CD22 Ligands on a Natural <i>N</i>‑Glycan Scaffold Efficiently Deliver Toxins to B‑Lymphoma Cells

CD22 is a sialic acid-binding immunoglobulin-like lectin (Siglec) that is highly expressed on B-cells and B cell lymphomas, and is a validated target for antibody and nanoparticle based therapeutics. However, cell targeted therapeutics are limited by their complexity, heterogeneity, and difficulties in production. We describe here a chemically defined natural <i>N</i>-linked glycan scaffold that displays high affinity CD22 glycan ligands and outcompetes the natural ligand for the receptor, resulting in single molecule binding to CD22 and endocytosis into cells. Binding affinity is increased by up to 1500-fold compared to the monovalent ligand, while maintaining the selectivity for hCD22 over other Siglecs. Conjugates of these multivalent ligands with auristatin and saporin toxins are efficiently internalized via hCD22 resulting in killing of B-cell lymphoma cells. This single molecule ligand targeting strategy represents an alternative to antibody- and nanoparticle-mediated approaches for delivery of agents to cells expressing CD22 and other Siglecs

The Galactosamine Residue in Mycobacterial Arabinogalactan Is α‑Linked

Previous studies have demonstrated that cell wall arabinogalactan from mycobacteria possesses a single galactosamine (GalN) residue. This moiety, which is one of the rare natural occurrences of galactosamine lacking an acetyl group on the nitrogen, has been identified as a pendant substituent attached to a highly branched arabinofuranose residue in the arabinan core. However, the stereochemistry by which the GalN residue is linked to the polysaccharide remains unknown. We report here the synthesis of two tetrasaccharides, <b>1</b> and <b>2</b>, consisting of GalN attached through either an α- or β-linkage to a trisaccharide fragment of mycobacterial arabinan. These molecules represent the first synthetic GalN-containing oligosaccharides, and the preparation of both targets was achieved from a single donor species by modulation of the reaction solvent. Comparison of the NMR spectra of <b>1</b> and <b>2</b> with those obtained from a sample derived from the natural glycan revealed that the GalN residue in the polysaccharide is attached via an α-linkage

Additional file 1 of GBC: a parallel toolkit based on highly addressable byte-encoding blocks for extremely large-scale genotypes of species

Additional file 1: Table S1. The basic compression performance comparison between GBC and alternative tools. Table S2. The comparison of GBC’s compression and decompression speed under multiple threads in the 1000GP3 dataset. Table S3. The data query performance comparison between GBC and alternative tools. Table S4. The comparison of LD calculation speed between GBC and alternative tools in the 1000GP3 and SG10K datasets. Table S5. The file management performance comparison between GBC and alternative tools. Table S6. BEG and MBEG coding tables for genotypes of diploid species

Additional file 3 of GBC: a parallel toolkit based on highly addressable byte-encoding blocks for extremely large-scale genotypes of species

Additional file 3: Note 1. A detailed description of GTB file. Note 2. The generation method of simulation genotypes. Note 3. The format of the contig file. Note 4. Examined programs

Additional file 2 of GBC: a parallel toolkit based on highly addressable byte-encoding blocks for extremely large-scale genotypes of species

Additional file 2: Fig. S1. Using CLM algorithm to achieve ordered output during parallel computation of large-scale data. Fig. S2. Optimized file management (sorting and merging) based on GTB

Additional file 4 of GBC: a parallel toolkit based on highly addressable byte-encoding blocks for extremely large-scale genotypes of species

Additional file 4. Review history

Synthesis of Biologically Active <i>N</i>- and <i>O</i>‑Linked Glycans with Multisialylated Poly‑<i>N</i>‑acetyllactosamine Extensions Using <i>P. damsela</i> α2‑6 Sialyltransferase

Sialosides on <i>N</i>- and <i>O</i>-linked glycoproteins play a fundamental role in many biological processes, and synthetic glycan probes have proven to be valuable tools for elucidating these functions. Though sialic acids are typically found α2-3- or α2-6-linked to a terminal nonreducing end galactose, poly-LacNAc extended core-3 <i>O</i>-linked glycans isolated from rat salivary glands and human colonic mucins have been reported to contain multiple internal Neu5Acα2-6Gal epitopes. Here, we have developed an efficient approach for the synthesis of a library of <i>N</i>- and <i>O</i>-linked glycans with multisialylated poly-LacNAc extensions, including naturally occurring multisialylated core-3 <i>O</i>-linked glycans. We have found that a recombinant α2-6 sialyltransferase from <i>Photobacterium damsela</i> (Pd2,6ST) exhibits unique regioselectivity and is able to sialylate internal galactose residues in poly-LacNAc extended glycans which was confirmed by MS/MS analysis. Using a glycan microarray displaying this library, we found that Neu5Acα2-6Gal specific influenza virus hemagglutinins, siglecs, and plant lectins are largely unaffected by adjacent internal sialylation, and in several cases the internal sialic acids are recognized as ligands. Polyclonal IgY antibodies specific for internal sialoside epitopes were elicited in inoculated chickens

Specificity of wild type and mutant H7 HAs on glycan arrays and binding to chicken and human trachea epithelium.

<p>Glycan binding analyses of Sh2 H7N9 HA wild type and several mutants that confer human-type receptor binding: G228S, K193T G228S, V186K K193T G228S, V186G K193T G228S, with human Cal/04/09 2009 pandemic H1N1 HA as a control. (A) ELISA-like assay using sialoside polymers. The mean signal and standard error were calculated from six independent replicates; white open circles represent α2–3 linked sialylated di-LacNAc (3’SLNLN), black closed circles represent α2–6 linked sialylated di-LacNAc (6’SLNLN), and non-sialylated di-LacNAc (LNLN) are represented in asterisks. (B) The glycan array mean signal and standard error were calculated from six independent replicates; α2–3 linked sialosides are shown in white bars (glycans 11 to 79 on the x axis) and α2–6 linked sialosides in black (glycans 80 to 135). Glycans 1 to 10 are non-sialylated controls (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006390#ppat.1006390.s001" target="_blank">S1 Table</a>). (C) Tissue binding to either chicken or human tracheal sections is observed by HRP-staining. The sialoside array, ELISA-like assay, and tissue binding experiments are representative of three independent assays performed with different batches of HA proteins.</p

Avidity of Sh2 (WT) and Sh2 V186K-K193T-G228S variant HA for N-linked glycan receptors assessed by glycan ELISA.

<p>Sh2 (upper panels) binds strongly to avian-type (α2–3) receptors (left, white open shapes) with weaker binding to human-type (α2–6) receptors (right, black closed shapes). Sh2 V186K-K193T-G228S (lower panels) shows vastly reduced avidity for avian N-glycans and increased selectivity for extended glycan receptors to human receptors. Assays are conducted with biantennary, N-linked glycans (N) with one to four LacNAc (LN, Galβ1-4GlcNAc) repeats terminated with sialic acid (S) in α2–3 or α2–6 linkage (SLN_1-4-N). An asialo, mono-LacNAc (LacNAc-biotin, LN-L) was used as a negative binding control.</p

Amino acid variation in the receptor binding pocket of influenza HAs and impact of K193T mutation on receptor conformation.

<p>(A) Variation at HA positions that are known to mediate the switch in receptor binding specificity for human H1, H2 and H3 pandemic viruses and corresponding avian viruses of H1, H2, H3 and H5 subtypes in comparison with human H7N9. Red indicates amino acids involved in either human- or avian-type receptor specificity, blue indicates amino-acid positions that are mutated to the amino acids found in human H3N2 and H2N2 viruses. (B) Projection of the receptor glycan from the binding pocket. The receptor analog 6’SLNLN (α2–6 linked sialylated di-LacNAc; NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc) is modeled in the WT H7 with K193 (dark gray), and the mutant H7 with V186K K193T G228S (light gray). In the WT, K193 causes the receptor to project further away from the 190 helix. Symbols in the sugar rings are the conventions for the Symbol Nomenclature For Glycans (SNFG) where sialic acid is the purple cubic diamond, galactose is the yellow sphere and GlcNAc is the blue cube.</p