Lectin-Seq: A method to profile lectin-microbe interactions in native communities.

Soluble human lectins are critical components of innate immunity. Genetic models suggest that lectins influence host-resident microbiota, but their specificity for commensal and mutualist species is understudied. Elucidating lectins' roles in regulating microbiota requires an understanding of which microbial species they bind within native communities. To profile human lectin recognition, we developed Lectin-Seq. We apply Lectin-Seq to human fecal microbiota using the soluble mannose-binding lectin (MBL) and intelectin-1 (hItln1). Although each lectin binds a substantial percentage of the samples (10 to 20%), the microbial interactomes of MBL and hItln1 differ markedly in composition and diversity. MBL binding is highly selective for a small subset of species commonly associated with humans. In contrast, hItln1's interaction profile encompasses a broad range of lower-abundance species. Our data uncover stark differences in the commensal recognition properties of human lectins.

Lectin Fingerprinting Distinguishes Antibody Neutralization in SARS-CoV-2.

Enveloped viruses co-opt host glycosylation pathways to decorate their surface proteins. As viruses evolve, emerging strains can modify their glycosylation patterns to influence host interactions and subvert immune recognition. Still, changes in viral glycosylation or their impact on antibody protection cannot be predicted from genomic sequences alone. Using the highly glycosylated SARS-CoV-2 Spike protein as a model system, we present a lectin fingerprinting method that rapidly reports on changes in variant glycosylation state, which are linked to antibody neutralization. In the presence of antibodies or convalescent and vaccinated patient sera, unique lectin fingerprints emerge that distinguish neutralizing versus non-neutralizing antibodies. This information could not be inferred from direct binding interactions between antibodies and the Spike receptor-binding domain (RBD) binding data alone. Comparative glycoproteomics of the Spike RBD of wild-type (Wuhan-Hu-1) and Delta (B.1.617.2) variants reveal O-glycosylation differences as a key determinant of immune recognition differences. These data underscore the interplay between viral glycosylation and immune recognition and reveal lectin fingerprinting to be a rapid, sensitive, and high-throughput assay to distinguish the neutralization potential of antibodies that target critical viral glycoproteins.

Human oral lectin ZG16B acts as a cell wall polysaccharide probe to decode host-microbe interactions with oral commensals.

The oral microbiome is critical to human health and disease, yet the role that host salivary proteins play in maintaining oral health is unclear. A highly expressed gene in human salivary glands encodes the lectin zymogen granule protein 16 homolog B (ZG16B). Despite the abundance of this protein, its interaction partners in the oral microbiome are unknown. ZG16B possesses a lectin fold, but whether it binds carbohydrates is unclear. We postulated that ZG16B would bind microbial glycans to mediate recognition of oral microbes. To this end, we developed a microbial glycan analysis probe (mGAP) strategy based on conjugating the recombinant protein to fluorescent or biotin reporter functionality. Applying the ZG16B-mGAP to dental plaque isolates revealed that ZG16B predominantly binds to a limited set of oral microbes, including Streptococcus mitis, Gemella haemolysans, and, most prominently, Streptococcus vestibularis. S. vestibularis is a commensal bacterium widely distributed in healthy individuals. ZG16B binds to S. vestibularis through the cell wall polysaccharides attached to the peptidoglycan, indicating that the protein is a lectin. ZG16B slows the growth of S. vestibularis with no cytotoxicity, suggesting that it regulates S. vestibularis abundance. The mGAP probes also revealed that ZG16B interacts with the salivary mucin MUC7. Analysis of S. vestibularis and MUC7 with ZG16B using super-resolution microscopy supports ternary complex formation that can promote microbe clustering. Together, our data suggest that ZG16B influences the compositional balance of the oral microbiome by capturing commensal microbes and regulating their growth using a mucin-assisted clearance mechanism.

Human intelectin-1 (ITLN1) genetic variation and intestinal expression.

Intelectins are ancient carbohydrate binding proteins, spanning chordate evolution and implicated in multiple human diseases. Previous GWAS have linked SNPs in ITLN1 (also known as omentin) with susceptibility to Crohn's disease (CD); however, analysis of possible functional significance of SNPs at this locus is lacking. Using the Ensembl database, pairwise linkage disequilibrium (LD) analyses indicated that several disease-associated SNPs at the ITLN1 locus, including SNPs in CD244 and Ly9, were in LD. The alleles comprising the risk haplotype are the major alleles in European (67%), but minor alleles in African superpopulations. Neither ITLN1 mRNA nor protein abundance in intestinal tissue, which we confirm as goblet-cell derived, was altered in the CD samples overall nor when samples were analyzed according to genotype. Moreover, the missense variant V109D does not influence ITLN1 glycan binding to the glycan ß-D-galactofuranose or protein-protein oligomerization. Taken together, our data are an important step in defining the role(s) of the CD-risk haplotype by determining that risk is unlikely to be due to changes in ITLN1 carbohydrate recognition, protein oligomerization, or expression levels in intestinal mucosa. Our findings suggest that the relationship between the genomic data and disease arises from changes in CD244 or Ly9 biology, differences in ITLN1 expression in other tissues, or an alteration in ITLN1 interaction with other proteins.

Recognition of microbial glycans by soluble human lectins.

Human innate immune lectins that recognize microbial glycans can conduct microbial surveillance and thereby help prevent infection. Structural analysis of soluble lectins has provided invaluable insight into how these proteins recognize their cognate carbohydrate ligands and how this recognition gives rise to biological function. In this opinion, we cover the structural features of lectins that allow them to mediate microbial recognition, highlighting examples from the collectin, Reg protein, galectin, pentraxin, ficolin and intelectin families. These analyses reveal how some lectins (e.g., human intelectin-1) can recognize glycan epitopes that are remarkably diverse, yet still differentiate between mammalian and microbial glycans. We additionally discuss strategies to identify lectins that recognize microbial glycans and highlight tools that facilitate these discovery efforts.

Discovery of Enzymatic Targets of Transcriptional Activators via in Vivo Covalent Chemical Capture.

The network of activator protein-protein interactions (PPIs) that underpin transcription initiation is poorly defined, particularly in the cellular context. The transient nature of these contacts and the often low abundance of the participants present significant experimental hurdles. Through the coupling of in vivo covalent chemical capture and shotgun LC-MS/MS (MuDPIT) analysis, we can trap the PPIs of transcriptional activators in a cellular setting and identify the binding partners in an unbiased fashion. Using this approach, we discover that the prototypical activators Gal4 and VP16 target the Snf1 (AMPK) kinase complex via direct interactions with both the core enzymatic subunit Snf1 and the exchangeable subunit Gal83. Further, we use a tandem reversible formaldehyde and irreversible covalent chemical capture approach (TRIC) to capture the Gal4-Snf1 interaction at the Gal1 promoter in live yeast. Together, these data support a critical role for activator PPIs in both the recruitment and positioning of important enzymatic complexes at a gene promoter and represent a technical advancement in the discovery of new cellular binding targets of transcriptional activators.

TRIC: Capturing the direct cellular targets of promoter-bound transcriptional activators.

Transcriptional activators coordinate the dynamic assembly of multiprotein coactivator complexes required for gene expression to occur. Here we combine the power of in vivo covalent chemical capture with p-benzoyl-L-phenylalanine (Bpa), a genetically incorporated photo-crosslinking amino acid, and chromatin immunoprecipitation (ChIP) to capture the direct protein interactions of the transcriptional activator VP16 with the general transcription factor TBP at the GAL1 promoter in live yeast.

Sequence context and crosslinking mechanism affect the efficiency of in vivo capture of a protein-protein interaction.

Protein-protein interactions (PPIs) are essential for implementing cellular processes and thus methods for the discovery and study of PPIs are highly desirable. An emerging method for capturing PPIs in their native cellular environment is in vivo covalent chemical capture, a method that uses nonsense suppression to site specifically incorporate photoactivable unnatural amino acids (UAAs) in living cells. However, in one study we found that this method did not capture a PPI for which there was abundant functional evidence, a complex formed between the transcriptional activator Gal4 and its repressor protein Gal80. Here we describe the factors that influence the success of covalent chemical capture and show that the innate reactivity of the two UAAs utilized, (p-benzoylphenylalanine (pBpa) and p-azidophenylalanine (pAzpa)), plays a profound role in the capture of Gal80 by Gal4. Based upon these data, guidelines are outlined for the successful use of in vivo photo-crosslinking to capture novel PPIs and to characterize the interfaces.

Fine-tuning multiprotein complexes using small molecules.

Multiprotein complexes such as the transcriptional machinery, signaling hubs, and protein folding machines are typically composed of at least one enzyme combined with multiple non-enzymes. Often the components of these complexes are incorporated in a combinatorial manner, in which the ultimate composition of the system helps dictate the type, location, or duration of cellular activities. Although drugs and chemical probes have traditionally targeted the enzyme components, emerging strategies call for controlling the function of protein complexes by modulation of protein-protein interactions (PPIs). However, the challenges of targeting PPIs have been well documented, and the diversity of PPIs makes a "one-size-fits-all" solution highly unlikely. These hurdles are particularly daunting for PPIs that encompass large buried surface areas and those with weak affinities. In this Review, we discuss lessons from natural systems, in which allostery and other mechanisms are used to overcome the challenge of regulating the most difficult PPIs. These systems may provide a blueprint for identifying small molecules that target challenging PPIs and affecting molecular decision-making within multiprotein systems.

Caught in the act: covalent cross-linking captures activator-coactivator interactions in vivo.

Currently there are few methods suitable for the discovery and characterization of transient, moderate affinity protein-protein interactions in their native environment, despite their prominent role in a host of cellular functions including protein folding, signal transduction, and transcriptional activation. Here we demonstrate that a genetically encoded photoactivatable amino acid, p-benzoyl-l-phenylalanine, can be used to capture transient and/or low affinity binding partners in an in vivo setting. In this study, we focused on ensnaring the coactivator binding partners of the transcriptional activator VP16 in S. cerevisiae. The interactions between transcriptional activators and coactivators in eukaryotes are moderate in affinity and short-lived, and due in part to these characteristics, identification of the direct binding partners of activators in vivo has met with only limited success. We find through in vivo photo-cross-linking that VP16 contacts the Swi/Snf chromatin-remodeling complex through the ATPase Snf2(BRG1/BRM) and the subunit Snf5 with two distinct regions of the activation domain. An analogous experiment with Gal4 reveals that Snf2 is also a target of this activator. These results suggest that Snf2 may be a valuable target for small molecule probe discovery given the prominent role the Swi/Snf complex family plays in development and in disease. More significantly, the successful implementation of the in vivo cross-linking methodology in this setting demonstrates that it can be applied to the discovery and characterization of a broad range of transient and/or modest affinity protein-protein interactions.