Chemoenzymatic Synthesis of Sulfated N-Glycans Recognized by Siglecs and Other Glycan-Binding Proteins.

Sulfated N-glycans are present in many glycoproteins, which are implicated in playing important roles in biological recognition processes. Here, we report the systematic chemoenzymatic synthesis of a library of sulfated and sialylated biantennary N-glycans and assess their binding to Siglecs and glycan-specific antibodies that recognize them as glycan ligands. The combined use of three human sulfotransferases, GlcNAc-6-O-sulfotransferase (CHST2), Gal-3-O-sulfotransferase (Gal3ST1), and keratan sulfate Gal-6-O-sulfotransferase (CHST1), resulted in asymmetric and symmetric branch-selective sulfation of the GlcNAc and/or Gal moieties of N-glycans. The extension of the sugar chain using α-2,3- and α-2,6-sialyltransferases afforded the sulfated and sialylated N-glycans. These synthetic glycans with different patterns of sulfation and sialylation were evaluated for binding to selected Siglecs and sulfoglycan-specific antibodies using glycan microarrays. The results confirm previously documented glycan-recognizing properties and further reveal novel specificities for these glycan-binding proteins, demonstrating the utility of the library for assessing the specificity of glycan-binding proteins recognizing sulfated and sialylated glycans.

Chemical immunology: Recent advances in tool development and applications.

Immunology was one of the first biological fields to embrace chemical approaches. The development of new chemical approaches and techniques has provided immunologists with an impressive arsenal of tools to address challenges once considered insurmountable. This review focuses on advances at the interface of chemistry and immunobiology over the past two decades that have not only opened new avenues in basic immunological research, but also revolutionized drug development for the treatment of cancer and autoimmune diseases. These include chemical approaches to understand and manipulate antigen presentation and the T cell priming process, to facilitate immune cell trafficking and regulate immune cell functions, and therapeutic applications of chemical approaches to disease control and treatment.

Demonstrating the utility of sugar-phosphate phosphatases in coupled enzyme assays: galactose-1-phosphate uridylyltransferase as proof-of-concept.

During our biochemical characterization of select bacterial phosphatases belonging to the haloacid dehalogenase superfamily of hydrolases, we discovered a strong bias of Salmonella YidA for glucose-1-phosphate (Glc-1-P) over galactose-1-phosphate (Gal-1-P). We sought to exploit this ability of YidA to discriminate these two sugar-phosphate epimers in a simple coupled assay that could be a substitute for current cumbersome alternatives. To this end, we focused on Gal-1-P uridylyltransferase (GalT) that is defective in individuals with classical galactosemia, an inborn disorder. GalT catalyzes the conversion of Gal-1-P and UDP-glucose to Glc-1-P and UDP-galactose. When recombinant YidA was coupled to GalT, the final orthophosphate product (generated from selective hydrolysis of Glc-1-P by YidA) could be easily measured using the inexpensive malachite green reagent. When this new YidA-based colorimetric assay was benchmarked using a recombinant Duarte GalT variant, it yielded kcat/Km values that are ~2.5-fold higher than the standard coupled assay that employs phosphoglucomutase and glucose-6-phosphate dehydrogenase. Although the simpler design of our new GalT coupled assay might find appeal in diagnostics, a testable expectation, we spotlight the GalT example to showcase the untapped potential of sugar-phosphate phosphatases with distinctive substrate-recognition properties for measuring the activity of various metabolic enzymes (e.g. trehalose-6-phosphate synthase, N-acetyl-glucosamine-6-phosphate deacetylase, phosphofructokinase).