Chuanhao Peng

Glycans form the outermost molecular interface of cells across all kingdoms of life, mediating recognition between organisms and their immune systems. Hypothetical mirror organisms would display glycans of reversed chirality, potentially impairing molecular recognition and contributing to proposed immune-evasion risks. Assessing DNA-encoded multivalent glycan arrays in settings ranging from purified proteins to mice, we found widespread recognition of mirror-image glycans by extant glycan-binding proteins, including recognition of L-glucose and L-galactose by the human immune receptor DC-SIGN. Mirror glycans were also selectively recognized by human immune cells and natural IgM antibodies and exhibited stereochemistry-dependent biodistribution in mice. Thus, glycan recognition can cross the chiral divide, revealing an unexpected molecular interface through which existing life could recognize and interact with hypothetical mirror life.

Recent synthesis of essential enzymes, such as DNA and RNA polymerases with opposite chirality, has boosted the feasibility of creating mirror-image life. Such life, if ever produced, will undoubtedly be coated by a dense display of glycans (glycoproteins, glycolipids, polysaccharides) built from enantiomers of common monosaccharides. Recognition of mirror-image glycans by extant glycan-binding proteins (GBPs) may be critical for colonization by or immune response to mirror life organisms. We evaluated recognition of enantiomers of common glycans by a diverse set of purified GBPs (plant and human derived), antibodies (including IgM from human plasma), mammalian cells (including immune cells), and organs in live animals. We found that GBP binding to enantiomers of naturally prevalent glycans is widespread. Notably, L-glucose and L-galactose interact with fucose-binding lectins, including DC-SIGN, a C-type lectin expressed on immune cells. These interactions can be inhibited by soluble “natural” glycan ligands and enantiomeric ones confirming specificity. Binding of L-glycans to diverse immune cell repertoires revealed preferences for specific glycan enantiomers. IgM antibodies from human serum showed donor-specific recognition of L-glycans. We propose that the recognition of L-glycans by extant GBPs arises from their co-evolution over millennia with the L-glycans that are present in the glycocalyx of many microorganisms.

Glycan-microbe interactions are central to gut colonization and host-microbiota communication. Here, we apply a DNA-encoded Liquid Glycan Array (LiGA) to quantify interactions between live gut bacteria and multivalent natural or mirror glycans. LiGA comprises glycosylated M13 bacteriophage bearing silent DNA barcodes that encode glycan identity and density. Using LiGA, we profiled glycan binding across 16 Limosilactobacillus reuteri strains isolated from murine, porcine, poultry, and human hosts, and then extended the approach to profile glycan binding of taxonomically diverse bacteria from three phyla Bacillota, Bacteroidota, Pseudomonadota consisting of nine different species. Recent discussion of mirror-image microorganisms raise a question whether these microorganisms could interact with present-day life by engaging naturally chiral glycans. We demonstrated that this question can be assessed by testing the binding of mirror-image glycans to natural bacteria. Evaluation of enantiomers of common glycan revealed cross-chiral recognition by Escherichia coli and L. reuteri , indicating that these bacteria can used mirror-image glycans to engage for adhesion and potential colonization. By symmetry the same arguments extends to mirror microorganisms and glycans of naturally chirality. We established that LiGA enables efficient characterization of bacterial glycan binding and provides new insights into intestinal microbial ecology.

We test the performance of the multiplexed liquid glycan array (LiGA) technology in serological assays. Specifically, we use LiGA to detect ABO blood group antibodies in human serum. This LiGA, which we name ABO-LiGA, contains ABO blood group trisaccharide glycans with an ethylazido aglycone conjugated to groups of ten multi-barcoded M13 particles carrying dibenzocyclooctyne (DBCO) on p8 proteins. ELISA clonal binding assays to anti-A/B antibodies confirmed the functional performance of ABO-clones and aligned with next-generation sequencing (NGS) of the mixed clones. Multiple DNA-barcoded technical replicates in LiGA allow for quantification of reproducibility and robustness as determined by the Z’-score using NGS. We then tested ABO-LiGA for specific detection of IgG and IgM anti-A and anti-B IgG and IgM antibodies in human serum samples. Comparison of antibody binding responses in sera from 31 healthy donors to ABO-LiGA with an ABO-Luminex-based method revealed consistent responses to LiGA-ABO but also minor deficiencies of ABO-LiGA such as low robustness of the current assay format and a limited ability to detect low intensity antibody responses. Some results point to undesired interactions of serum antibodies with small-footprint glycans conjugated to phage via the bulky DBCO moiety. This report illuminates the path for future development of LiGA-based serological assays and suggests the need to develop alternative methods for conjugating glycans to phage to avoid liabilities of the hydrophobic DBCO moiety.