Glycosaminoglycan chains of keratan sulfate proteoglycans appear to be physiologically significant by pairing with tissue lectins. Here, we used NMR spectroscopy and molecular dynamics (MD) simulations to characterize interactions of corneal keratan sulfate (KS), its desulfated form, as well as di-, tetra- (N-acetyllactosamine and lacto-N-tetraose) and octasaccharides with adhesion/growth-regulatory galectins, in particular galectin-3 (Gal-3). The KS contact region involves the lectin canonical binding site, with estimated KD values in the low µM range and stoichiometry of ~ 8 to ~ 20 galectin molecules binding per polysaccharide chain. Compared to Gal-3, the affinity to Gal-7 is relatively low, signaling preferences among galectins. The importance of the sulfate groups was delineated by using desulfated analogs that exhibit relatively reduced affinity. Binding studies with two related di- and tetrasaccharides revealed a similar decrease that underscores affinity enhancement by repetitive arrangement of disaccharide units. MD-based binding energies of KS oligosaccharide-loaded galectins support experimental data on Gal-3 and -7, and extend the scope of KS binding to Gal-1 and -9N. Overall, our results provide strong incentive to further probe the relevance of molecular recognition of KS by galectins in terms of physiological processes in situ, e.g. maintaining integrity of mucosal barriers, intermolecular (lattice-like) gluing within the extracellular meshwork or synaptogenesis.
Bibliographical noteFunding Information:
KHM is grateful to the Ludwigs-Maximillians-Universitaet (LMU) Center for Advanced Study, as well as to the Alexander von Humboldt Stiftung, for financial support during his sabbatical stay at LMU in Munich, Germany. KHM also holds a van der Laar Visiting Professorship in Structural Biology at Maastricht University, The Netherlands. KHM acknowledges that NMR instrumentation was provided with funds from the National Science Foundation (BIR-961477), the University of Minnesota Medical School, and the Minnesota Medical Foundation. NLBP is grateful for partial support of this work from the U.S. National Institutes of Health (1R01 M090280), and their Common Fund Glycosciences Program (1U01 GM116248). HJG acknowledges support by the Deutsche Forschungsgemeinschaft (SFB 1123/A2) and COST Action CA18103 (InnoGly). The authors gratefully acknowledge the valuable input of the reviewers.
© 2020, The Author(s).