroscopy. The structure of the major one is DELTAGlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-omicron-Ser and that of the minor is DELTAGlcAbeta1-4GlcNAc(6-omicron-sulfate)alpha1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-omicron-Ser. The novel 6-omicron-sulfated GlcNAc residue was demonstrated to occur in the vicinity of the carbohydrate-protein linkage region. The Gal residues were nonsulfated, being in contrast to the sulfated Gal structures recently discovered in the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans. Less