Conformational analysis of complex oligosaccharides: the CICADA approach to the uromodulin O-glycans Gianluca Cioci, a, * Alain Rivet, a Jaroslav Ko ca a,b and Serge P erez a, * a Centre de Recherches sur les Macromol ecules V eg etales, CNRS and Joseph Fourier University, IFR 2607, BP 53, F-38041 Grenoble, France b National Centre for Biomolecular Research, Masaryk University, CZ-61137 Brno, Czech Republic Received 29 September 2003; accepted 12 December 2003 Abstract—Uromodulin is the pregnancy-associated Tamm–Horsfall glycoprotein, with the enhanced ability to inhibit T-cell pro- liferation. Pregnancy-associated structural changes mainly occur in the O-glycosylation of this glycoprotein. These include up to 12 glycan structures, made up of an unusual core type 2 sequence terminated with one, two, or three sialyl Lewis x sequences; this type of O-glycans could serve as E- and P-selectin ligands. The present work focuses on the most complex one; a tetradecamer made up of a type 2 core carrying three sialyl Lewis x branches. Five different monosaccharides are assembled by 14 glycosidic linkages. The conformational behavior of the constituting disaccharide segments was evaluated using the flexible residue procedure of the MM3 molecular mechanics procedure. For each disaccharide, the adiabatic energy surface, along with the local energy minima were established. All these results were used for the generation, prior to complete optimization of the tetradecamer. This was followed by a complete exploration of conformational hyperspace throughout the use of the single coordinate method as implemented in the CICADA program. Despite the potential flexibility of the tetradecasaccharide, only four conformational families occur, accounting for more than 95% of the total low energy conformations. For each family, the molecular properties (electrostatic, lipophilicity, and hydrogen potential) were studied. The shape of the tetradecasaccharide is best described as a flat ribbon, flanked by three branches having terminal sialyl residues. Two of the branches interact through nonbonded interactions, bringing further energy stabilization, and limiting the conformational flexibility of the sialyl residues. Only one branch maintains the original conformational features of sialyl Lewis x . This O-glycan can be seen as a fascinating example of ÔdendrimericÕ structure, where the spatial arrangement of three S-Le x epitopes may favor its complementary ÔpresentationsÕ for the interactions with E- and P-selectins. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Core 2 branched O-glycans; Sialyl Lewis x 1. Introduction Tamm–Horsfall (THp) is the major glycoprotein pro- duced by the kidney, 1;2 where it is expressed via a glycosylphosphatidylinositol (GPI) anchor on the endothelium of the thick ascending limb of the loop of the Henle. This 94kDa glycoprotein can be released from its membrane anchor through proteases or phos- pholipase and is excreted in urine. The physiological roles of THp are still under dispute, and many hypotheses have been formulated. THp could act as an inhibitor of microbial infection of the urinary tract and of antigen-specific T-cell proliferation. Also, the aggre- gation and gel formation capabilities of THP could play important roles in several physiological and patholo- gical states of the kidney. Pregnancy-associated THp is called uromodulin. 3 This is an immunosuppressive molecule whose ability to inhibit T-cell proliferation is increased 13-fold during pregnancy. 4 THp exhibits extremely large glycosylation microheterogeneities, as over 150 glycan structures that are mainly sialylated and/or sulfated to varying extents have been found on seven glycosylation sites. 5 The carbohydrate part can account for 28% of the total weight of the THp. Mass * Corresponding authors. Tel.: +33-4-76037630; fax: +33-4-76037629; e-mail: gianluca.cioci@cermav.cnrs.fr 0008-6215/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2003.12.022 Carbohydrate Research 339 (2004) 949–959 Carbohydrate RESEARCH