Neoglycoproteins with the Synthetic Complex Biantennary Nonasaccharide or Its α2,3/α2,6-Sialylated Derivatives:  Their Preparation, Assessment of Their Ligand Properties for Purified Lectins, for Tumor Cells in Vitro, and in Tissue Sections, and Their Biodistribution in Tumor-Bearing Mice

Abstract

Neoglycoproteins were prepared with chemoenzymatically synthesized complex biantennary N-glycan derivatives the nonreducing ends of which bear typical sequences found in glycoproteins. A chemically obtained biantennary heptasaccharide-azide was reduced and acylated with a 6-aminohexanoyl spacer. Elongation of the deprotected heptasaccharide using glycosyltransferases yielded a biantennary nonasaccharide with terminal galactose residues and two undecasaccharides terminating with alpha 2,6- or alpha 2,3-linked sialic acid. The free amino group of the spacer of these oligosaccharides was converted into an isothiocyanate. Its subsequent coupling to bovine serum albumin gave neoglycoproteins with a yield of 2.4-3.6 glycan chains per carrier molecule. This versatile synthetic pathway allows employment of a wide variety of complex-type glycans, which can be introduced to various test systems in vitro and in vivo to evaluate potential biomedical applications. Solid-phase assays with biotinylated sugar receptors revealed discriminatory binding properties of the three neoglycoproteins, especially for the mistletoe lectin. This direct assay system is preferable to the measurement of inhibitory capacities with respect to model ligands. Ligand type- and cell type-dependent quantitative differences in the binding properties of the probes were detected by FACScan analyses with a panel of tumor cell lines and by monitoring of staining in tissue sections for small cell and non-small-cell lung cancer and mesotheliomas. Biodistribution of iodinated neoglycoproteins in mice gave a prolonged presence of the sialylated probes in serum. Relative to the nonasaccharide, the uptake, especially of the iodinated neoglycoprotein with alpha 2,3-sialylated ligand chains, was clearly elevated in mice for kidneys and Ehrlich tumors. On the basis of the documented feasibility of these applications, it is concluded that the further elaboration of glycan chain variants by the described synthetic approach in combination with the given test panel is warranted to evaluate the potential of complex glycan chain-carrying neoglycoproteins for diagnostic and therapeutic purposes.

Assessing the inhibitory potency of galectin ligands identified from combinatorial (glyco)peptide libraries using surface plasmon resonance spectroscopy

“…2B). The value of R max (14.7 RU) was also obtained from this plot and was used to calculate F im by using the equation [24,25] and that respective SPR data are known to vary with loading densities [26,27].…”

Negative Regulation of Neuroblastoma Cell Growth by Carbohydrate-dependent Surface Binding of Galectin-1 and Functional Divergence from Galectin-3

“…Polyclonal antibodies against human galectin-1 were raised in rabbits and purified from serum by protein A-Sepharose 4B chromatography (38). For assessment of parameters of galectin binding to (neo)glycoproteins (lactosylated albumin, asialofetuin, and Mac-2-binding protein, which was kindly provided by R. Timpl, Munich, Germany), we carried out solid phase assays using the ligand-exposing protein as a surface-immobilized matrix as described (39). Iodination of galectins and antibodies was performed as outlined previously (22).…”

From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans

“…Tumor cells of different histogenetic origin were tested with the set of neoglycoproteins under identical experimental conditions (André et al, 1997(André et al, , 2007aUnverzagt et al, 2002). A dependence of carbohydratemediated cell binding on the structural parameters of the glycan chain is evident (Figure 4).…”

“…Monitoring biodistribution of neoglycoproteins, especially blood and liver contents, is thus a sensitive diagnostic tool to recognize an influence of N-glycan core substitution on lectin-binding properties, with clinical perspectives. Systematic experimental series with neoglycoproteins presenting the different N-glycans have thus been carried out (André et al, 1997(André et al, , 2007aUnverzagt et al, 2002).…”