Revealing biomedically relevant cell and lectin type-dependent structure–activity profiles for glycoclusters by using tissue sections as an assay platform

Abstract

Introducing tissue sections for testing glycocluster activity as inhibitors of lectin binding close to in vivo conditions.

Calorimetric Analysis of the Interplay between Synthetic Tn Antigen-Presenting MUC1 Glycopeptides and Human Macrophage Galactose-Type Lectin

“…Soluble recombinant hMGL was purified by affinity chromatography on a column of homemade lactose-Sepharose 4B, as described previously. 21 , 27 …”

“…Protein was isolated from inclusion bodies by steps that involve solubilization, refolding, and purification. The obtained product was then ascertained for purity as described previously. , To obtain the full-length cDNA sequence of the extracellular domain of hMGL, specific oligonucleotides were designed on the basis of the published sequence in the GSDB, DDBJ, EMBL, and NCBI databases with accession number D50532 . The PCR-based amplification was directed using sense primer 5′-CC GGATCC ­TGGTG­ACCCT­GAG­AAC-3′ and antisense primer 5′-CC GGATCC ­GGGTG­GTCCC­AC­CAA-3′ with an internal Bam HI restriction site (underlined).…”

“…Washed pellets were solubilized with 100 mL of 2 M ammonium hydroxide under stirring for 6 h at rt and then dialyzed against 20 mM MOPS buffer (pH 7.0) containing 20 mM CaCl 2 and 0.5 M NaCl at 4 °C. Soluble recombinant hMGL was purified by affinity chromatography on a column of homemade lactose-Sepharose 4B, as described previously. , …”

“…We first prepared the complete panel of Thr-modified glycopeptides of the MUC1 tandem-repeat motif. In this context, it is noteworthy that comparative analysis of staining profiles of Gal N Ac-specific lectins from plants (DBA and SBA) or snails (HPA) with that of hMGL in mucin-rich murine jejunum had disclosed differences so that extrapolations between lectins of the same nominal monosaccharide specificity should be done with caution …”

“…In this context, it is noteworthy that comparative analysis of staining profiles of GalNAcspecific lectins from plants (DBA and SBA) or snails (HPA) with that of hMGL in mucin-rich murine jejunum had disclosed differences so that extrapolations between lectins of the same nominal monosaccharide specificity should be done with caution. 27 Having made the glycopeptides with site-specific glycosylation in systematic permutations to yield mono-to triglycosylated products, available in the first part of our study, we then performed a systematic ITC analysis. It includes examining the effect of glycan site variations at constant valency.…”