Strategy for the sequence analysis of heparin

被引：35

作者：

Liu, J ^{[1
]}

Desai, UR ^{[1
]}

Han, XJ ^{[1
]}

Toida, T ^{[1
]}

Linhardt, RJ ^{[1
]}

机构：

[1] UNIV IOWA,COLL PHARM,DIV MED & NAT PROD CHEM,IOWA CITY,IA 52242

来源：

GLYCOBIOLOGY | 1995年 / 5卷 / 08期

关键词：

heparin; fluorescent labelling; PAGE; sequence analysis;

D O I：

10.1093/glycob/5.8.765

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 Delta UAp(1 --> 3)-beta-D-Galp(1 --> 3)-beta-D-Galp(1 --> 4)-beta-Xylp-(1 --> O)-Ser-NDBP (Delta UAp 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, Delta UAp2S(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 H-1-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(or 6OH)(1 --> 4)-alpha-L-IdoAp2S(1 --> 4)-alpha-D-GlcNp2S6S(or 6OH) (1 --> 4)-alpha-L-IdoAp2S(1 --> 4) 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.

引用

页码：765 / 774

页数：10

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