Abstract
The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments dp6-dp24 corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s20,w of HS dp6-dp24 showed a small rotor speed dependence, where similar s20,w values of 0.82-1.26 S (absorbance optics) and 1.05-1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6-dp24 gave radii of gyration RG values from 1.03 to 2.82 nm, cross-sectional radii of gyration RXS values from 0.31 to 0.65 nm, and maximum lengths L from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5,000 to 12,000 conformationally randomized HS structures gave best fit dp6-dp24 molecular structures that were longer and more bent than their equivalents in heparin. Alternative fits were obtained for HS dp18 and dp24, indicating their higher bending and flexibility. We conclude that HS displays bent conformations that are significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin.
Highlights
The polysaccharide heparan sulfate (HS) exhibits key physiological roles
A slight decrease in the s20,w values of the absorbance and interference data were observed with increase of rotor speed; this indicated that the s20,w values depend on the rotor speed (Fig. 4, A and B)
A commonly used model compound for HS, consists of lengthy S domains, made up largely of the repeating trisulfated disaccharide shown in Fig. 1B, separated by much smaller, unsulfated NA domains
Summary
The polysaccharide heparan sulfate (HS) exhibits key physiological roles. Results: Analytical ultracentrifugation and x-ray scattering revealed extended but bent HS solution structures. Small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments dp6 – dp corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Given the importance of understanding the HS solution structure, we have used a multidisciplinary approach to determine molecular structures for HS based on the combination of three methods, namely analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling [21, 22] This approach is well established for solution structure determinations of large multidomain complement and antibody proteins and was recently applied to small heparin oligosaccharide fragments [19, 23]. This present study supersedes the 2011 study, which has been withdrawn
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