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Glycobiology vol 5 no 8 pp. 765-774, 1995
© 1995

research-article

Strategy for the sequence analysis of heparin

Jian Liu, Umesh R. Desai, Xue-Jun Han, Toshihiko Toida and Robert J. Linhardt1

Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa Iowa City, IA 52242, USA

1To whom correspondence should be addressed

Received on June 5, 1995; revised on July 14, 1995; accepted on July 16, 1995

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)-ß-D-Galp(1->3)-ß-D-Galp(1->4)-ß-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)-ß-D-GlcAp(1->3)-ß-D-Galp(1->3)-ß-D-Galp(1->4)-ß-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(or 6OH)(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)-ß-D-GlcAp(1->3)-ß-D-Galp(1->3)-ß-D-Galp(1->4)-ß-Xyl-AGA.

heparin fluorescent labelling PAGE sequence analysis


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