Full-Length Galectin-3 Is Required for High Affinity Microbial Interactions and Antimicrobial Activity

Abstract

While adaptive immunity enables the recognition of a wide range of microbial antigens, immunological tolerance limits reactively toward self to reduce autoimmunity. Some bacteria decorate themselves with self-like antigens as a form of molecular mimicry to limit recognition by adaptive immunity. Recent studies suggest that galectin-4 (Gal-4) and galectin-8 (Gal-8) may provide a unique form of innate immunity against molecular mimicry by specifically targeting microbes that decorate themselves in self-like antigens. However, the binding specificity and antimicrobial activity of many human galectins remain incompletely explored. In this study, we defined the binding specificity of galectin-3 (Gal-3), the first galectin shown to engage microbial glycans. Gal-3 exhibited high binding toward mammalian blood group A, B, and αGal antigens in a glycan microarray format. In the absence of the N-terminal domain, the C-terminal domain of Gal-3 (Gal-3C) alone exhibited a similar overall binding pattern, but failed to display the same level of binding for glycans over a range of concentrations. Similar to the recognition of mammalian glycans, Gal-3 and Gal-3C also specifically engaged distinct microbial glycans isolated and printed in a microarray format, with Gal-3 exhibiting higher binding at lower concentrations toward microbial glycans than Gal-3C. Importantly, Gal-3 and Gal-3C interactions on the microbial microarray accurately predicted actual interactions toward intact microbes, with Gal-3 and Gal-3C displaying carbohydrate-dependent binding toward distinct strains of Providentia alcalifaciens and Klebsiella pneumoniae that express mammalian-like antigens, while failing to recognize similar strains that express unrelated antigens. While both Gal-3 and Gal-3C recognized specific strains of P. alcalifaciens and K. pneumoniae, only Gal-3 was able to exhibit antimicrobial activity even when evaluated at higher concentrations. These results demonstrate that while Gal-3 and Gal-3C specifically engage distinct mammalian and microbial glycans, Gal-3C alone does not possess antimicrobial activity.

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

“…Although immunological tolerance reduces the probability of autoimmunity, it creates a gap in adaptive immunity toward microbes that decorate themselves in blood group-like antigens. Our prior results demonstrated that Gal-3, Gal-4, and Gal-8 possess the ability to bind and kill microbes that exhibit key features of mammalian blood-group antigens, suggesting that these galectins may fill this gap by targeting microbes that utilize molecular mimicry ( Stowell et al., 2010 , 2014 ; Wu et al., 2021b ). However, the extent to which other galectins likewise possess the ability to bind and kill microbes that express blood group-like antigens remained relatively unknown.…”

“…The apparent discrepancy between Gal-7 binding on the CFG glycan microarray and actual interactions with microbial glycans, including those observed on the microbial glycan microarray, is unique. Prior studies examining Gal-1, Gal-3, Gal-4, and Gal-8 demonstrated that CFG microarray findings largely predicted actual interactions with similar glycan determinants when expressed on microbes ( Stowell et al., 2010 , 2014 ; Wu et al., 2021b ). Although the glycan motifs on microbes certainly possess the ability to mimic mammalian blood group antigens, they are distinct.…”

“…Subtle, but important, differences in the mode of glycan binding may in part be responsible for differences in the recognition of microbial and mammalian blood group antigens by different galectin family members. For example, Gal-3, Gal-4, and Gal-8 display high affinity for mammalian blood group antigens when presented as a single terminal blood group unit, suggesting that intrinsic affinity for the blood group modification on a LacNAc backbone may support substantial binding affinity ( Stowell et al., 2010 ; Wu et al., 2021b ). In contrast, Gal-7 not only failed to bind blood group antigens with high affinity in general, but only bound the H antigen when presented on an extended polyLacNAc structure (iH).…”

“…Labeled Gal-3 or Gal-7 were purified again by lactosyl-sepharose column to remove possible inactive protein that may have formed during labeling process. Bound Gal-7 was eluted with 100 mM lactose in PBS plus 2-mercaptoethanol (2-ME) and then a PD-10 gel filtration column was used to remove 2-ME and lactose prior to the use of Gal-7 for experiments ( Blenda et al., 2022 ; Stowell et al., 2007 , 2008b ; Wu et al., 2021b , 2021c ). The molecular weight of monomeric Gal-7 is 15 kDa.…”

“…However, defining the binding specificity of carbohydrate binding proteins was historically challenging because of a lack of suitable tools and sufficient glycan libraries. Using microarrays populated with hundreds of mammalian glycans, including blood group antigens, our recent studies demonstrated that several members of the galectin family, including galectin-3 (Gal-3), galectin-4 (Gal-4), and galectin-8 (Gal-8), possess the ability to bind blood group A and B with high affinity ( Stowell et al., 2010 , 2014 ; Wu et al., 2021b ), strongly suggesting that these galectins may be uniquely poised to provide innate immunity against blood group molecular mimicry. Consistent with this, Gal-3, Gal-4, and Gal-8 not only engage blood group antigens on the glycan microarray but also specifically recognize and kill microbes that likewise express blood group-like determinants ( Stowell et al., 2010 , 2014 ; Wu et al., 2021b ).…”