Strategy for the sequence analysis of heparin

Area of Science:

• Biochemistry
• Glycobiology
• Analytical Chemistry

Background:

• Proteoglycans' biological activities are primarily mediated by glycosaminoglycan (GAG) components.
• Existing methods for sequencing GAGs, unlike those for proteins and nucleic acids, are unsatisfactory.
• Heparin, a complex GAG, requires advanced sequencing techniques for structural elucidation.

Purpose of the Study:

• To develop and validate a novel strategy for sequencing the glycosaminoglycan (GAG) chains of heparin.
• To characterize the linkage region and internal sequences of heparin GAGs.
• To enable detailed structural analysis of heparin for a better understanding of its biological functions.

Main Methods:

• Labeling of porcine mucosal heparin's core protein remnants with N-4-(6-dimethylamino-2-benzofuranyl) phenyl (NDBP)-isothiocyanate.
• Enrichment of NDBP-heparin using phenyl-Sepharose chromatography.
• Enzymatic digestion with heparin lyases (I, III) and chemical treatment (lithium hydroxide) followed by labeling with 7-amino-1,3-naphthalene disulphonic acid (AGA).
• Structural characterization using 1H-NMR and gradient polyacrylamide gel electrophoresis (PAGE).

Main Results:

• Characterization of the NDBP-linked tetrasaccharide at the linkage region: deltaUAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp-(1-->O-Ser-NDBP.
• Isolation and structural determination of NDBP-octasaccharides using heparin lyase I, including 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.
• Deduction of a predominant heparin sequence near the protein core attachment site using AGA-Xyl-heparin sequenced by gradient PAGE: -D-GlcNp2S6S(or 6OH)(1-->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S(or60H)...-beta-Xyl-AGA.

Conclusions:

• The developed strategy provides a robust method for sequencing heparin GAG chains.
• The study elucidates specific structural features of heparin, including the linkage region and internal sequences.
• This advancement facilitates a deeper understanding of heparin's structure-function relationships in its diverse biological activities.