Abstract
The heparan sulfates (HS) are hypervariable linear polysaccharides that act as membrane co-receptors for growth factors, chemokines, and extracellular matrix proteins. In most instances, the molecular basis of protein recognition by HS is poorly understood. We have sequenced 75% of the sulfated domains (S-domains) of fibroblast HS, including all of the major ones. This analysis revealed tight coupling of N- and 2-O-sulfation and a low frequency but precise positioning of 6-O-sulfates, which are required functional groups for HS-mediated activation of the fibroblast growth factors. S-domain sequencing was conducted using a novel and highly sensitive method based on a new way of reading the sequence from high performance liquid chromatography separation profiles of metabolically labeled HS-saccharides following specific chemical and enzymatic scission. The implications of the patterns seen in the sulfated domains for better understanding of the synthesis and function of HS are discussed.
Highlights
Many biological macromolecules have information encoded in their primary structure
S-domains generated by heparinase III digestion were separated by Bio-Gel P10 gel filtration and strong anion-exchange (SAX)-HPLC to yield a range of oligosaccharide species differing in length, sugar sequence, and sulfation pattern
The last piece of information to be extracted from the profiles is the identity of the residues in the reducing terminal disaccharide of dp6a. ⌬UA-GlcNAc was seen in the disaccharide analysis of the hexasaccharide, and iduronidase generates a peak in the position of free GlcNAc from the nonsulfated disaccharides
Summary
(Received for publication, October 28, 1998, and in revised form, January 28, 1999). Catherine L. High affinity binding sites have been identified for bFGF [25, 26] These sites are of relatively simple structure, being mainly composed of 4 –5 repeat units of IdoUA(2S)-GlcNS, but intriguingly, these sequences fail to elicit a biological response to bFGF unless substituted by one or more 6-O-sulfate groups [27, 28]. All the major S-domains falling within the size range of hexa- to octasaccharides, together with a proportion of the decasaccharides have been sequenced without selection by ligand binding This approach has yielded new and intriguing information on the molecular structure of HS
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