Highly Sensitive Sequencing of the Sulfated Domains of Heparan Sulfate

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.Many biological macromolecules have information encoded in their primary structure. In proteins, the amino acid sequence determines the secondary and tertiary structural characteristics of the folded protein, whereas in DNA and RNA the nucleotide sequence is the means of storing coded information that can be read by the transcriptional and translational machinery of the cell. Information for ligand binding and activation is also present within the structure of complex saccharides (1, 2). Heparan sulfate (HS) 1 is one of a class of polysaccharides known as glycosamino glycans, and recent research indicates that it expresses important protein recognition domains within its primary sequence (3, 4). However, unlike nucleic acids and protein, there is no established method available to read this primary information.The near ubiquitous occurrence and strategic positioning of heparan sulfate proteoglycans, both at the surface of most mammalian cells and in the extracellular matrix, is a good indicator of their role in cell-cell recognition and cell-matrix adhesion (5, 6). Heparan sulfate proteoglycans are key sites of interaction and signaling when cells form focal adhesions on extracellular matrix substrates such as fibronectin (7). A very extensive range of growth factors and cytokines, including key angiogenic factors such as basic fibroblast growth factor (bFGF) (8, 9), hepatocyte growth factor (10), and vascular endothelial growth factor (11), have been shown to not only bind HS in vitro but to require its presence as a membrane coreceptor for optimal activation of their cognate, high affinity signaling receptors. The similarity between the co-receptor role of HS and the effect of heparin in facilitating the inactivation of thrombin by antithrombin is indicative of many biological processes being driven by HS catalysis. It may therefore be possible to exploit an understanding of HS-growth factor interactions to modulate the effects of these ligands (12) in a similar way to the current clinical use of heparin. Although there is a sub...

The Interactions of Hepatocyte Growth Factor/Scatter Factor and Its NK1 and NK2 Variants with Glycosaminoglycans Using a Modified Gel Mobility Shift Assay

“…1A). Although molecular heterogeneity in HS oligosaccharides (and probably DS) increases with increasing fragment length (40), there is a wider charge variation and a lower overall charge/ mass ratio within the smaller dp2-4 populations, which could explain their distinctive behavior. The minimal level of detection of AMAC-labeled oligosaccharides was ϳ10 pmol (Fig.…”

Biosynthetic Oligosaccharide Libraries for Identification of Protein-binding Heparan Sulfate Motifs

“…In HS biosynthesis 2-O-sulfation generally precedes 6-Osulfation, and the distribution of the latter substituents appears to be quite strictly regulated (12,42). Thus, whereas excessively 6-O-sulfated sequences such as that displayed by fragment a are rarely seen in nonmanipulated systems, 2-Oand 6-O-sulfated structures of g and h type may be detected in authentic HS (8,42).…”

Use of Sulfated Linked Cyclitols as Heparan Sulfate Mimetics to Probe the Heparin/Heparan Sulfate Binding Specificity of Proteins

“…The usual approach has been to isolate HS fragments that bind to particular proteins and then determine the structure of the reactive fragments (32,33). This is a difficult procedure as there are often multiple binding motifs in HS of low abundance and thus obtaining detailed structural information, particularly of lengthy HS fragments, is a complex process.…”