Liu J, Desai U R, Han X J, Toida T, Linhardt R J
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, USA.
Glycobiology. 1995 Dec;5(8):765-74. doi: 10.1093/glycob/5.8.765.
The versatile biological activities of proteoglycans are mainly mediated by their glycosaminoglycan (GAG) components. Unlike proteins and nucleic acids, no satisfactory method for sequencing GAGs has been developed. This paper describes a strategy to sequence the GAG chains of heparin. Heparin, prepared from animal tissue, and processed by proteinases and endoglucuronidases, is 90% GAG heparin and 10% peptidoglycan heparin (containing small remnants of core protein). Raw porcine mucosal heparin was labelled on the amino termini of these core protein remnants with a hydrophobic, fluorescent tag [N-4-(6-dimethylamino-2-benzofuranyl) phenyl (NDBP)-isothiocyanate]. Enrichment of the NDBP-heparin using phenyl-Sepharose chromatography, followed by treatment with a mixture of heparin lyase I and III, resulted in a single NDBP-linkage region tetrasaccharide, which was characterized as deltaUAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp -(1-->O-Ser-NDBP (deltaUAp is 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid). Several NDBP-octasaccharides were isolated when NDBP-heparin was treated with only heparin lyase I. The structure of one of these NDBP-octasaccharides, deltaUAp2S(1-->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp(1-->3)-beta-D- Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp-(1-->O-Ser NDBP (S is sulphate, Ac is acetate), was determined by 1H-NMR and enzymatic methods. Enriched NDBP-heparin was treated with lithium hydroxide to release heparin, and the GAG chain was then labelled at xylose with 7-amino-1,3-naphthalene disulphonic acid (AGA). The resulting AGA-Xyl-heparin was sequenced on gradient PAGE using heparin lyase I and heparin lyase III. A predominant sequence in heparin at the protein core attachment site was deduced to be -D-GlcNp2S6S(or 6OH)(1-->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S (or60H) (1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNp2S6S( or 6OH)(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpAc (1- ->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-++ +GlcAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xyl-AGA.
蛋白聚糖的多种生物活性主要由其糖胺聚糖（GAG）成分介导。与蛋白质和核酸不同，目前尚未开发出令人满意的GAG测序方法。本文描述了一种对肝素的GAG链进行测序的策略。从动物组织中制备并经蛋白酶和内切葡糖醛酸酶处理后的肝素，90%是GAG肝素，10%是肽聚糖肝素（含有核心蛋白的小片段）。将粗制的猪黏膜肝素在这些核心蛋白片段的氨基末端用疏水性荧光标签[N-4-(6-二甲基氨基-2-苯并呋喃基)苯基（NDBP）-异硫氰酸酯]进行标记。使用苯基-琼脂糖色谱法对NDBP-肝素进行富集，随后用肝素酶I和III的混合物处理，得到了一种单一的NDBP连接区四糖，其结构被鉴定为δUAp(1→3)-β-D-Galp(1→3)-β-D-Galp(1→4)-β-Xylp-(1→O-Ser-NDBP（δUAp是4-脱氧-α-L-苏-己-4-烯吡喃糖醛酸）。当只用肝素酶I处理NDBP-肝素时，分离出了几种NDBP-八糖。其中一种NDBP-八糖的结构，δUAp2S(1→4)-α-D-GlcNpAc(1→4)-α-L-IdoAp(1→4)-α-D-GlcNpAc6S(1→4)-β-D-GlcAp(1→3)-β-D-Galp(1→3)-β-D-Galp(1→4)-β-Xylp-(1→O-Ser NDBP（S是硫酸根，Ac是乙酰基），通过1H-NMR和酶法确定。用氢氧化锂处理富集后的NDBP-肝素以释放肝素，然后用7-氨基-1,3-萘二磺酸（AGA）在木糖处对GAG链进行标记。所得的AGA-Xyl-肝素在梯度聚丙烯酰胺凝胶电泳上使用肝素酶I和肝素酶III进行测序。推导出肝素在蛋白核心附着位点的主要序列为-D-GlcNp2S6S(或6OH)(1→4)-α-L-IdoAp2S-(1→4)-α-D-GlcNp2S6S(或60H)(1→4)-α-L-IdoAp2S(1→4)-α-D-GlcNp2S6S(或6OH)(1→4)-α-L-IdoAp2S(1→4)-α-D-GlcNpAc(1→4)-α-L-IdoAp(1→4)-α-D-GlcNpAc6S(1→4)-β-D-++ +GlcAp(1→3)-β-D-Galp(1→3)-β-D-Galp(1→4)-β-Xyl-AGA。
