Biology subjects

Klassen, J. S.

Publications and source records attributed to Klassen, J. S..

3 recordsLinked to original sources

bioRxiv2021

Carbohydrate sulfation as a mechanism for fine-tuning Siglec ligands

The immunomodulatory family of Siglecs recognize sialic acid-containing glycans as self, which is exploited in cancer for immune-evasion. The biochemical nature of Siglec ligands remains incompletely understood with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs (CHST1, CHST2, or CHST4) significantly enhances the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, pharmacological blockade of N- and O-glycan maturation reveals a cell type-specific pattern of importance for either class of glycan. Production of a highly homogenous CD33 (Siglec-3) fragment enabled a mass spectrometry-based binding assay to determine 10-fold and 3-fold enhanced affinity for Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4GlcNAc and Neu5Ac2-3Gal{beta}1-4(6-O- sulfo)GlcNAc, respectively, over Neu5Ac2-3Gal{beta}1-4GlcNAc. CD33 showed significant additivity in affinity (36-fold) for the disulfated ligand, Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4(6-O-sulfo)GlcNAc. Moreover, overexpression of both CHST1 and CHST2 in cells greatly enhanced the binding of several Siglecs, including CD33. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a modification that is a general mechanism for tuning Siglec ligands on cells, including in cancer.

immunology↗

bioRxiv2021

Sialic acid-Dependent Binding and Viral Entry of SARS-CoV-2

Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 M) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral coreceptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

biochemistry↗

bioRxiv2020

A Versatile Soluble Siglec Scaffold for Sensitive and Quantitative Detection of Glycan Ligands

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. A growing connection between Siglecs and human disease motivates improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively. Using these Siglec-Fc proteins, Siglec ligands were systemically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. Additional features enable the production of small, homogenous Siglec fragments and development of a quantitative ligand-binding mass spectrometry assay. Using this assay, the ligand specificities of several Siglecs were clarified. For CD33, we demonstrate that it recognizes both 2-3 and 2-6 sialosides in solution and on cells, which has implications for its link to Alzheimers disease susceptibility. These soluble Siglecs reveal the abundance of their glycan ligands on host cells as self-associated molecular patterns.

biochemistry↗