ROA study of chondroitin sulphate and its building blocks

 

V. Profant1*, Ch. Johannessen2, E.W. Blanch2, P. Bouř3, V. Baumruk1

 

1Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, 121 16, Prague, Czech Republic

2Faculty of Life Sciences, Manchester Interdisciplinary Biocentre,131 Princess Street, M1 7DN Manchester, United Kingdom

2Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo nám 2, 166 10, Prague, Czech Republic

profant@karlov.mff.cuni.cz

 

Glucoaminoglycans (GAGs) represent a class of linear carbohydrate polymers with essential roles in many biological processes, such as cell signalling and proliferation, angiogenesis and tumorigenesis etc. One of the most common GAGs is chondroitin sulphate, an important structural component of cartilage that provides much of its resistance to compression. Chondroitin sulphate is composed of repeating sulphated disaccharide units, formed by b-D-glucuronic acid (GlcA) and 2-acetyl-b-D-galactosamine (GalNAc), joined together by b(1 4) and b(1 3) glycosidic linkages [1]. Despite its biological importance little is still known about the secondary and tertiary structural properties of chondroitin sulphate and any effects related to the sulfation of its chain, as X-ray crystallography and NMR are difficult to apply to these samples.

In our experiment, we measured the Raman and Raman optical activity (ROA) spectra of chondroitin A sulphate and its building blocks; GlcA, 1-O-methyl-GlcA, GalNAc, GalNAc-4-O-sulphate and GalNAc-6-O-sulphate in a wide frequency range between 250 cm-1 and 1800 cm-1 and analyzed these with respect to the occurrence of specific spectral marker bands and the influence of sulphation. The technique of ROA [2,3], which is based on a different interaction of a specimen with right- and left-handed circularly polarized laser light, represented an ideal methodology for this type of observation due to its high sensitivity to the conformational stability and rigidity of pyranose rings of the saccharides, orientation of sugar hydroxyl groups and also secondary structure of the GAG’s backbone. There is also a strong link to previous experiments [4], which focused on the characterization of hyaluronan, another important GAG.

Focus was placed on the interconnection between experiments and simulations: We performed ab initio calculations of Raman and ROA transitions and their intensities in order to obtain a more accurate interpretation of recorded spectra and observed phenomena. The initial geometries of most of the probable conformers were acquired from molecular dynamic simulations and quantum mechanical computations were performed using the Gaussian09 program suite, where implementation of analytical gradient calculations of optical activity tensors has lead to a significant decrease of computational time.

1.     T.E. Hardingham, A.J. Fosang, FASEB Journal, 6 (1992) 861-870.

2.     P.W. Atkins, L.D. Barron, Mol. Phys., 16 (1969) 453-466.

3.     L.D. Barron, M.P. Boggard, A.D. Buckingham, Nature, 241 (1973) 113-114.

4.    N.R. Yaffe, A. Almond, E.W. Blanch, J. Am. Chem. Soc., 132 (2010) 10654-10655.

 

The work was supported by the Grant Agency of the Czech Republic (grant No. P208/11/0105), by the Charles University in Prague (projects SVV-2012-265304), by the British Biological Sciences Research Council (grant No. BB/H023763/1) and by the Engineering and Physical Sciences Research Council Windfall Scheme.