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

Abstract

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.

ABO blood group antigens and differential glycan expression: Perspective on the evolution of common human enzyme deficiencies

“…There is uncertainty as to the origin of so called “natural antibodies.” Conflicting data exist regarding the potential role of plant materials, microbes, genetic elements, and even transient blood group A and B expression, in the development of naturally occurring antibody formation, in general, and formation of anti-ABO(H) antibodies, in particular ( Bernstein, F., 1925 , F S, 1934 ; Furuhata, 1969 ; Hayakawa et al., 1999 ; New et al., 2020 ; Springer, 1956 ; Springer et al., 1961 ; Storry and Olsson, 2009 ; Wagner, 1955 ; Watkins, 2001 ). Innate immune factors appear to specifically target microbes decorated in blood group carbohydrate antigens ( Arthur et al., 2015b ; Blenda et al., 2022 ; Ho et al., 2022 ; Stowell et al., 2010 , 2014 , 2014 ; Wu et al., 2021b , 2022 , 2022 ), suggesting that a unique interplay may exist between blood group positive microbes, innate immunity, and the development of naturally occurring anti-blood group antibodies. However, studies using germ-free mice suggest that exogenous antigen stimulation is not required for naturally occurring antibody formation ( Bos et al., 1989 ; Bunker et al., 2017 ; Haury et al., 1997 ; Hooijkaas et al., 1984 , 1985 ).…”

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

“…These results strongly suggest that while the overall trends in bindings specificity were similar for Gal-8 and Gal-8C, Gal-8 exhibited higher affinity for the CPS of several S. pneumoniae strains compared with Gal-8C. Given the relative similarities in glycan recognition at the highest concentrations tested, these results also suggest that establishing binding isotherms may provide more accurate information regarding the relatively affinity of Gal-8 toward individual strains of S. pneumoniae 39 .…”

“…To ascertain the relative affinity of Gal-8 binding toward each of these microbial glycans, we analyzed binding isotherms generated following analysis of each concentration tested as recently outlined 34 , 37 , 38 . Using this approach, the observed K D of Gal-8 toward Sp 14 and Sp 33F was very similar, around 1.2 ± 0.05 μM and 0.85 ± 0.05 μM, respectively (Fig.…”

“…As noted, the synthetic complexity of microbial glycans prohibits a detailed analysis of the unique glycan determinants that are ultimately responsible for dictating differences in binding affinity observed by Gal-8 toward unique microbial glycans. This can be further complicated by challenges associated with accurately defining glycan or microbial printing efficiency that can make it difficult to interpret relative binding preferences when results are obtained at a single concentration 38 . By examining galectin binding over a range of concentrations a relative affinity can be obtained that may provide additional information regarding the overall preference of a given galectin for a distinct microbial strain 38 .…”

“…This can be further complicated by challenges associated with accurately defining glycan or microbial printing efficiency that can make it difficult to interpret relative binding preferences when results are obtained at a single concentration 38 . By examining galectin binding over a range of concentrations a relative affinity can be obtained that may provide additional information regarding the overall preference of a given galectin for a distinct microbial strain 38 . However, array results ultimately require confirmation with intact microbes.…”