Transient interactions among glycoconjugates underlie developmental, immunological and metastatic recognition. Glycan-mediated interactions have low binding affinities and rapid dissociation rates. As a result, these complexes dissociate when removed from their cellular context, complicating characterization. Photocrosslinkers introduce a covalent bond between glycoconjugates and their binding partners, allowing physiologically relevant complexes to be isolated. This protocol describes metabolic incorporation of a diazirine photocrosslinker into sialic acids in cellular glycoconjugates. Subsequent irradiation results in photocrosslinking of sialic acid to neighboring macromolecules, providing a photochemical 'snapshot' of binding events. As photocrosslinking sugars are light activated, these reagents have the potential to be used for temporally and/or spatially restricted crosslinking. We provide instructions for the synthesis of photocrosslinking sugar precursors, cell culture for metabolic incorporation, flow cytometry to evaluate metabolic incorporation, photoirradiation and analysis of the crosslinked complexes. Synthesis of photocrosslinking sugars requires 4-6 d, and photocrosslinking experiments can be completed in an additional 6 d.

Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photocrosslinking approach in which a diazirine photocrosslinker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogs in which the length of the linker between the pyranose ring and the diazirine was varied. These analogs were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogs could be incorporated into both α2–3 and α2–6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently crosslinked to their interaction partners. We demonstrate that all three diazirine-modified analogs were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogs were less efficiently metabolized, yet could still function as effective crosslinkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogs to glycans other than sialosides, providing fidelity to selectively incorporate the crosslinker into sialylated molecules. These compounds are non-toxic and display only minimal growth inhibition at the concentrations required for crosslinking studies. This report provides essential information for the deployment of photocrosslinking analogs to capture and study ephemeral, yet essential, sialic acid-mediated interactions.

Exopolysaccharides act as mediators of cross-talk between probiotics and the host. Here, we found that EPS derived from probiotic Lactobacillus casei WXD030 strain (L-EPS) could modulate immune responses in vitro and in vivo. L-EPS could significantly enhance the proliferation and phagocytic activity as well as induce the production of NO, TNF-α, IL-1β and IL-6 in RAW264.7 cells. Furthermore, L-EPS could induce the maturation of BMDCs. In addition, L-EPS could largely increase the titres of OVA-specific antibodies and markedly enhanced T cell proliferation. Notably, L-EPS also increased expression of IL-4 and INF-γ expression in CD4 + T cells. Consistently, when used as an adjuvant in vivo with the foot-and-mouth disease vaccine, L-EPS largely enhanced the FMDVspecific antibody production. Collectively, these results suggested that L-EPS derived from probiotic L. casei strain had adjuvant activity, which may be a safe and efficacious adjuvant candidate suitable for a wide spectrum of prophylactic and therapeutic vaccines.

Liver diseases alter the gut microbiota, but several lactic acid bacteria can reduce the degree of liver damage. The present study investigated whether Lactobacillus buchneri TCP016 reduces the degree of liver damage by modifying the gut microbiota via its exopolysaccharides (EPSs). First, it was illustrated that the main EPS (EPS016; molecular weight = 8.509 × 104 Da) comprised rhamnose, xylose, glucosamine, glucuronic acid, galactose, galacturonic acid, glucose, and mannose in molar ratios of 9.2:3.9:3.8:2.8:2.1:2.0:1.6:1.0. Our data showed that EPS016 alleviated the increase in plasma and hepatic enzyme and cytokine levels, increased superoxide dismutase and glutathione activity, and alleviated bacterial translocation to the liver and mesenteric lymph nodes in vivo. Furthermore, EPS016 ameliorated intestinal mucosal injury and gut flora dysbiosis, thereby decreasing the enrichment of Helicobacteraceae, Lachnospiraceae, and Enterobacteriaceae and increasing the abundance of Lactobacillus, Rikenellaceae, Bacteroidaceae, Bacteroidales_S24-7_group, and Prevotellaceae. These findings indicated that EPS016 inhibits lipopolysaccharides/d-galactosamine-induced liver injury and improves the modification of the gut microbiota.

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