Donor specificity in the glycosylation of Tamm-Horsfall glycoprotein: conservation of the Sda determinant in pairs of twins.

The content of the Sd(a) determinant in urinary human Tamm-Horsfall glycoprotein (THp) has been reported to be donor-specific. This feature was further addressed by investigating THp from genetically identical individuals. To this end, THp was isolated from the urine of two monozygotic pairs of twins (A and B). The four samples (THp A1, A2, B1, and B2) were subjected to endo-beta-galactosidase from Bacteroides fragilis leading to the liberation of the Neu5Ac(alpha2-3)Gal (beta1-4)GlcNAc(beta1-3)Gal and Neu5Ac(alpha2-3)[GalNAc(beta1-4)] Gal(beta1-4)GlcNAc(beta1-3)Gal (Sd(a) epitope) motifs, both located at the nonreducing termini of complex type N-glycans. The isolated mixtures of oligosaccharides were analyzed for the absolute and relative amounts of the two oligosaccharides. The obtained data clearly indicate that in THp A1 and A2, and in THp B1 and B2, the molar ratios of the tetra- and Sd(a) pentasaccharide are identical for a pair of twins. This conservation of molar ratios points to an identical relative expression of beta-1,4-N-acetylgalactosaminyltransferase activity involved in the biosynthesis of the Sd(a) determinant. Apparently, the degree of conversion of the tetrasaccharidic Sd(a) precursor into the final pentasaccharidic Sd(a) form can be considered to result from a very closely related pattern of glycosylation for genetically homogeneous individuals.

Epitope mapping of sialyl Lewis(x) bound to E-selectin using saturation transfer difference NMR experiments.

A complex between sialyl Lewisx (alpha-D-Neu5Ac-[2-->3]- beta-D-Gal-[1-->4]-[alpha-L-Fuc-(1-->3)]-beta-D-GlcNAc-O-[CH2]8 COOMe) and E-selectin was studied using saturation transfer difference (STD) nuclear magnetic resonance (NMR) experiments. These experiments allow the identification of the binding epitope of a ligand at atomic resolution. A semiquantitative analysis of STD total correlation spectroscopy spectra provides clear evidence that the galactose residue receives the largest saturation transfer. The protons H4 and H6 of the galactose residue are in especially close contact to the amino acids of the E-selectin binding pocket. The fucose residue also receives a significant saturation transfer. The GlcNAc and Neu5Ac residues, with the exception of H3 and H3' of Neu5Ac, were found to interact weakly with the protein surface. These findings are in excellent agreement with a recently published X-ray structure and with the earlier findings from syntheses and activity assays. To further characterize the binding pocket of E-selectin, an inhibitory peptide, Ac-TWDQLWDLMK-CONH2, was synthesized and the binding to E-selectin studied utilizing transfer nuclear Overhauser effect spectroscopy (trNOESY) experiments. Finally, competitive trNOESY experiments were performed, showing that the synthetic peptide is a competitive inhibitor of sialyl Lewisx.