Innate immune Galectin-7 specifically targets microbes that decorate themselves in blood group-like antigens

Numerous studies have highlighted the utility of glycan microarray analysis for the elucidation of protein-glycan interactions. However, most current glycan microarray studies analyze glycan binding protein (GBP)-glycan interactions at a single protein concentration. While this approach provides useful information related to a GBP’s overall binding capabilities, extrapolation of true glycan binding preferences using this method fails to account for printing variations or other factors that may confound relative binding. To overcome this limitation, we examined glycan array binding of three galectins over a range of concentrations to allow for a more complete assessment of binding preferences. This approach produced a richer data set than single concentration analysis and provided more accurate identification of true glycan binding preferences. However, while this approach can be highly informative, currently available data analysis approaches make it impractical to perform binding isotherms for each glycan present on currently available platforms following GBP evaluation. To overcome this limitation, we developed a method to directly optimize the efficiency of assessing association constants following multi-GBP concentration glycan array analysis. To this end, we developed programs that automatically analyze raw array data (kdMining) to generate output graphics (kaPlotting) following array analysis at multiple doses. These automatic programing methods reduced processing time from 32.8 h to 1.67 min. Taken together, these results demonstrate an effective approach to glycan array analysis that provides improved detail and efficiency when compared to previous methods.

An Automated Approach to Assess Relative Galectin-Glycan Affinity Following Glycan Microarray Analysis

Polymorphonuclear neutrophils (PMNs) play a critical role in clearing invading microbes and promoting tissue repair following infection/injury. However, dysregulated PMN trafficking and associated tissue damage is pathognomonic of numerous inflammatory mucosal diseases. The final step in PMN influx into mucosal lined organs (including the lungs, kidneys, skin, and gut) involves transepithelial migration (TEpM). The β2-integrin CD11b/CD18 plays an important role in mediating PMN intestinal trafficking, with recent studies highlighting that terminal fucose and GlcNAc glycans on CD11b/CD18 can be targeted to reduce TEpM. However, the role of the most abundant terminal glycan, sialic acid (Sia), in regulating PMN epithelial influx and mucosal inflammatory function is not well understood. Here we demonstrate that inhibiting sialidase-mediated removal of α2-3–linked Sia from CD11b/CD18 inhibits PMN migration across intestinal epithelium in vitro and in vivo. Sialylation was also found to regulate critical PMN inflammatory effector functions, including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition reduces bacterial peptide–mediated CD11b/CD18 activation in PMN and blocks downstream intracellular signaling mediated by spleen tyrosine kinase (Syk) and p38 MAPK. These findings suggest that sialylated glycans on CD11b/CD18 represent potentially novel targets for ameliorating PMN-mediated tissue destruction in inflammatory mucosal diseases.

http://insight.jci.org/articles/view/167151/files/pdf

Sialylation regulates neutrophil transepithelial migration, CD11b/CD18 activation, and intestinal mucosal inflammatory function

Abstract Microbial glycan microarrays (MGMs) populated with purified microbial glycans have been used to define the specificity of host immune factors toward microbes in a high throughput manner. However, a limitation of such arrays is that glycan presentation may not fully recapitulate the natural presentation that exists on microbes. This raises the possibility that interactions observed on the array, while often helpful in predicting actual interactions with intact microbes, may not always accurately ascertain the overall affinity of a host immune factor for a given microbe. Using galectin-8 (Gal-8) as a probe, we compared the specificity and overall affinity observed using a MGM populated with glycans harvested from various strains of Streptococcus pneumoniae to an intact microbe microarray (MMA). Our results demonstrate that while similarities in binding specificity between the MGM and MMA are apparent, Gal-8 binding toward the MMA more accurately predicted interactions with strains of S. pneumoniae, including the overall specificity of Gal-8 antimicrobial activity. Taken together, these results not only demonstrate that Gal-8 possesses antimicrobial activity against distinct strains of S. pneumoniae that utilize molecular mimicry, but that microarray platforms populated with intact microbes present an advantageous strategy when exploring host interactions with microbes. 

https://www.nature.com/articles/s41598-023-27964-y.pdf

Whole microbe arrays accurately predict interactions and overall antimicrobial activity of galectin-8 toward distinct strains of Streptococcus pneumoniae

Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His6-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His6-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His6-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His6-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His6-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.

/pdf/characterization-of-galectin-fusion-proteins-with-207o2o15.pdf

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

Purification of Recombinant Galectins from Different Species Using Distinct Affinity Chromatography Methods.

Evaluation of the Bactericidal Activity of Galectins.

Alkylation of Galectin-1 with Iodoacetamide and Mass Spectrometric Mapping of the Sites of Incorporation.

Blood Group A Enhances SARS-CoV-2 Infection

/pdf/blood-group-a-enhances-sars-cov-2-infection-2fug6z5w.pdf

A. Paul

Papers

Purification of Recombinant Galectins from Different Species Using Distinct Affinity Chromatography Methods.

Evaluation of the Bactericidal Activity of Galectins.

Alkylation of Galectin-1 with Iodoacetamide and Mass Spectrometric Mapping of the Sites of Incorporation.

Blood Group A Enhances SARS-CoV-2 Infection

Blood Group A Enhances SARS-CoV-2 Infection