STRATEGY FOR THE SEQUENCE-ANALYSIS OF HEPARIN

The versatile biological activities of proteoglycans are mainly mediat ed by their glycosaminoglycan (GAG) components, Unlike proteins and nu cleic acids, no satisfactory method for sequencing GAGs has been devel oped, This paper describes a strategy to sequence the GAG chains of he parin. Heparin, prepared from animal tissue, and processed by proteina ses and endoglucuronidases, is 90% GAG heparin and 10% peptidoglycan h eparin (containing small remnants of core protein). Raw porcine mucosa l heparin was labelled on the amino termini of these core protein remn ants with a hydrophobic, fluorescent tag [N-4-(6-dimethylamino-2-benzo furanyl) phenyl (NDBP)-isothiocyanate]. Enrichment of the NDBP-heparin using phenyl-Sepharose chromatography, followed by treatment with a m ixture of heparin lyase I and III, resulted in a single NDBP-linkage r egion tetrasaccharide, which was characterized as Delta UAp(1 --> 3)-b eta-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). S everal NDBP-octasaccharides were isolated when NDBP-heparin was treate d with only heparin lyase I. The structure of one of these NDBP-octasa ccharides, 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- Gal p(1 --> 3)-beta-D-Galp(1 --> 4)-beta-Xylp-(1 --> O)-Ser NDBP (S is sul phate, 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 wa s sequenced on gradient PAGE using heparin lyase I and heparin lyase I II, A predominant sequence in heparin at the protein core attachment s ite 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)-al pha-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.