Intelectin-2 is a broad-spectrum antimicrobial lectin

Domain: chemistry
Published: 2026-01-13
Editorial outcome: minor_revision

Abstract
Mammals regulate the localization, composition, and activity of their native microbiota at colonization sites. Lectins residing at these sites influence microbial populations, but their functional roles are often unclear. Intelectins are found in chordates at mucosal barriers, but their functions are not well characterized. In this study, we find that mouse intelectin-2 (mItln2) and human intelectin-2 (hItln2) engage and crosslink mucins via carbohydrate recognition. Moreover, both lectins recognize microbes within native microbial communities, including gram-positive and gram-negative isolates from the respiratory and gastrointestinal tracts. This ability to engage mammalian and microbial glycans arises from calcium-coordinated binding of carbohydrate residues within mucus and microbial surfaces. Microbes, but not human cells, bound by mItln2 or hItln2, suffer a loss of viability. These findings underscore the crucial antimicrobial role of mammalian intelectin-2 in mucosal defense, where it plays offensive (microbial killing) and defensive (mucus crosslinking) roles in regulating microbial colonization.

Introduction
Mucosal surfaces of the mammalian respiratory and gastrointestinal tracts are replete with microorganisms seeking to colonize these host tissues. Under homeostatic conditions, microbial communities flourish and contribute to host health. Simultaneously, exposure to opportunistic pathogens, allergens, and helminths can injure host tissues and lead to infection. Host tissues must therefore respond to an ever-changing landscape of immunological challenges. In response to microbial colonization, host cells secrete a suite of soluble factors that regulate the localization, composition, and activity of microbial communities. Among these factors are lectins, carbohydrate-binding proteins that bind glycans on host and microbial cells1–4. The intelectins, or X-type lectins, are secreted at mucosal surfaces and, though purported to play a role in host defense against microbes, aspects of their function are still unclear5–11. Human and mouse intelectin-1 (Itln1) are constitutively expressed in the intestines and exclusively recognize a set of microbial glycans12–16. Itln1 has been suggested to facilitate phagocytic uptake of microbes by neutrophils and regulate the localization of mucolytic microbes8,10,13. In addition to Itln1, humans and certain mouse strains possess a second intelectin gene, intelectin-2 (Itln2), which has poorly characterized ligand specificity and unclear biological function6,17–20. Mouse intelectin-2 (mItln2) has been reported to be upregulated during nematode infections. Human intelectin-2 (ITLN2, henceforth hItln2) is constitutively expressed and restricted to the small intestine. Moreover, in the small intestine of ileal Crohn’s disease (CD) patients, the expression of hItln2 is decreased while it is increased in the colonic tissue of patients with colonic CD and ulcerative colitis (UC). Given that intelectin-2 is implicated in inflammatory processes and disorders involving microbial dysbiosis, we investigated its ligand specificity and function15,21–26.

Herein, we report the characterization of mItln2 and hItln2. Using a novel mouse model with an intact intelectin locus, we found that mItln2 is selectively and potently induced by T-helper type 2 (Th2) stimuli. We show that mItln2 and hItln2 are indeed lectins, as they recognize select glycans with terminal β-D-galactopyranose residues, which are found on mammalian and microbial glycans. Regarding the host, these lectins cross-link mucins in a glycan-dependent manner. Regarding microbes, these lectins bind a range of gram-positive and gram-negative bacteria. Moreover, we found that mItln2 exerts direct microbicidal effects. HItln2 binding also limits microbial growth, in this case through agglutination, and its activity is intriguingly more potent at lower pH and salt conditions. Taken together, our findings reveal the roles of Itln2 in host defense—through mucin cross-linking and broad-spectrum antimicrobial activity—underscoring the multifaceted roles of intelectins in host–microbe interactions.

Results
MItln2 expression is induced by IL-4 and IL-13 in enteroids
In Th2 inflammation, microbial-, helminth-, and allergen-derived toxins and proteases compromise the integrity of host mucosal barriers27–29. Such damage can increase the risk of infection by opportunistic pathobionts and prompt the host to activate wound repair and defense pathways. There is evidence that mammalian intelectins can be induced during Th2-type immune responses, including during intestinal helminth infections and asthma, but many details are unclear6,7,17,20,23,24,30.

We used a mouse model to probe the regulation of intelectin expression during inflammation. The widely used strains for studying inflammation, such as C57BL/6 and its sub-strains (e.g., C57BL/10), encode a single intelectin gene, Itln1, resulting from a large 420-kb deletion across the intelectin locus9,17,18,31,32. However, most wild-derived and conventional laboratory mouse strains encode six intelectin paralogs: Itln1, Itln2, Itln3, Itln4, Itln5, and Itln618,31. To better understand intelectin regulation, we generated a congenic C57BL/6 mouse model (B6.C-Itln1-6) that encodes a complete Itln1-6 locus derived from the BALB/c strain18,33. Stimulation of enteroids from B6.C-Itln1-6 mice with type 2 cytokines (IL-4 and IL-13) resulted in a six-fold increase in total Itln expression compared to a two-fold increase in wild-type C57BL/6 enteroids (Fig. 1A, B). In tissue and unstimulated enteroids from B6.C-Itln1-6 mice, most (≥94%) of total intelectin mRNA was Itln1, with 2–5% being Itln2 (Fig. 1C, D). Following IL-4 and IL-13 treatment, the relative abundance of Itln2 increased to 30% of total transcript reads, indicating a 40-fold upregulation (Fig. 1D; Supplementary Table 1). This finding aligns with previous report that Itln2 is robustly induced in the small intestine of BALB/c mice infected with Nippostrongylus brasiliensis, Trichuris muris, and Trichinella spiralis, organisms that promote Th2 inflammation6,17,20. As Stat6 is critical for Th2 inflammation, we examined the promoter of mItln2. In the BALB/c mouse genome, the mItln2 promoter region (−2000 base pairs to −1 base pair upstream of transcription start site) has a putative Stat6-binding motif, further supporting our observation of Th2-dependent expression of mItln2 (Supplementary Fig. 1A)34. No other intelectins (Itln3-6) were detected in small intestinal tissue or enteroids at baseline or after type 2 cytokine stimulation. Type-1 associated cytokines were not evaluated in the enteroids as these cytokines, including interferon gamma, can affect the viability of goblet and Paneth cells35,36. Instead, we selected the type 17/22 inflammatory cytokine, IL-22, as a control because IL-22 can stimulate the production of antimicrobial proteins, including Reg family proteins37. In these assays, IL-22 showed little or no effect on mItln2 expression. These results establish that Th2-type cytokines selectively induce mItln2 (Fig. 1E). These data suggest a role for mItln2 distinct from that of other intelectins.

MItln2 recognizes select glycans with terminal β-D-galactopyranose residues
Mouse Itln2 shares greater than 80% sequence identity with the homologous protein Itln1 (Supplementary Fig. 1B). This similarity prompted us to analyze the structure and function of mItln2. To this end, we generated a recombinant form of mItln2 containing a StrepII tag that facilitated purification and detection (Supplementary Fig. 1C, D). We found that strep-mItln2 was glycosylated and properly folded with a melting temperature of approximately 53°C (Supplementary Fig. 1E–I; Supplementary Fig. 2A). Circular dichroism analysis further revealed that mItln2 exhibits a mixed alpha-helical and beta-sheet secondary structure, suggesting a comparable folded structure as Itln1 (Supplementary Fig. 1E). Although mItln2 lacks the two cysteines present in hItln1 that form a disulfide-linked homotrimer (Supplementary Fig. 1B)12,34, treatment of mItln2 with the bifunctional homo-crosslinker bis-sulfosuccinimidyl suberate gave rise to a laddered mixture of monomer, dimer, trimer, and hexamer species, suggesting non-covalent oligomerization of mItln2 (Supplementary Fig. 2B). Dynamic light scattering also showed that mItln2 occupies a variable distribution of oligomeric species in solution (Supplementary Fig. 2C). Similarly, the structural prediction of mItln2 trimer using AlphaFold 3 yielded highly confident structures in which the ligand-binding domain from each monomer is facing the same side, as in the previously resolved hItln1 structures (Supplementary Fig. 2d)2,38. Together, these data indicate that mItln2 is a stably folded, glycosylated lectin that assembles into non-covalent oligomers. Additionally, the observed laddered oligomerization is consistent with other lectins that form higher-order quaternary structures to achieve avid binding to their cognate ligands1,2.

Human and mouse Itln1 were previously shown to have identical binding sites and bind exclusively to microbial carbohydrates, including β-D-galactofuranose (β-Galf), the five-membered ring isomer of galactose12,14. Despite the high sequence similarities of mItln2 with mItln1 and hItln1, the ligand-binding domain of mItln2 differs in a single residue (W288A) (Fig. 2A; Supplementary Fig. 1B). The aromatic box formed by W288 and Y297 is crucial for Itln1’s specificity for microbial monosaccharides. The parsimonious binding site change between mItln1 and mItln2 suggests that the latter might exhibit different ligand specificities than mItln1 and hItln1, leading to differences in microbial binding13.

To assess whether mItln2 is a glycan-binding protein, we screened the recombinant protein on multiple mammalian and microbial glycan arrays to ensure a robust analysis (Supplementary Fig. 3A). Array hits and intensities cannot be directly compared because of differences in their ligand composition, surface-immobilization chemistries, and imaging instrumentation; however, all arrays revealed hits containing terminal galactopyranose residues. Microbial glycan analysis yielded hits including yeast and plant-like galactomannan (Davanat) and bacterial galactose-containing capsular polysaccharides (Fig. 2B, C; Supplementary Data 1)39. In addition, mItln2 bound mammalian glycans on the National Consortium for Functional Glycomics (NCFG) and commercial glycan arrays, including several oligosaccharides from N- and O-glycans displaying terminal α- or β-D-galactopyranose or 6-SO3-galactopyranose (Fig. 2B, C; Supplementary Fig. 3B, C; Supplementary Data 1). Moreover, the NCFG glycan array showed binding primarily to bi- and tri-antennary ligands with three or more repeating N-acetyllactosamine (LacNAc) units, which are composed of β-D-galactopyranose (β-Galp) and β-D-N-acetyl-glucosamine (β-GlcNAc). While the commercial mammalian glycan array also showed mItln2 binding to terminal Galp, there was greater hit diversity regarding the extent of branching, number of LacNAc repeats, and identity of terminal Galp-containing disaccharides (e.g., Galβ1-3GalNAc). Notably, the binding profile of mItln2 had no overlap with hItln1, indicating these related lectins had distinct specificity. We previously reported that hItln1, like mItln1, binds to acyclic vicinal (1,2)-diol moieties present on microbial but not mammalian glycans (Supplementary Fig. 3D)2,12.

A limitation of glycan array data is its lack of information on relative affinities or ranked binding preferences. The apparent binding signal intensities within an array may reflect differences in glycan density, presentation context, or other technical factors rather than true affinity differences. We therefore validated the glycan array results through complementary biochemical approaches. To quantify the array results and assess mItln2 minimal binding groups, we used an enzyme-linked lectin assay (ELLA). In this assay, biotinylated carbohydrates were immobilized on streptavidin-coated plates and increasing concentrations of mItln2 were added. Plates were washed to remove unbound lectin, and bound mItln2 was detected with anti-StrepII-HRP antibody (Supplementary Fig. 3E, F). Consistent with the array results, these experiments demonstrated that mItln2 binds β-N-acetyllactosamine (β-LacNAc), and β-6-SO3-N-acetyllactosamine (β-6SO3-LacNAc) (Supplementary Fig. 3G). Because β-LacNAc is a disaccharide composed of terminal β-Galp linked to β-GlcNAc, we also evaluated binding to individual monosaccharides to determine mItln2 specificity. ELLA results show that mItln2 does not bind β-GlcNAc or α-Galp and only recognizes β-Galp (Supplementary Fig. 3G). The lectin binding was dose-dependent, a hallmark of specific recognition. Additional experiments using biolayer interferometry (BLI) indicate that mItln2 selectively binds the pyranose form of galactose (β-Galp) but not the furanose form (β-Galf) recognized by Itln1 (Supplementary Fig. 3H–J). The apparent dissociation constant of mItln2 for β-Galp is approximately 1.5 μM (Fig. 2D; Supplementary Fig. 3K). Because mItln2 appears to accommodate both β-Galp and β-6SO3-Galp as ligands, we postulate that it uses calcium to coordinate binding to conserved hydroxyl groups on the pyranose ring of galactose, a feature shared by all glycan hits. Still, mItln2 does not bind every β-Galp-containing glycan on the array, indicating there may be additional subsites that contribute to affinity. The prevalence of bi- and tri-antennary ligands as top hits on the arrays also suggests that this kind of glycan may have favorable orientation or display for oligomeric mItln2 (Supplementary Fig. 2B, D). Notably, a single amino acid difference in the binding site of mItln2 allows this lectin to preferentially bind the pyranose form of galactose over the furanose form preferred by Itln1.

MItln2 cross-links mucin glycoproteins
During parasitic nematode infections, mucins are secreted by goblet cells into the intestinal and lung mucosa, where they can interact with newly expressed mItln217. Mucins are glycosylated with epitopes similar to those of the top mammalian glycan array hits for mItln2; therefore, we assayed mItln2 interactions with gastric and intestinal mucins. We first immobilized commercially available gastric mucin to nitrocellulose in a dot blot assay. Following incubation with mItln2, we observed robust binding to gastric mucus in the presence of calcium. EDTA addition abrogated binding, as expected, given that mItln2 and hItln1 are related members of the calcium-dependent X-type lectin family. These data indicate that mItln2 binds mucus by engaging mucin glycans (Supplementary Fig. 3L). To test glycan binding more directly, we added 10 mM galactose and found it could competitively displace this interaction, whereas 10 mM glucose could not (Supplementary Fig. 3M). These data demonstrate that mItln2 binds mucus in a glycan-dependent manner.

We next asked if we could characterize the binding profile of mItln2 using mucins isolated from tissues that support mItln2 expression. We tested mItln2 binding to MUC2, the predominant intestinal mucin in mammals; MUC5B, a salivary and bronchial mucin; and MUC5AC, a respiratory and gastrointestinal mucin that is induced during Th2 inflammation40. Purified porcine MUC2, MUC5AC, and MUC5B were immobilized to nitrocellulose, and assayed for mItln2 binding in the presence of calcium ions, EDTA, or 10 mM galactose. The lectin exhibited robust binding to all three mucins. These interactions were attenuated by the addition of EDTA or galactose competitor (Fig. 2E). These data suggest that mItln2 interacts with secreted mucins at its expression sites via recognition of Galp residues.

Certain lectins, such as galectins and trefoil factors, cross-link mucin to strengthen the mucus barrier41–43. In examining mucin-mItln2 interactions by confocal microscopy, we observed that fluorescently labeled MUC5AC co-localized in large aggregates with fluorescently labeled mItln2; this aggregation was not observed in the absence of the lectin (Fig. 2F). These observations suggest that mItln2 cross-links mucins. To examine this possibility directly, we utilized a kinetic spectroscopic assay to measure changes in light transmittance upon mixing lectin with mucin. Increasing amounts of mItln2 were titrated into solutions containing 0.01% MUC5AC, MUC5B, and MUC2 and evaluated for changes in percent transmission over time. We observed rapid and dose-dependent clustering of mucins by mItln2, which was abrogated by the addition of galactose (Fig. 2G; Supplementary Fig. 3N). Moreover, this activity was observed at concentrations similar to those previously reported for other mucus cross-linking lectins41. Using time-lapse confocal microscopy, we confirmed mucin cross-linking by mItln2; lectin–mucin complexes were observed to form and grow over time (Fig. 2H; Supplementary Fig. 3O). To test whether this cross-linking depends on carbohydrate binding, we used chemically defined mucin mimetic polymers displaying pendant β-lactose (a disaccharide with terminal β-Galp residue)44. As with mucins, mItln2 promoted robust polymer clustering (Fig. 2I), further indicating that mItln2 binds and cross-links mucins via carbohydrate recognition.

MItln2 is a microbicidal lectin
The epitope β-Galp is found widely across taxonomic classes of bacteria45,46. Therefore, we hypothesized that mItln2 would bind microbes. Flow cytometry and microscopy experiments indicate that mItln2 robustly bound mouse fecal bacteria, confirming that it interacts with microbes in native communities (Fig. 3A, B)13. In line with this finding, mItln2 bound numerous gram-positive and gram-negative isolates from native mouse fecal communities, including Limosilactobacillus reuteri, Lactobacillus murinus, Lactobacillus johnsonii, Bacillus paramycoides, Mucispirillum schaedleri and pathobionts, including Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis (Fig. 3C–E; Supplementary Fig. 4A–E). Moreover, mItln2 binding was calcium-dependent, an indication that it is glycan-mediated. Notably, mItln2 did not bind to all tested isolates, highlighting its specificity (Fig. 3F; Supplementary Fig. 4F–J). Together, these data demonstrate that mItln2 recognizes specific microbes that colonize tissues in which mItln2 is expressed.

We next evaluated the consequences of mItln2 binding to microbes. Analysis of mItln2-bound microbes by flow cytometry revealed that mItln2 treatment caused marked shifts in forward scatter (FSC) versus side scatter (SSC) plot, in a region associated with debris (Fig. 4A, B; Supplementary Fig. 5A–E). This effect depended on the lectin concentration, with higher mItln2 concentrations resulting in increased events in the debris region for both gram-negative and gram-positive binders. This increase in debris was not observed in non-binding isolates (Supplementary Fig. 5F–K) nor when EDTA was added (Supplementary Fig. 5B, C), suggesting that the effect is glycan-dependent. Moreover, treatment with equimolar amounts of hItln1 did not show this phenotype (Fig. 4A, B). Similar alterations in FSC vs. SSC profiles of microbes have been previously reported for other antimicrobial proteins, such as lysozyme, suggesting that mItln2 functions as an antimicrobial lectin47. Additionally, microscopy evaluation of mItln2-treated L. reuteri showed a loss of cell integrity and accumulation of ghost-like cells (empty bacterial cell envelopes) over time (Fig. 4C, D; Supplementary Movies 1 and 2). Together, these data suggest that mItln2 compromises the cellular integrity of bound bacteria and may influence cell viability.

To assess whether mItln2 has microbicidal activity, we used a colony-forming unit (CFU) assay. Bacterial isolates were grown to mid-log phase and treated with increasing concentrations of mItln2. Consistent with the flow cytometry and microscopy data, a significant reduction in the viability of L. reuteri and B. paramycoides occurred upon treatment with low micromolar concentrations of mItln2 (Fig. 4E; Supplementary Fig. 5L). These concentrations are in line with other antimicrobial proteins shown to kill microbes48,49. The cell-killing effect of mItln2 was confirmed using a plate reader-based growth recovery assay, which showed a significant delay in the growth recovery for mItln2-binding Lactobacillus isolates following mItln2 treatment (Supplementary Fig. 6A, B). Delayed growth recovery was not observed when non-binding isolates were exposed to mItln2. The effect of mItln2 was distinct from hItln1. Specifically, isolates recognized by hItln1 had no viability loss when treated with hItln1. Thus, mItln2 possesses microbicidal activity against specific gram-positive species, an effector function lacking in the related lectin hItln1.

To test the activity of mItln2 on gram-negative pathogenic bacteria, we used K. pneumoniae, a common opportunistic pathogen. We observed robust mItln2 binding and a shift in the FSC vs SSC plot for K. pneumoniae (Fig. 3E; Supplementary Fig. 5D). Furthermore, including mItln2 in the culture media led to dose-dependent growth inhibition of K. pneumoniae, with a minimum inhibitory concentration of 0.3 μM (Fig. 4F; Supplementary Fig. 5M). These data suggest a mechanistic rationale for the finding that Th2 inflammation in BALB/c mice is protective against K. pneumoniae infection50. Collectively, the results indicate that mItln2 is a broad-spectrum antimicrobial protein that can target opportunistic pathogens at mucosal surfaces.

MItln2 lyses bacterial cells
Other mammalian lectins have been implicated in microbial membrane perturbation or pore formation, processes that can kill cells and release their intracellular contents48,49. Given that mItln2 alters bacterial morphology and reduces viability, we postulated that mItln2 acts as a cytolytic agent by disrupting microbial membranes. In support of this hypothesis, we observed loss of DNA signal in bacteria stained with DNA intercalating dye (SYTO BC) following mItln2 treatment (Supplementary Fig. 7A; Supplementary Movie 3). We also found an increased uptake of propidium iodide, a DNA dye that cannot penetrate intact membranes (Supplementary Fig. 7B).

We employed a liposome disruption assay to better understand how mItln2 exerts its microbicidal activity (Supplementary Fig. 7C). In this approach, we generated 200 nm diameter liposomes composed of palmitoyl-oleoyl-phosphatidylcholine (POPC), 10 mol% cholesterol, and 10 mol% 1,2-dioleoyl-sn-glycero-3-phosphethanolamine-N-dibenzocyclooctyl (18:1 DBCO PE), as the latter has a functional handle to append specific glycans. Liposomes were functionalized with a β-lactose derivative (a disaccharide with a terminal β-Galp residue) and treated with increasing concentrations of lectins51. Exposure of lactose-functionalized liposomes to mItln2 led to a concentration-dependent increase in absorbance at 405 nm, indicative of membrane disruption (Fig. 4G). This process was glycan-dependent, as adding mItln2 to liposomes lacking a glycan ligand afforded no change in absorbance. Confocal microscopy further confirmed the glycan-dependent ability of mItln2 to disintegrate the lactose-functionalized liposomes encapsulating Texas red-dextran dye (Fig. 4H). Moreover, although mItln2 bound mammalian cells, propidium iodide uptake assays demonstrate that it did not compromise cellular membrane integrity, indicating that mItln2 specifically diminishes microbial viability (Supplementary Fig. 7D, E).

Unlike other cytolytic proteins reported, the disruption of liposomes by mItln2 depended on glycan binding and occurred at physiological pH (7.4) and osmolarity (150 mM NaCl). The liposome disruption and cell-killing activity did not require an acidic pH or low salt concentration (<50 mM). These findings reveal that the powerful cell-killing ability of mItln2 synergizes with its ability to bind and cluster mucins. Thus, mItln2 has dual roles during Th2 inflammation: it functions by cross-linking host mucins and by binding and killing microbes.

Human intelectin-2 (hItln2) is a galactose-binding antimicrobial lectin
Human intelectin-2 (hItln2) is constitutively expressed by Paneth cells in the small intestine and is detectable in the lungs19,52. A recent study demonstrated that hItln2 expression is even higher than hItln1 in healthy small intestinal tissue and that aberrant expression of hItln2 is observed in both the ileum and colon of CD patients19. While Th2 stimuli have been shown to upregulate hItln1 expression in the lungs, Th2 inflammation-dependent expression of hItln2 has not been directly demonstrated24. Our analysis of the promoter region (−2000 base pairs to −1 base pair upstream of transcription start site) of hItln2 indicated potential STAT6-binding motif; however, further experimental validation is required to confirm Th2-dependent upregulation of hItln2 (Supplementary Fig. 8A). Nevertheless, consistent with its constitutive intestinal secretion, hItln2 is readily detected in human fecal samples (Supplementary Fig. 8B). However, as with mItln2, the ligand specificity and biological function of hItln2 have not been clear.

To characterize hItln2, we expressed and purified recombinant StrepII-tagged hItln2. The production of recombinant hItln2 required optimization. We made two changes: an introduction of a glycosylation site (A177S) that is conserved in hItln1 and mItln2 and the removal of an unpaired cysteine near the C-terminus (C311G) that does not participate in trimerization and binding data suggests is amenable to substitution (Supplementary Fig. 8C)53. These modifications improved solubility and reduced aggregation, yielding soluble, stably folded protein with a melting temperature of approximately 54°C (Supplementary Fig. 8D). HItln2 formed covalent trimers and higher-order oligomers, as observed for native hItln2 in small intestinal tissue (Supplementary Fig. 8E, F)19. Consistent with our experimental data, the AlphaFold 3-predicted structure of hItln2 is a trimer with a similar ligand binding domains as hItln1 and conserved intermolecular disulfide linkages between C42 and C60 residues of the neighboring monomers (Supplementary Fig. 8G).

Like mItln2, the ligand binding site of hItln2 lacks the aromatic box present in Itln1 (Fig. 5A; Supplementary Fig. 1B). In hItln2, a serine at position 309 replaces the tyrosine at the corresponding site in hItln1. We reasoned that this more open binding pocket might accommodate pyranose sugars, as observed with mItln2. We next investigated if hItln2 and mItln2 share glycan targets. Using BLI to evaluate the binding of hItln2 with immobilized mono- and di-saccharides, we observed that hItln2 indeed binds mItln2 ligands (β-Galp and β-6SO3-LacNAc) but not the Itln1 ligand (β-Galf) (Fig. 5B–D). These data are supported by a recent report indicating a monomeric hItln2 variant lacking disulfides binds to Galp53. The apparent dissociation constant of hItln2 for β-Galp was approximately 3.9 μM (Supplementary Fig. 8H, I). Thus, mItln2 and hItln2 share common ligands. Moreover, the microbial glycan array analysis for hItln2 yielded hits containing Galp, but also primary amine-containing substituents (e.g., phosphoethanolamine (PEt)), suggesting that terminal amine-containing substituents may serve as an additional microbial ligand for hItln2 (Fig. 5E, F; Supplementary Data 2). These overlapping yet distinct ligand recognition properties could be due to other differences in the binding site residues of mItln2 and hItln2 (mItln2/hItln2: E274/Q286, A288/W300, and Y297/S309).

Because mItln2 and hItln2 bind to β-Galp, we reasoned that they might interact with similar physiological substrates. Supporting this hypothesis, we found that hItln2 binds intestinal mucins, MUC2, and MUC5AC in a calcium ion-dependent manner (Fig. 5G, H). Spectroscopic analysis of hItln2–mucin interactions showed mucin cross-linking by hItln2, but hItln2 activity was more challenging to detect via microscopy (Supplementary Fig. 8J–L). The minor differences in mucin selectivity observed between mItln2 and hItln2 may be due to differences in mucin glycosylation patterns (density, modification, and accessibility) or the influence of adjacent glycans on lectin selectivity. For example, porcine MUC2 displays glycans reported to be shorter and less extended than in MUC5AC and MUC5B54. Moreover, mItln2’s non-covalent oligomerization state (monomer, dimer, trimer, hexamer) as well as its pronounced binding to extended, branched LacNAc array ligands compared to disulfide-bonded hItln2 may also contribute to the observed differences.

Like mItln2, hItln2 also recognized microbes within a human fecal sample (Fig. 6A, B) and a range of gram-positive and gram-negative bacterial isolates that includes the opportunistic pathogens Escherichia coli and Enterococcus faecalis. These cell-binding properties were inhibited by the chelator EDTA (Supplementary Fig. 9A–F), indicating that hItln2 interacts with glycans through its calcium ion. Unlike mItln2, hItln2-treated microbes formed large cellular aggregates, which could be disrupted with EDTA, findings consistent with glycan-dependent agglutination (Supplementary Fig. 9B, D).

We investigated if hItln2 functions as an antimicrobial protein based on several key characteristics: (1) abundant expression at sites of microbial colonization; (2) secretion from canonically anti-microbial Paneth cells; and (3) cationic nature (isoelectric point of 8.3), and (4) lengthened N-terminal sequence (relative to hItln1) that is predicted to form an alpha-helix (Supplementary Fig. 9G). We first evaluated if hItln2 could bind the pathobiont Staphylococcus aureus, a common gram-positive opportunistic pathogen. Increasing evidence suggests that S. aureus can colonize the human gut and, under the right conditions, can disseminate to other host tissues via translocation across the intestinal mucosa and epithelium55. We reasoned that hItln2 might encounter this organism in the gastrointestinal tract. Consistent with this hypothesis, hItln2 showed robust binding to and pronounced agglutination of methicillin-resistant S. aureus (MRSA) by flow cytometry and microscopy (Fig. 5C, D, Supplementary Fig. 9H).

To evaluate the antimicrobial properties of hItln2, we performed CFU assays on hItln2-treated S. aureus. To this end, MRSA was grown to mid-log phase and then exposed to increasing amounts of hItln2. Cells were then vigorously mixed, serially diluted in assay buffer with thorough mixing between dilutions, plated on growth substrate, and left overnight at 37°C before counting colonies. As we found for mItln2, hItln2 treatment decreased CFU counts for MRSA, indicating reduced bacterial viability (Fig. 6E). This result was also obtained after treatment of other gram-positive bacteria recognized by hItln2 (Supplementary Fig. 9I). HItln2 also bound and inhibited the growth of the gram-negative pathobiont K. pneumoniae in a dose-dependent manner, with minimum inhibitory concentration of 0.3 μM (Fig. 6F, G, Supplementary Fig. 9J). Together, these data suggest that hItln2 inhibits microbial viability and growth through the antimicrobial effector function of agglutination. Like mItln2, hItln2 did not compromise the membrane integrity of mammalian cells (Supplementary Fig. 10A, B).

Expression of hItln2 in healthy subjects is primarily restricted to the small intestine19. Our initial characterization of hItln2 activity was performed under ileum-like conditions: neutral pH and physiological ionic strength. However, hItln2 is present in the colons of patients with colonic CD and UC due to Paneth cell metaplasia19. The colonic environment in CD and UC patients can range from acidic to neutral pH (pH 4–7) and has altered ionic strength owing to aberrations in electrolyte uptake and secretion during inflammation56–58. Moreover, other small intestinal-derived antimicrobial proteins are reported to be active only under acidic pH and low ionic strength48,59–61. To address these variables, we investigated hItln2 activity at low pH and ionic strength.

Even under these stress conditions, hItln2 remained well-folded and retained binding to β-Galp and β-6SO3-LacNAc (Fig. 7A; Supplementary Fig. 11A, B). We also confirmed that hItln2 bound to mucins and microbes under these conditions (Fig. 7B–D; Supplementary Fig. 11C–E); however, EDTA addition did not abrogate these interactions, suggesting a shift to an alternate binding mode. Moreover, under these conditions, hItln2 mediated rapid and robust formation of mucin and microbial aggregates (Fig. 7E, F; Supplementary Fig. 11F, G). Similarly, CFU analysis showed a dramatic reduction in the growth of the pathobionts, S. aureus, B. paramycoides, and E. coli, confirming that the agglutination-mediated antimicrobial function of hItln2 was enhanced under conditions that represent intestinal inflammation (Fig. 7G, H; Supplementary Fig. 11H–J).

Methods
The research in this paper complies with all ethical regulations set by the Institutional Review Board (MIT) and the Institutional Animal Care and Use Committee (IACUC, UC-Davis). Human stool samples used in the flow cytometry test of human intelectin-2 were obtained under a protocol approved by the Massachusetts Institute of Technology [Institutional Review Board (IRB) protocol ID no. 1510271631]. The participants provided informed consent, and all experiments complied with the review board regulations. Human stool samples were collected for a previously published study13. Banked stool sample from the aforementioned study was used in our study. Patient samples were selected based on diagnosis regardless of sex and gender. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of California, Davis.

Congenic C57BL/6NTac.BALB/cAnNTac-Itln1:6 (B6.C-Itln1-6) mouse model
Male BALB/cAnNTac mice, encoding a full Itln1-6 locus18, were crossed with female C57BL/6NTac mice that encode a single intelectin gene, Itln1 (parental strains obtained from Taconic Biosciences, Germantown, NY). Male offspring, positive for the Itln1-6 locus (F1/N1: heterozygous), were crossed with pure C57BL/6NTac female mice for an additional nine generations (N10: ~99.9% C57BL/6NTac background). Experimental tissues and enteroids were derived from 10-week-old heterozygous mice. Animals were humanely euthanized under deep anesthesia with ketamine/xylazine (100/10 mg/kg).

Genotyping
DNA was isolated from tissue samples by HotSHOT method66. Briefly, samples were incubated in alkaline lysis buffer (25 mM NaOH, 0.2 mM EDTA, pH 12.0) overnight in a water bath at 65°C. Following digestion, an equivalent volume of neutralization buffer (40 mM Tris-HCl, pH 5.0) was added to the digest solution. The Itln1-6 haplotype was identified using primers designed to amplify both Itln1 and Itln6, where the Itln1 allele (i.e., wild-type C57BL/6N; deletion) generates a 305 bp product, and the Itln6-containing allele generates an 88 bp product. Genotyping primers: F: 5’-TATTCCTGTCTCAGCTCCTAG-3’, and R: 5’-GTCAGGTAAAKCCAGAAGG-3’ (K=G or T). Polymerase chain reaction (PCR) was performed using an Applied Biosystems thermocycler (Waltham, MA). The PCR products were amplified using Taq DNA polymerase (New England Biolabs, Cat# M0273S). Thermocycler conditions: one cycle: 95°C (30 s), 35 cycles: (95°C [30 s], 62°C [30 s], and 70°C [30 s]), and additional extension for 5 min at 70°C.

Enteroid culture
Small intestinal crypts were isolated as previously described with minor modification67. Briefly, 10 cm of distal small intestine was flushed with Dulbecco’s PBS (DPBS), dissected longitudinally, scraped with a glass coverslip to remove villi, and minced into ~1 cm pieces. Intestinal fragments were incubated at 4°C under gentle rocking (60 rpm) for 45 min in ice-chilled 2 mM EDTA (DPBS) dissociation solution. The intestinal pieces were allowed to settle at the bottom of the conical tube by gravity, and the dissociation solution was decanted. Following a second 45 min incubation, intestinal pieces were vigorously shaken for ~3 min in dissociation solution to release intestinal crypts. The resulting crypt-containing suspension was passed through a 70 μm filter to exclude villi fragments, pelleted, and enumerated. The crypts were plated on 24-well Costar® culture dishes (StemCell Technologies, Cat# 38017) as 50 μL suspensions of 50% v/v growth factor reduced matrigel (Corning, Cat# 356231) in murine Intesticult™ Organoid Growth Media (StemCell Technologies, Cat# 06005), supplemented with 50 µg/mL gentamycin (Sigma-Aldrich, Cat# G1397). Enteroids were passaged every 12 days following standard procedures (StemCell Technologies, document# 28223), and complete media was replenished every three days. For cytokine treatment, enteroids were cultured for nine days prior to 72 h treatment with recombinant (PeproTech, Cranbury, NJ) murine IL-4 (Cat# 214-14, 20 ng/mL), IL-13 (Cat# 210-13, 10 ng/mL), or IL-22 (Cat# 210-22; 20 ng/mL).

RNA extraction, cDNA synthesis, and RT-qPCR
RNA extraction from intestinal tissue was performed using the guanidine thiocyanate/cesium chloride gradient method, as previously reported18,68. For enteroids, media was removed from the 24-well culture plates following cytokine treatment, and 500 μL of TRIzol™ reagent (Thermo Fisher Scientific, Cat# 15596026) was added directly to the Matrigel domes. RNA was extracted as outlined by the manufacturer with minor modifications. Following RNA precipitation and decanting of isopropyl alcohol, resulting pellets were resuspended in 250 mL of 70% molecular grade ETOH (30% v/v DEPC H2O) with 10 mL of 3 M sodium acetate, and incubated overnight at −80°C. Thereafter, the RNA was again pelleted by centrifugation, washed with 80% ETOH, and resuspended in DEPC H2O to determine concentration by ultraviolet absorbance (Nanodrop™) spectroscopy. cDNA synthesis was performed as previously reported, using 1–3 mg of isolated RNA with the SuperScript™ III First-Strand Synthesis kit (Thermo Fisher Scientific, Cat# 18080051), followed by column purification using the QIAquick PCR purification kit (Qiagen, Cat# 28104)18. RT-qPCR was performed using a Roche Lightcycler® 2.0 under conditions parallel to those previously reported18,68. Primers: Actb forward 5’-GGCTGTATTCCCCTCCATCG-3’, Actb reverse 5’-CCAGTTGGTAACAATGCCATGT-3’. Itln1 forward 5’-ACCGCACCTTCACTGGCTTC-3’, Itln1 reverse 5’-CCAACTTTCCTTCTCCGTATTTC-3’. Itln-common forward 5’-GCCTCAGCAGAGAAAGGTTCC-3’, Itln-common reverse 5’-GAAGGTCTGGTAGATGACACCATTC-3’. Clca1 forward 5’-CCTGACTCCTGACTTCTTAGC-3’, Clca1 reverse 5’-TGAACACCTCACTGCTTGG-3’. Reg3g forward 5’-CCTCAGGACATCTTGTGTC-3’, Reg3g reverse 5’-TCCACCTCTGTTGGGTTCA-3’18,68.

Next-generation sequencing of Itln paralogs
A subset of PCR reactions, generated by RT-qPCR using the Itln-common primers, were pooled (B6.C-Itln1-6: distal small intestine; n=3 animals, and enteroids; n=6 independent samples/treatment), column purified (QIAquick PCR purification kit) and submitted for Illumina sequencing (GENEWIZ Amplicon-EZ, Azenta Life Sciences). Sequence data (i.e., unique reads) were binned and then aligned to the reference mRNA for the individual Itlns (Itln1, Itln2, Itln3, Itln4, Itln5, and Itln6) retrieved from the BAC contig of the 129S7 strain (GeneBank #HM370554), which like BALB/c (parental donor strain for the B6.C-Itln1-6 congenic mouse model), encodes a full Itln1-6 locus18. Percentage total and individual Itln1-6 read count (Fig. 1C, D, Supplementary Table 1) was generated following two selective filters of the raw data: (1) 283 nt (i.e., expected PCR product size), and (2) the minimum recorded unique read count was set at 2000, representing ≤0.2% of total reads (median total read count of experimental groups = 1.23 million; Supplementary Table 1).

Cloning of StrepII-tagged mouse intelectin-2 and StrepII-tagged human intelectin-2
Forward primer (5’GCGTTTAAACTTAAGCTTCACCATGACCCAACTGGGCTTCCTG3’) and reverse primer (5’CCACCACACTGGACTAGTGGATCCTCATTAGCGATAAAACAGAAGCACAGC3’) were used to amplify strep-mItln2 (Accession number AAO60215, https://www.uniprot.org/uniprotkb/Q80ZA0/entry) from pFastBac-Strep-mItln2 vector (Kiessling lab, unpublished). Forward primer (5’-AGTTAAGCTTCACCATGCTGTCCATGCTGAGGACAATGACC-3’) and Reverse primer (GCTCGGATCCTCATTATCTATAGAACAAGAGTACAGCCGCCTCCGTT) were used to amplify hItln2 cDNA (AY358905) (Kiessling Group, unpublished). The resulting PCR products were inserted into pcDNA4 using Gibson ligation. For hItln2, StrepII-tag was inserted using two-step Quikchange mutagenesis with the following primers: (5’GCAGCAGCCTCTTCTtggagccatccgcagtttgaaaagtcttctCTTGAGATGCTCTCG3’) and (5’CGAGAGCATCTCAAGagaagacttttcaaactgcggatggctccaAGAAGAGGCTGCTGC3’). Plasmid sequences were verified using Sanger sequencing (Quintara Biosciences) with pCMV forward (5’CGCAAATGGGCGGTAGGCGTG3’) and BGH reverse primers (5’CTAGAAGGCACAGTCGAGG3’).

Recombinant protein expression and purification
Recombinant hItln1, mItln2, and hItln2 with N-terminal Strep-tag II were expressed by transient transfection of suspension-adapted HEK293 cells, following established procedures12. HEK293 cells were maintained at a density of 1×106 cells/mL in DMEM medium (Thermo Fisher, Cat# 11995) supplemented with 10% heat-inactivated FBS, 1x penicillin-streptomycin, 1x L-glutamine, and 1x non-essential amino acids in spinner flasks at 37°C, 5% CO2. Cells were passaged every 2–3 days, with the addition of Pluronic-F68 (Thermo Fisher). Transfection was carried out at 1×106 cells/mL in the growth medium using Lipofectamine 2000 (Thermo Fisher, Cat# 11668030) as per the manufacturer’s protocol. Six hours post-transfection, the culture medium was replaced with FreeStyle F17 expression medium (Thermo Fisher, Cat# A1383501), supplemented with 1x penicillin-streptomycin, 1x L-glutamine, 1x non-essential amino acids. Transfected cells were cultured for up to 3 days, and the expression medium was harvested by centrifugation and sterile filtration.

To verify protein expression, expression media was combined with 6x Laemmli buffer containing dithiothreitol, boiled at 95°C for 10 min, and run on 4–15% TGX gel (Bio Rad). Proteins were transferred to PVDF membrane, blocked in 5% milk in TBST (Tris-Buffered Saline with Tween-20), and blotted for the presence of mItln2 using 1:5000 rabbit anti-Itln2 (Proteintech, Cat# 11770-1-AP) and goat-anti-rabbit HRP (1:10,000, Jackson, Cat# AB_2313567). hItln2 was detected on protein expression blots using hItln2 specific antibody (Bevins Lab, 1:5000) and goat-anti-rabbit HRP (1:10,000, Jackson, Cat# AB_2313567)19.

Purification of StrepII-tagged lectins followed established procedures12. The harvested expression medium was subjected to avidin (IBA, Cat# 2-0204-015) treatment, resulting in a final concentration of 0.084 mg/mL, as per the IBA protocol. Protein was captured on Strep-Tactin Superflow High-Capacity resin (IBA, Cat# 2-1208-002), which was pre-equilibrated with HEPES/Ca buffer (20 mM HEPES, 10 mM CaCl2, 150 mM NaCl, pH 7.4). Subsequently, the resin was washed twice with HEPES/EDTA buffer (20 mM HEPES, 1 mM EDTA, 150 mM NaCl, pH 7.4). The StrepII-tagged lectins were then eluted with 5 mM d-desthiobiotin (Sigma-Aldrich) in HEPES/EDTA buffer and concentrated using 10,000-molecular weight cutoff (MWCO) Vivaspin 6 centrifugal filter (Sartorius).

All proteins underwent buffer exchange to HEPES/EDTA for storage except for StrepII-hItln2 (UC/CD conditions). Purification of hItln2 in low pH and low salt (UC/CD condition) was identical, except that the purified protein was buffer exchanged into PIPES/EDTA buffer (10 mM PIPES, 25 mM NaCl, 1 mM EDTA, pH 5.5). Protein concentrations were determined by absorbance at 280 nM, with extinction coefficients and molecular weights calculated for the monomeric form of each protein (without the signal peptide) using the Protparam tool. StrepII-tagged hItln1 had ε=79,925 cm−1 m−1 and an estimated molecular mass of 34,224 Da (monomer). StrepII-tagged mItln2 had ε=69,830 cm−1 m−1 and an estimated molecular mass of 34,212 Da. StrepII-tagged hItln2 had ε=69,955 cm−1 m−1 and an estimated molecular mass of 34,436 Da (monomer).

Detection of hItln2 in human fecal samples
Human fecal samples from three independent donors were kindly provided by the Alm lab (MIT)13. Fecal samples were supplied as homogenates in glycerol and stored at −80°C. To evaluate the secretion of hItln2 in the intestinal lumen, human fecal homogenates were scraped from frozen glycerol stock and thawed on ice. The homogenate was centrifuged at 100×g for 1 min to pellet fibrous material. The supernatant was transferred to a new tube and pelleted at 3000×g, 5 min. The pellet was washed twice with PBS, and then masses of pellets were measured. Pellets were suspended in PBS to achieve a concentration of 40 mg/mL. 20 mL of each sample was mixed with 6x Laemmli buffer containing DTT (Dithiothreitol), boiled at 95°C, and run on 4–15% TGX Stain-Free gel (Bio-Rad). Total protein was visualized using stain-free settings on ChemiDoc XP (BioRad). Proteins were transferred to PVDF, blocked in 5% milk in TBST, and blotted for hItln2 protein using an anti-hItln2 antibody (Bevins Group, 1:5000) and Goat-anti-rabbit HRP (1:10,000, Jackson, Cat# AB_2313567)19.

Treatment of mItln2 with PNGase F
To examine N-glycosylation, 20 μg of recombinant mItln2 was subjected to PNGase F (NEB, Cat# P0704S) treatment according to the manufacturer’s recommendations. Following glycosidase treatment, samples were mixed with 6x Laemmli buffer containing dithiothreitol (DTT), boiled at 95°C, and run on 4–15% TGX stain-free gel (Bio-Rad) with molecular weight standards (Bio-Rad). Gel was imaged on ChemiDoc MP Imaging system using stain-free imaging settings.

Circular dichroism (CD) analysis
Recombinant mItln2 was purified for CD analysis as described above, except all washes, elution, and buffer exchanges were performed with PBS buffer. CD spectra from 180 nm to 260 nm with 1 nm wavelength steps were collected on a Jasco H-1500 (Jasco Inc) at 25°C. Measurements were conducted with 500 μg/mL of mItln2 (in PBS buffer) using a 1 mm cuvette, with PBS buffer serving as the blank. Averaging time was 5 s, settling time was 0.33 s, and readings were taken in triplicate. Thermal denaturation steps were run from 25°C to 95°C with 5°C temperature steps. Scans were averaged, corrected by subtracting the blank, and plotted in GraphPad Prism to calculate melting temperature and visualize spectra. The calculated mean molar ellipticity (θ) is plotted as a function of wavelength (nm) for the protein.

Differential scanning fluorometry (DSF) and dynamic light scattering (DLS)
Particle light scattering and change in intrinsic fluorescence intensity of recombinant lectins at 330 nm and 350 nM in response to temperature changes were measured using a NanoTemper Prometheus NT.48 instrument. Capillaries were filled with ~10 μL of recombinant lectins (in HEPES/EDTA buffer or PIPES/EDTA buffer), placed into the sample holder, and the temperature was increased from 25°C to 90°C. Typical lectin stock concentrations for testing were 5–20 μM. The fluorescence intensity ratio at 350 nm and 330 nm was plotted as a function of temperature, and its first derivative was calculated using the manufacturer’s software. The melting temperatures were calculated as the inflection point of the ratio curve. DLS was measured at 25°C prior to melting temperature analysis. The hydrodynamic radii were determined using buffer-only capillaries as a baseline. The DLS and DSF experiments were conducted using two replicates for each sample.

Chemical cross-linking of proteins
For mItln2 cross-linking, 1 mg/mL of mItln2 (in HEPES/EDTA buffer) was combined with BS3 (Thermo Fisher, Cat# 21580) cross-linker in HEPES/EDTA buffer to achieve different final concentrations (0.1 mM, 0.25 mM, 0.5 mM, 1.25 mM, 2.5 mM, and 5 mM). Cross-linking was performed at RT for 30 min. Subsequently, protein mixtures were denatured by adding an SDS loading buffer with DTT. Samples were then heated at 95°C for 5 min, separated by SDS-PAGE, and imaged with ChemiDoc MP Imaging system using SYPRO Ruby protein stain or TGX Stain Free technology.

Enzyme-linked lectin assay (ELLA)
To assay mItln2 binding to different carbohydrates, a 96-well MaxiSorp ELISA plate (Thermo Fisher, Cat# 442404) was coated with 0.5 μg of streptavidin (Agilent, Cat# SA26) in PBS per well. Subsequently, the wells were loaded with 5 μM of biotinylated carbohydrates (in PBS) for 1 h at RT. After blocking with 5% BSA in HEPES/Ca buffer, the plate was incubated with various concentrations of recombinant lectins in HEPES/Ca/BSA/T buffer (20 mM HEPES, 10 mM CaCl2, 150 mM NaCl, 0.1% BSA, 0.1% Tween-20) for 2 h at RT. Following washes with the HEPES/Ca/BSA/T buffer, the wells were treated with anti-StrepMAB-Classic HRP conjugate antibody (1:10,000, IBA, Cat# 2-1509-001) in HEPES/Ca/BSA/T buffer for 2 h at RT. Then, the plates were washed with HEPES/Ca/BSA/T buffer, and the bound StrepII-tagged lectins were detected colorimetrically using 1-Step Ultra TMB-ELISA (Thermo Fisher). The reaction was quenched by adding an equal volume of 2 M sulfuric acid. Plates were read at 450 nm on Molecular Devices SpectraMax 5. Data were analyzed using GraphPad Prism.

Mucin dot blot
Purified porcine MUC2, MUC5AC, and MUC5B were generously provided by the Ribbeck Lab at MIT. Partially purified gastric mucus was purchased from Sigma (Cat# M1778). Mucins were hydrated overnight in deionized water, 4°C, with rotation to achieve a stock concentration of 10 mg/mL. Mucins and gastric mucus were serially diluted in 20 mM HEPES (pH 7.4) and 150 mM NaCl buffer to generate working stocks for downstream assays. For individual mucins, 1 μg of hydrated mucins were spotted onto nitrocellulose and allowed to dry. After blocking with 5% BSA in TBS-T for 1 h at RT, 0.5–1 μM recombinant lectins in HEPES/Ca/BSA/T buffer or HEPES/EDTA/BSA/T buffer (20 mM HEPES, 1 mM EDTA, 150 mM NaCl, 0.1% BSA, 0.1% Tween-20) were applied to blot overnight at 4°C. To test the glycan specificity of lectin–mucin interactions, the blots were incubated with the lectins in combination with 10 mM galactose in HEPES/Ca/BSA/T buffer. For UC/CD conditions, the blot was incubated with hItln2 in PIPES/Ca/BSA/T buffer (10 mM PIPES, pH 5.5, 25 mM NaCl, 10 mM CaCl2, 0.1% BSA, 0.1% Tween-20) or PIPES/EDTA/BSA/T buffer (10 mM PIPES, pH 5.5, 25 mM NaCl, 1 mM EDTA, 0.1% BSA, 0.1% Tween-20). Blots were washed in TBS-T and treated with anti-StrepMAB-Classic HRP conjugate antibody (1:10,000, IBA, Cat# 2-1509-001) in the appropriate buffer. Subsequently, the blot was washed three times with TBS-T and developed in the ChemiDoc MP Imaging system using ECL Prime reagent (Amersham).

For gastric mucus binding, serial dilutions of mucus were spotted on nitrocellulose and blocked as described above. Membranes were incubated with 0.5 μM strepII-mItln2 in HEPES/Ca/BSA/T alone or in combination with 10 mM glucose or 10 mM galactose. Blots were incubated for 1 h at RT or overnight at 4°C, processed as described above, and imaged using autoradiography film (Cole-Palmer) and developing reagents (Kodak).

Mucin and glycopolymer agglutination assay
To test the agglutination effect of mItln2, we adapted an assay from Järvå et al.41. 1% (w/v) mucins or glycopolymer were first diluted to 0.35% (w/v) in HEPES/Ca or PIPES/Ca2+ buffer. 1 µL mucin or glycopolymer was aliquoted into each well of a 384-well plate (black/clear bottom). Using a multichannel pipette, 34 µL of increasing concentrations of Itln2 in HEPES/Ca or PIPES/Ca buffer or buffer alone was added simultaneously to wells and briefly mixed to achieve a final concentration of 0.01% (w/v) mucin. The plate was immediately monitored for changes in absorbance at 405 nm (A405 nm) using Molecular Devices SpectraMax 5 via a kinetic assay, taking reads every 10 s for 30 min at RT. Triplicates were averaged and plotted in GraphPad Prism. Data is displayed as changes in A405 nm as a function of time.

Imaging of lectin-mediated mucin agglutination
Purified porcine MUC2, MUC5AC, and MUC5B were hydrated in deionized water overnight at 4°C, with rotation to prepare stock concentration of 10 mg/mL. Mucins were labeled with Alexa Fluor 568 NHS ester (Thermo Fisher, Cat# A20006) as per the manufacturer’s recommendations. Labeled mucins were pelleted at 10,000×g for 10 min and washed thrice with excess PBS to remove unreacted fluorophore. Mucins were resuspended to a working concentration of 1% (w/v) in HEPES/Ca or PIPES/Ca buffer. Mucins were plated in a lysine-coated 96-well imaging plate, which was followed by the addition of solutions containing 5 µM lectin, StrepMAB-Classic DY649 (IBA, Cat# 2-1569-050, 1:250) in HEPES/Ca/BSA/T or PIPES/Ca/BSA/T. Mucins treated with only buffers were used as a control. The final mucin concentration in the assay was 0.01% (w/v). Following treatment with lectins, the plate was centrifuged briefly at 1000×g for 1 min and immediately imaged on Molecular Devices IXM HC confocal microscope. Images were acquired at the start of the experiment and every 15 min after that for a total of 2 h. Images were analyzed in Fiji, where settings required for the brightest images were applied to all samples. Signal appears lower for untreated mucins for this reason.

Synthesis of biotin-carbohydrates and glycopolymers
Synthesis of biotinylated carbohydrates (β-Galf-biotin and β-Galp-biotin) and 60% lactose-functionalized trans-poly(norbornene) polymer, which were synthesized in-house, has been described previously12,44. Other biotinylated carbohydrates (α-Galp, β-LacNAc, β-GlcNAc, α-Neu5Ac, β-6-SO3-LacNAc) were purchased from GlycoTech.

Biolayer interferometry (BLI)
Lectin binding to biotinylated carbohydrates was assessed using the Octet Red BLI instrument (ForteBio). Biotinylated carbohydrate was loaded onto streptavidin biosensor (ForteBio) for 120 s as a 5 μM solution in PBS buffer. The sensor was subsequently washed in PBS for 60 s, and a baseline was established in HEPES/Ca/BSA/T or PIPES/Ca/BSA/T buffer for 120 s. Various concentration of recombinant lectins (in HEPES/Ca/BSA/T or PIPES/Ca/BSA/T buffer) was then associated for 600 s followed by dissociation in HEPES/Ca/BSA/T or PIPES/Ca/BSA/T buffer for 600 s. The shake rate was maintained at 1000 rpm throughout the experiment, and the binding assays were performed at 30°C. Data were normalized through background subtraction using the response from biotin-loaded streptavidin. The results were analyzed using GraphPad Prism.

To determine the dissociation constant for lectin with biotinylated carbohydrates, we conducted the BLI experiments with various lectin concentrations using the same protocol as above. The response at equilibrium was plotted against the concentration of lectin, and the resulting curve was fit to a one-site total non-linear regression equation to determine the equilibrium dissociation constant (GraphPad Prism).

Assay of Itln2 on microbial and mammalian glycan array
Lectin binding to mammalian glycans was assessed using the Consortium for Functional Glycomics (CFG) version 5.5 microarray and RayBiotech slides (Cat# GA-Glycan-300-1). Binding to microbial glycans was assessed using the CFG microbial glycan microarray version 2 (MGM). Each glycan sample on the microarray had six replicates. The microarray slides were first rehydrated for 5 min in HEPES/Ca/BSA/T buffer. Subsequently, the slides were incubated with 25 μg/mL of StrepII-tagged mItln2 in HEPES/Ca/BSA/T buffer for 1 h at RT. The slides were then washed with HEPES/Ca/BSA/T buffer and incubated with StrepMAB-Classic DY-549 (1:250, IBA, Cat# 2-1566-050) in HEPES/Ca/BSA/T buffer for 1 h at RT. Afterward, the array underwent washing steps using HEPES/Ca/BSA/T buffer, HEPES/Ca buffer, and deionized water. Finally, the bound lectin in the microarray was detected using a fluorescent scanner. The results are presented as relative fluorescence units obtained by averaging the background-subtracted signals for the four replicate spots (after excluding the highest and lowest values from the six replicates), with error bars representing the SD of the averaged values. The microbial glycan array was interpreted using the key from the original publication39. Of note, hItln2 was assayed for binding on the mammalian glycan arrays (CFG v5.5) but, for unclear reasons, the lectin did not display binding. Alternative approaches (dot blot, BLI, and microscopy) showed that mItln2 and hItln2 have similar binding patterns.

The differences in fluorescence intensities between experiments reflect different time points of data collection and differences in the glycan array slides and fluorescence scanners used. The mItln2 mammalian glycan array data (Fig. 2B) was collected in 2016 using ProScanArray Scanner (Perkin Elmer) instrument at the core facility. The microbial glycan array data (Fig. 2B) and the RayBiotech mammalian array data (Supplementary Fig. 2B) for mItln2 were collected in 2021 in-house using a Genepix 4400A scanner (Molecular Devices). Similarly, the hItln2 microbial glycan array data (Fig. 5E) was collected in 2024 using a Genepix 4400A scanner (Molecular Devices) instrument at the core facility. During the 2024 run, the core facility noted that the scanner in use during that time had a maximum lower relative fluorescence unit (RFU) of approximately 4000 compared to 60,000 on the previous scanner, resulting in lower RFU values. Moreover, in 2024, the core facility also changed protocols and began regenerating and reusing slides; the long-term effects on fluorescence intensity are unclear69.

Assay for lectin binding to fecal samples
Freshly collected murine fecal samples were homogenized in PBS. Human fecal homogenates were scraped from frozen glycerol stock and thawed on ice. The homogenate was centrifuged at 100×g for 1 min to pellet fibrous material. The supernatant was transferred to a new tube and pelleted at 3000×g, 5 min. The pellet was washed twice with PBS, and the optical density at 600 nm (OD600) was measured. To test Itln2 binding to fecal microbiota, the homogenate was diluted to OD600 of 0.2 and treated with 0.6 μM of StrepII-tagged mItln2, or StrepMAB-Classic DY549 antibody (1:250, IBA, Cat# 2-1566-050), or 1 μM StrepII-hItln2, Streptactin XT-DY549 (1:250, IBA, Cat# 2-1565-050) and SYTO BC (1:1000, Thermo Fisher, Cat# S34855) in HEPES/Ca/BSA/T buffer for 2 h at 4°C. To test Ca2+-dependent binding of Itln2, staining was carried out in HEPES/EDTA/BSA/T buffer and used as a control. For the hIltn2 in UC/CD condition, the homogenate was treated with 1 μM StrepII-hItln2, Streptactin XT-DY549 (1:250, IBA, Cat# 2-1565-050) and SYTO BC (1:1000, Thermo Fisher, Cat# S34855) in PIPES/Ca/BSA/T buffer or PIPES/EDTA/BSA/T buffer for 2 h at 4°C. Untreated fecal samples and the fecal samples treated solely with StrepMAB-Classic DY549 antibody, StrepTactin XT-549, or SYTO BC were used as additional controls. Following staining, the cells were analyzed on LSR II, LSR Fortessa HTS flow cytometer or FACS Symphony flow cytometer. Flow data were analyzed with FlowJo.

For the microscopy experiment, the fecal samples were processed identically to the flow experiment described earlier. Following staining, the samples were transferred to a lysine-coated 96-well black/clear bottom Plate (Thermo Fisher, Cat# 165305) and centrifuged at 500×g for 1 min. Imaging was performed using the RPI Spinning Disk Confocal microscope or Molecular Devices IXM HC confocal microscope. Image analysis was performed using Fiji.

Assay for lectin binding to microbial isolates
Bacterial isolates Bacillus paramycoides (RJX001), Bacillus paramycoides (RJX007), L. reuteri (RJX004), L. murinus (RJX002), and L. johnsonii (RJX009) were isolated from mouse stool by Xavier Lab (Broad Institute). Lactobacillus reuteri (DSM 20016) and Escherichia coli DH5α were obtained from Prof. Federico Rey, courtesy of Dr. Robert Kerby (UW-Madison) and NEB, respectively. B. paramycoides (RJX001), B. paramycoides (RJX007), L. reuteri (DSM 20016 and RJX004), L. murinus (RJX002), and L. johnsonii (RJX009) were grown anaerobically in a chamber with AnaeroGen sachet (Thermo Fisher) at 37°C for 16 to 24 h without shaking. B. paramycoides (RJX001 and RJX007) were grown in CHG medium [brain heart infusion (37 g L−1, BD) supplemented with 1% vitamin K1-hemin (ATCC), D-(+)-cellobiose (1 g L−1, Sigma-Aldrich), D-(+)-fructose (1 g L−1, Sigma-Aldrich), D-(+)-maltose (1 g L−1, Sigma-Aldrich), and L-(+)-cysteine (1 g L−1, Sigma-Aldrich). L. reuteri (DSM 20016 and RJX004), L. murinus (RJX002), and L. johnsonii (RJX009) were grown in MRS medium (BD). E. coli was grown in Luria Broth (LB) at 37°C for 16 to 24 h with shaking at 200 rpm.

To test lectin binding, cells were revived from glycerol stock in their respective buffers and grown overnight in appropriate aerobic or anaerobic conditions. Cultures were harvested by centrifugation and washed with PBS twice before OD600 measurement. Staining was performed at OD600 of 0.2 for all samples. For staining, cells were treated with various concentrations of StrepII-tagged lectins, StrepMAB-Classic DY549 antibody (1:250, IBA, Cat# 2-1566-050) or Streptactin XT-DY549 (1:250, IBA, Cat# 2-1565-050), and SYTO BC (1:1000, Thermo Fisher, Cat# S34855) in HEPES/Ca/BSA/T buffer for 2 h at 4°C. To test the Ca2+-dependent binding of lectins, staining was carried out in HEPES/EDTA/BSA/T buffer and used as a control. For UC/CD conditions, cells were treated with lectins in PIPES/Ca/BSA/T or PIPES/EDTA/BSA/T buffers. Unstained bacteria, bacteria treated only with StrepMAB-Classic DY549 antibody or Streptactin XT-DY549, and bacteria treated only with SYTO BC were used as additional controls. Following staining, the cells were diluted five-fold and analyzed on LSR Fortessa HTS flow cytometer or FACS Symphony flow cytometer. Data were analyzed with FlowJo.

For the microscopy experiment, the microbial isolates were processed identically to the flow experiment described earlier. Following staining, the samples were transferred to a lysine-coated 96-well black/clear bottom Plate (Thermo Fisher, Cat# 165305) and centrifuged at 500×g for 1 min. Imaging was performed using Molecular Devices ImageXpress Micro Confocal Microscope, RPI spinning disc confocal, Olympus FV1200 Laser Scanning Confocal Microscope. Image analysis was performed using Fiji.

Assay for lectin binding to pathogenic isolates (S. aureus, K. pneumoniae, and S. pneumoniae)
Staphylococcus aureus USA300Lac (MRSA) and Klebsiella pneumoniae UCI60 were generously shared by Xavier and Hung groups (Broad Institute), respectively. Cells were grown in suspension of LB at 37°C for 16 to 24 h with shaking at 200 rpm. S. pneumoniae (Klein) Chester serotypes 70 (ATCC 10370) were obtained from ATCC. Cells were grown in suspension of Todd Hewitt broth (BD) with 0.5% yeast (BD) without shaking at 37°C under 5% CO2 for 24 h.

To test lectin binding, cells were harvested by centrifugation, washed with PBS, and fixed in 4% formaldehyde in PBS for 1 h at RT. Fixation was neutralized with Tris buffer (pH 7.4) at a working concentration of 50 mM. Of note, previous studies have compared lectin binding to live versus formaldehyde-fixed bacteria and found that fixation had minimal impact on lectin binding patterns, suggesting that the surface glycans are preserved during fixation53. After washing cells with PBS, OD600 was measured, and staining was performed at OD600 of 0.2. For staining, fixed cells were treated with various concentrations of StrepII-tagged lectins, StrepMAB-Classic DY549 antibody (1:250, IBA, Cat# 2-1566-050) or Streptactin XT-DY549 (1:250, IBA, Cat# 2-1565-050), and SYTO BC (1:1000, Thermo Fisher, Cat# S34855) in HEPES/Ca/BSA/T buffer for 2 h at 4°C. To test Ca2+-dependent binding of lectins, staining was carried out in HEPES/EDTA/BSA/T buffer and used as a control. For UC/CD conditions, cells were treated in PIPES/Ca/BSA/T or PIPES/EDTA/BSA/T buffers. Unstained bacteria, bacteria treated only with StrepMAB-Classic DY549 antibody or Streptactin XT-DY549, and bacteria treated only with SYTO BC were used as additional controls. Following staining, the fixed cells were diluted five times without washing and analyzed on LSR Fortessa HTS flow cytometer or FACS Symphony flow cytometer. Data were analyzed with FlowJo.

For the microscopy experiment, the microbes were processed identically to the flow experiment as described earlier. Following staining, the fixed samples were transferred to a lysine-coated 96-well black/clear bottom Plate (Thermo Fisher, Cat# 165305) and centrifuged at 500×g for 1 min. Imaging was performed using Molecular Devices ImageXpress Micro Confocal Microscope or Olympus FV1200 Laser Scanning Confocal Microscope. Image analysis was performed using Fiji.

Colony-forming unit (CFU) Assays
L. reuteri (DSM 20016) was cultured anaerobically in MRS medium at 37°C for 16 h without shaking. B. paramycoides RJX001 grown anaerobically in a chamber with AnaeroGen sachet (Thermo Fisher) at 37°C for 16 to 24 h without shaking. S. aureus USA300Lac (MRSA) was cultured in LB medium at 37°C for 16 h with shaking at 200 rpm. Upon reaching the mid-logarithmic phase, the cells were harvested by centrifugation and washed with PBS before measuring OD600. Subsequently, cells were diluted to OD600 of 0.2 and treated with various concentrations of StrepII-tagged lectins in HEPES/Ca/BSA/T or PIPES/Ca/BSA/T buffer for 4 h (at 4°C for L. reuteri and S. aureus and 37°C for B. paramycoides). Following incubation with lectins, cells were serially diluted in sterile HEPES/Ca (10^1–10^5) or PIPES/Ca buffer and plated on appropriate agar plates (MRS for L. reuteri, CHG for B. paramycoides, or LB for S. aureus) and incubated overnight at 37°C. L. reuteri and B. paramycoides plates were grown in Oxoid Anaero Jar 2.5 L anaerobic chamber with AnaeroGen 2.5 L sachet (Thermo Fisher, AN0025A). Surviving bacterial colonies were counted, and CFU/mL was calculated. Data is displayed as a percentage relative to the control sample (cells treated with HEPES/Ca/BSA/T buffer or PIPES/Ca/BSA/T buffer without lectins).

Suspension-culture growth assay for K. pneumoniae
For lectin-mediated growth inhibition assays, K. pneumoniae UCI60 was cultured directly from glycerol stock in LB media overnight at 37°C with shaking. Cells were back-diluted in fresh LB and grown to mid-log phase (OD600 of 0.4–0.7). Cells were harvested by centrifugation, washed with PBS, and the OD600 was measured. Cells were then diluted in M9 growth media (1x M9 salts, 0.1% glucose, 100 µM CaCl2, 2 mM MgSO4) to achieve a 2x stock of cells (OD600 = 0.001). 2x cells were mixed with 2x stocks of increasing concentration of lectins in M9 media in a final volume of 50 μL. The plate was covered with BreathEasy gas-permeable film (Diversified Biotech, BEM-1) and allowed to grow overnight, 18 h, at 37°C in SpectraMax M5 plate reader. OD600 nm reads were taken every 15 min. Triplicate samples were averaged, and the mean OD600 nm was plotted as a function of time (GraphPad Prism).

To determine the minimum inhibitory concentration (MIC) for lectins, the OD600 measurements at 960 min for different lectin-treated conditions were normalized to OD600 measurement using the no-lectin treatment conditions. The resulting normalized OD600 was used to determine % of bacterial growth and fitted to the modified Gompertz functions to obtain MIC value as described before70.

Suspension-culture growth assay for Lactobacilli
As Lactobacilli do not grow in M9 minimal media, an alternate growth recovery assay was designed to assay lectin-mediated growth assay. L. reuteri (DSM 20016 and RJX004), L. murinus (RJX002), and L. johnsonii (RJX009) were cultured anaerobically in MRS medium at 37°C for 16 h without shaking. Cells were harvested by centrifugation, washed with PBS, and OD600 measured. Subsequently, the cells were diluted to OD600 of 0.2 and treated with various concentrations of StrepII-tagged lectins in HEPES/Ca/BSA/T buffer in a sterile 96-well round-bottom plate. Following treatment, cells were diluted five-fold in MRS broth, sealed with film, and allowed to recover overnight on SpectraMax M5 plate reader, 37°C without shaking, taking OD600 reads every 15 min. The growth assay was run in triplicate, and the data was averaged. Data was displayed as mean OD600 changes plotted as a function of time.

Time-lapse microscopy of L. reuteri
L. reuteri (DSM 20016) was cultured anaerobically in MRS medium at 37°C for 16 h without shaking. To assess the impact of lectin on L. reuteri, the bacterial cells were harvested by centrifugation and washed with PBS before OD600 measurement. Subsequently, the cells were diluted to OD600 of 0.2 and treated with various concentrations of StrepII-tagged lectins in HEPES/Ca/BSA/T buffer in a lysine-coated 96-well black/clear bottom Plate (Thermo Fisher, Cat# 165305). Imaging was performed using a 40x water immersion objective in Molecular Devices ImageXpress Micro Confocal Microscope at 10 min intervals over 6 h. Images were processed in Fiji.

Assay for propidium iodide uptake in B. paramycoides
B. paramycoides RJX001, grown anaerobically in a chamber with AnaeroGen sachet (Thermo Fisher) at 37°C for 16 to 24 h without shaking, was harvested at mid-logarithmic phase by centrifugation and washed with PBS before measuring OD600. Subsequently, cells were diluted to OD600 of 0.2 and treated with various concentrations of StrepII-tagged mItln2 in HEPES/Ca/BSA/T or HEPES/EDTA/BSA/T buffer for 4 h at 37°C. Following incubation with lectins, cells were treated with propidium iodide (Thermo Fisher, Cat# P3566) for 20 min and analyzed using FACS Symphony flow cytometer.

Assay for lectin binding and propidium iodide uptake in mammalian cells
HEK-293T cells were maintained in DMEM medium (Thermo Fisher, Cat# 11995) supplemented with 10% heat-inactivated FBS, 1x penicillin-streptomycin, 1x L-glutamine, and 1x non-essential amino acids. To assay lectin binding, the HEK-293T cells were dissociated with EDTA and washed with DPBS. Subsequently, cells (100 K/staining condition) were washed with HEPES/Ca or HEPES/EDTA buffer and then incubated with various concentrations of StrepII-tagged lectins and StrepMAB-Classic DY-549 (1:250, IBA, Cat# 2-1566-050) in HEPES/Ca/BSA/T or HEPES/EDTA/BSA/buffer for 3 h at 4°C. Following incubation with lectins, the cells were treated with 1 μL of Propidium iodide (Thermo Fisher, Cat# P3566) for 20 min at 4°C. The cells were then washed with the respective buffer twice and analyzed using an Attune flow cytometer.

Liposome preparation
Palmitoyloleoylphosphatidylcholine (POPC) was obtained as a 25 mg/mL solution in CHCl3 from Avanti Polar Lipids (Cat# 850457C). 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (18:1 DBCO PE) was obtained as a 10 mg/mL solution in CHCl3 from Avanti Polar Lipids (Cat# 870129C). The lipid solutions were stored at −20°C and used within 3 months. Cholesterol was obtained as a white solid from Sigma-Aldrich (Cat# C8667).

Prior to preparing a lipid film, the lipid solutions were warmed to ambient temperature for at least 30 min to prevent condensation from contaminating the solution and degrading the lipid film. 330 µL of POPC, 75 µL of 18:1 DBCO PE, and 100 µL of a 4 mg/mL cholesterol solution in CHCl3 were added to a glass scintillation vial. The solvent was removed with a gentle stream of nitrogen, and the resulting lipid film was stored under a high vacuum for a minimum of 12 h prior to use.

The dried lipid film was rehydrated with 1 mL of 150 mM NaCl, 20 mM HEPES, 10 mM CaCl2, pH 7.4 containing 200 μg/mL Texas Red-dextran conjugate MW 3000 Da (Thermo Fisher, Cat# D3329), and vortexed vigorously for approximately 3 min to form a suspension of multilamellar vesicles (MLVs). To obtain a sufficient quantity of large unilamellar vesicles (LUVs), at least three independent lipid film preparations were pooled together for the subsequent formation of LUVs. The resulting lipid suspension was pulled into a Hamilton (Reno, NV) 1 mL gas tight syringe and the syringe was placed in an Avanti Polar Lipids Mini-Extruder (610000). The lipid solution was then passed through a 200 mm pore size hydrophilic polycarbonate filter (Avanti Polar 610006) 21 times, the newly LUV suspension being collected in the syringe that did not contain the original suspension of MLVs to prevent the carryover of MLVs into the LUV solution. The newly formed LUVs were purified via size-exclusion chromatography (SEC) using Sephadex G-50 (Sigma-Aldrich G50150) and functionalized with 5 mM β-Lactose-PEG3-azide (Sigma-Aldrich SMB00404-25MG) with shaking at 4°C overnight via a strain-promoted azido-alkynyl cycloaddition (SPAAC). The lactose-functionalized liposomes were then purified by SEC to remove unreacted glycan ligands.

Liposome membrane disruption assay
Liposomes were diluted to OD 0.1 in 150 mM KCl, 20 mM HEPES, 10 mM CaCl2, pH 7.4 buffer. Absorbance (405 nm) was measured with a SpectraMax microplate reader (Molecular Devices). 50 µL of the liposome suspension was added to a 96-well plate, and initial measurement was recorded. Then, 25 µL of mItln2 or vehicle (buffer only) was added to each well, and absorbance was continuously read for 45 min, with measurements taken every 30 s.

For the microscopy experiment to visualize liposomal structural integrity, following measurement of absorbance over time following lectin addition, samples were imaged on an RPI Spinning Disc Confocal. Image analysis was performed using Fiji.

STAT6 motif analysis
The promoter sequence for mItln2 in BALB/c mouse genome (Accession: GCA_921997145.2, https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_921997145.1/) was retrieved from Ensemble (projects.ensembl.org/mouse_genomes/). The promoter sequence for hItln2 was retrieved from GRCh38/hg38 assembly (genome.ucsc.edu/). Then, the promoter regions (–2000 base pairs to −1 base pair relative to the transcription start site) of mItln2 and hItln2 were scanned for the presence of STAT6 motif [obtained from JASPAR (jaspar.elixir.no/)] using FIMO version 5.5.8 with default parameters34. The sequences that matched the STAT6 motif, along with their log-odds score (score for the match), p-value, and q-value (false-discovery rate) are reported.

Statistical analysis
Statistical analysis of the data was performed using GraphPad Prism 9. Unpaired Student’s t-test was used when comparing two independent samples. One-way ANOVA with Dunnett’s multiple comparison test was used when comparing three or more samples. Three biological replicates were used, except when indicated otherwise. ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Software
Protein structures and overlays were generated with PyMol (The PyMol Molecular Graphics System, Version 3.0 Schrodinger, LLC). Putative structures of mItln2 and hItln2 were generated with AlphaFold 338. Flow cytometry data was analyzed using FlowJo (FlowJo™ v10.8 for Mac Software jm). Imaging data was analyzed using Fiji71. GraphPad Prism version 10.1.1 for Mac was used for data analysis and graph generation (GraphPad Software, Boston, MA, USA, www.graphpad.com). Sequencing data was analyzed using SnapGene (www.snapgene.com). Carbohydrate structures were generated using ChemDraw version 20.1 for Mac (Revvity Signals Software). References were cited using Zotero version 6.0.37 (www.zotero.org). Schematics in figures were generated with BioRender.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In their manuscript entitled \"Intelectin-2 is a broad-spectrum antimicrobial lectin\", Dugan et al. report an in-depth functional\ncharacterization of murine and human intelectin-2 (Itln2). The authors provide evidence for an interaction of both mItln2 and\nhItln2 with different host mucin isoforms as well as a variety of gram-negative and gram-positive bacteria, thus suggesting a\nrole for Itln2 in microbial recognition. Moreover, they convincingly show direct antimicrobial effects of Itln2, thereby indicating\na relevance for Itln2 in mucosal defense. In summary, the manuscript presents novel and important mechanistic insights into\nthe role of intelectins in host-microbe interactions.\nOverall, the present manuscript contains a wealth of experimental data that allow for elucidating the glycan preferences of\nboth mItln2 and hItln2. It builds on previous studies that reported an upregulated expression of mouse Itln2 during nematode\ninfection and postulated a role for Itln2 in inflammatory processes. By using an innovative combination of biophysical\ntechniques, glycan arrays, biochemistry, and microbiology methods, the authors demonstrate a dual role of Itln2 for host\nprotection, i.e., via binding to host-derived mucins on the one hand and recognition and killing of bacteria on the other hand.\nThe manuscript is well written and of interest to a wide readership.\nHowever, specific points need to be addressed to justify and/or support some conclusions drawn by the authors (see\n\"Specific points\" below).\nSpecific points:\n1.) Figure 1: Using enteroids from a congenic C57BL/6 mouse model encoding the complete Itln1-6 locus, the authors show\nan induced Itln2 expression upon IL4 and IL-13 stimulation, according to previous studies reporting Itln2 induction during\nTh2 responses. In this assay, Th1 cytokines and Th1-inducing inflammatory stimuli should be included to assess the\nspecificity of the Itln2 upregulation by Th2 cytokines. Moreover, it will be interesting to see whether Itln2 is also induced upon\nstimulation of the enteroids with killed bacteria and/or bacteria-derived PAMPs.\n2.) Figures 2 and 5; Supplementary Tables 2 and 3: The authors determine the glycan specificity of mItln2 and hItln2 using\ndifferent glycan arrays. They observe some hits for microbial glycans as well as mammalian glycans. However, the glycan\narray results require further explanation. For instance, it looks as if the RayBiotech mammalian glycan array yielded several\n(potentially unspecific?) hits, which seems to be in partial contrast to the CFG mammalian glycan array. Moreover, the\nfluorescence intensities are quite low in some instances (e.g., Figure 5E). It will be helpful and facilitate readability if the\nauthors discuss the sometimes counterintuitive glycan array results in more detail and give further explanations.\n3.) Figures 2E, 5G (mucin binding of mItln2 and hItln2): The authors show a direct binding of mucins to mItln2 and hItln2 and\nconvincingly confirm these interactions by different methods, including dot blot, fluorescence microscopy, and spectroscopic\nassays. However, how specific are the interactions of mItln2 with the different mucin isoforms? Are there any mucin isoforms\nthat are not recognized by mItln2? Can the authors provide an explanation for the differential binding of hItln2 (e.g., why\nMUC5B is not bound by hItln2)?\n4.) Supplementary Figure 6: To optimize hItln2 expression, the authors mutated some amino acid residues for an improved\nsolubility and reduced aggregation. Could these modifications have affected the binding properties of hItln2?\n5.) According to the Methods section, binding studies to microbial isolates were done with live bacteria, while lectin binding\nto pathogenic isolates was determined using formaldehyde-fixed bacteria. Did the fixation procedure affect the binding\nbehavior of the bacteria (did the authors compare Itln2 binding to live vs. fixed bacteria)?\n6.) Titration of Itln2 for determination of antimicrobial activity: In some cases, the authors used different Itln2 concentrations\nfor bacteria binding and killing assays, which renders comparisons of antimicrobial activities of one Itln2 against different\nbacteria species and/or among mItln2 and hItln2 difficult. This should be made consistent by titration of both lectins over a\nwider concentration range. For instance, in Figure 4B, it looks as if only the highest concentration of 5 µM mItln2 is active\nagainst L. reuteri (i.e. no activity up to 2.5 µM), whereas in a different assay for microbicidal activity, even the lowest\nconcentration of 1 µM Itln2 exhibited a maximum antimicrobial effect. Thus, Ki values should be measured to determine the\neffective antimicrobial concentrations of mItln2 and hItln2.\n7.) The authors found that hItln2 retained binding to beta-galactopyranose and additional ligands at low pH and ionic\nstrength (Figure 7). Were similar experiments performed with mItln2 as well? EDTA addition did not abrogate the interaction\nof hItln2 with mucins and microbes at low pH and low salt concentrations, which is an interesting but somewhat unexpected\nfinding. The authors suggest an “alternate binding mode” (p.7) as a potential explanation. Does that mean that the\norientation of the carbohydrate ligands in their interaction with Itln2 is expected to be completely different under these\nconditions? Does such an altered binding mode have an impact on the specificity of the recognized microbes (i.e., is\nbacterial recognition of hItln2 altered, as compared with Supplementary Figure 7)?", "reviewer_2": "This is a fine manuscript describing an animal lectin for which no activity could be assigned previously. The extensive study\nbrings molecular characterization, specificity study with some measurement of affinity, and functional role against pathogen\ninfection. The manuscript reads very well, but, presumably because of space limitation, lacks some better description of the\nlarge amount of experimental data.\nMajor points\nThe oligomeric state of the proteins is quickly discussed and some more description of experimental data would be helpful. If\nmItln2 is composed of a mixture of different oligomers, does this mean that only part of the sample is correctly associated ?\nwhat about the effect on activity ? As for hItln2, it is stated that it occurs as a covalent trimer, but supp. Fig 6e is more in\nevidence of a hexamer (Supp Fig 6d should be explicated). A Cys involved in oligomerization has been mutated, what is the\neffect on oligomerization ?\nThe glycan array data are of high importance. They provide the basis of the lectin specificity that was unknown for\nintelectin2. Fig 2B should include identification (labeling) of the highly positive ligands. Some more discussion about the\nobservation would be of interest. In the mammalian array, results pointing to N-glycans and to O-glycans should be\ndiscussed : biantennary glycan with LacNAc repeats are excellent ligands, specially those with 3 LacNAc repeats (540 and\n567) and no binding is observed with only 2 repeats. Triantennary is even better. More numerous LacNAc repeats result in\nweaker binding. This could be discussed in term of lectin topology. Similarly the binding of terminal sulfated Gal is not that\nsimple : it is efficient only when bound to 6SGlcNAc or 6SGlc by b1-4 linkage. Figure 2C is rather misleading: the two\ndisaccharides at the bottom are not recognized by the lectin.\nIt is not clear why hItln2 was not assayed on the mammalian array.\nThe excel files provided in supplemental data are not easy to understand (except for CFG mammalian). There is no\nidentification of the polysaccharides/microbes in the microbial analysis, and only code names for the RayBiotech data. This\nglycan array is never mentioned in the main text (Supp Fig 2b is cited with no explanation) and the strong discrepancy\nbetween SFG glycan array and RayBiotech is not discussed.\n3D-models are important for comparing intelectin-2 (mouse and human) with intelectin-1. In the present version, one model\nis obtained with Swissmodel and the other with Alpha-Fold3 without justification. The same procedure (either one or the\nother) should be used for both. The two methods also allows for modeling oligomeric state and it would be of interest to have\na view of the modelled trimers. Alignment of sequences is provided only for 2 sequences in Supp Fig1a.. It would be useful\nto have a global alignment for mouse and human intelectins (1 and 2). Finally docking galactose would not be much work\n(for example with Autodock) and will rationalize the effect of sulfate (but of course could be kept for further study).", "reviewer_3": "This manuscript provides important new insights into mucosal immunity by characterizing the intelectin-2 (Itln2) family in\nboth mice and humans. Building on the authors' prior work on intelectin-1 (Itln1), the study reveals distinct and\ncomplementary roles for Itln2 in host defense at mucosal surfaces. These lectins are expressed by epithelial cells in barrier\ntissues, and the current study focuses on their function in intestinal immunity.\nThe authors show that mouse Itln2 expression is induced by type 2 cytokines and that the protein exhibits antimicrobial\nactivity via direct bacterial killing and membrane disruption. In contrast, human ITLN2 primarily agglutinates bacteria. Both\nforms can cross-link mucins, suggesting a dual role in antimicrobial defense and enhancing mucosal barrier integrity.\nImportantly, the authors demonstrate that mItln2 binds terminal β-D-galactopyranose, a specificity distinct from mItln1 and\nhITLN1. This divergence is supported by structural data.\nOverall, the data are quite robust and the conclusions are well-supported. The work makes a significant contribution to our\nunderstanding of the diversity of antimicrobial proteins (AMPs) at epithelial surfaces. That said, I have a few specific\nquestions and suggestions that could further strengthen this already strong manuscript:\n1. The microarray data indicate that intelectins also bind to mammalian glycans. More discussion is warranted on how\ncarbohydrate structure enables discrimination between host and microbial targets. What prevents host cell targeting in vivo?\n2. Several known AMPs show preferential activity against log-phase bacteria. Has the antimicrobial activity of mouse Itln2\nbeen tested across different bacterial growth phases?\n3. The liposome disruption assays use mammalian-type lipid compositions, yet these membranes are disrupted by mItln2.\nCan the authors clarify why they think that these proteins don’t show toxicity to mammalian cell membranes?\n4. What is the estimated concentration of mouse Itln2 in the intestine or enteroids? This would help contextualize the in vitro\nfindings.\n5. The section on pH sensitivity could be clarified. While the authors note that low pH and low salt are linked to\ninflammation, gut epithelial surfaces also experience these conditions physiologically. Where are Itln2 proteins expressed relative to such\nenvironments? Notably, their ability to function at pH 7.4 and 150 mM NaCl is unusual for AMPs and could be emphasized.\n6. How do intelectins complement or differ from other AMPs? In other words, how are they solving a distinct “evolutionary\nchallenge”? Is it just that they are induced by type 2 cytokines? Their broad bacterial specificity would suggest that they are\nnot simply targeting a subset of bacteria that is “missed” by other AMPs.\n7. Since type 2 cytokines promote goblet cell hyperplasia and mucus production, the co-induction of antimicrobial intelectins\nthat also cross-link mucins is intriguing. This synergistic relationship could be highlighted more explicitly in the discussion.\n8. Given the role of type 2 cytokines, are STAT6 binding motifs present in the promoters of the mItln2 and hITLN2 genes?\n9. Do N-linked glycosylation sites on the recombinant proteins affect their oligomerization state or binding activity? Are any\nof the N-glycans capped with β-D-galactopyranose, potentially affecting specificity?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We thank the reviewers for their thoughtful, constructive comments, which have improved our manuscript. Our\nresponse to each comment is provided below.\nReviewer #1 (Remarks to the Author):\nIn their manuscript entitled \"Intelectin-2 is a broad-spectrum antimicrobial lectin\", Dugan et al. report an in-\ndepth functional characterization of murine and human intelectin-2 (Itln2). The authors provide evidence for an\ninteraction of both mItln2 and hItln2 with different host mucin isoforms as well as a variety of gram-negative\nand gram-positive bacteria, thus suggesting a role for Itln2 in microbial recognition. Moreover, they\nconvincingly show direct antimicrobial effects of Itln2, thereby indicating a relevance for Itln2 in mucosal\ndefense. In summary, the manuscript presents novel and important mechanistic insights into the role of\nintelectins in host-microbe interactions.\nOverall, the present manuscript contains a wealth of experimental data that allow for elucidating the glycan\npreferences of both mItln2 and hItln2. It builds on previous studies that reported an upregulated expression of\nmouse Itln2 during nematode infection and postulated a role for Itln2 in inflammatory processes. By using an\ninnovative combination of biophysical techniques, glycan arrays, biochemistry, and microbiology methods, the\nauthors demonstrate a dual role of Itln2 for host protection, i.e., via binding to host-derived mucins on the one\nhand and recognition and killing of bacteria on the other hand. The manuscript is well written and of interest to\na wide readership.\nHowever, specific points need to be addressed to justify and/or support some conclusions drawn by the\nauthors (see \"Specific points\" below).\nSpecific points:\n1. Figure 1: Using enteroids from a congenic C57BL/6 mouse model encoding the complete, Itln1-6 locus, the\nauthors show an induced Itln2 expression upon IL4 and IL-13 stimulation according to previous studies\nreporting Itln2 induction during Th2 responses. In this assay, Th1 cytokines and Th1-inducing inflammatory\nstimuli should be included to assess the specificity of the Itln2 upregulation by Th2 cytokines. Moreover, it will\nbe interesting to see whether Itln2 is also induced upon stimulation of the enteroids with killed bacteria and/or\nbacteria-derived PAMPs.\nResponse: We appreciate the reviewer’s request for more information on this point. In small intestinal tissue of\nconventionally housed/colonized mice, Itln2 transcript levels paralleled unstimulated (antibiotic-treated)\nenteroid cultures, indicating in the absence of stimuli, Itln2 expression is low. These data highlight the\ndifferences between the Itln2 and Itln1, which is constitutively expressed. Type1-associated cytokines, including\ninterferon gamma, are reported to affect the viability and functions of Goblet and Paneth cells\n(https://doi.org/10.1084/jem.20130753; https://doi.org/10.1172/jci.insight.121886). This information and the\ncited reports have been added to the Results section (page 2). We found that type 2 cytokines, IL-4 and IL-13\nselectively induce mItln2. As a control, the type 17/22 inflammatory cytokine, IL-22, had little or no effect on\nmItln2 expression. The effects of stimulating enteroids with microbes or microbe-derived PAMPs is an\ninteresting research question that would require extensive optimization of experimental conditions, making it an\nintriguing avenue for future research.\n2. Figures 2 and 5; Supplementary Tables 2 and 3: The authors determine the glycan specificity of mItln2 and\nhItln2 using different glycan arrays. They observe some hits for microbial glycans as well as mammalian\nglycans. However, the glycan array results require further explanation. For instance, it looks as if the\nRayBiotech mammalian glycan array yielded several (potentially unspecific?) hits, which seems to be in partial\ncontrast to the CFG mammalian glycan array. Moreover, the fluorescence intensities are quite low in some\ninstances (e.g., Figure 5E). It will be helpful and facilitate readability if the authors discuss the sometimes\ncounterintuitive glycan array results in more detail and give further explanations.\nResponse: The reviewer helpfully highlights the need for a clearer explanation of our glycan array data. We\nrevised the Results and Methods sections to address these concerns as follows:\n1\n1. Array differences: The CFG and RayBiotech mammalian glycan arrays contain different glycan\nepitopes and utilize different surface chemistries, which contribute to the observed differences in\nbinding patterns. The RayBiotech array includes some glycans likely to represent non-physiological\nepitopes. We added a discussion of these differences in the main text (page 3).\n2. Signal intensities: In running many glycan arrays, we and others have observed differences in\nfluorescence intensities between experiments. These variations in intensity arise from the differences in\nthe glycan array slides and fluorescence scanners used. The mItln2 CFG mammalian glycan array data\n(Figure 2B) was collected in 2016 using ProScanArray Scanner (Perkin Elmer) instrument at the core\nfacility. The microbial glycan array data (Figure 2B) and the RayBiotech mammalian array data\n(previously Figure S2B, now Figure S3B) for mItln2 was collected in 2021 in-house using Genepix\n4400A scanner (Molecular Devices). Similarly, the hItln2 microbial glycan array data (Figure 5E) was\ncollected in 2024 using a Genepix 4400A scanner (Molecular Devices) instrument at the core facility.\nDuring the 2024 run, the core facility noted that the scanner in use during that time had a maximum\nRFU of approximately 4,000 compared to 60,000 on previous scanner, resulting in lower Relative\nFluorescence Unit (RFU) values. Moreover, in 2024, the core facility also changed protocols and began\nregenerating and reusing slides; the long-term effects on fluorescence intensity are unclear\n((https://doi.org/10.1093/glycob/cwad091). Despite these intensity differences, the binding patterns\nremain consistent within each experiment and were validated with biolayer interferometry. We added\nthis information to the Methods section (page 15).\n3. Data interpretation: We also added a more detailed discussion of the glycan array results (page 3). We\nfocused on the highest-intensity and the most reproducible hits. These align with our biochemical\nvalidation experiments.\n3. Figures 2E, 5G (mucin binding of mItln2 and hItln2): The authors show a direct binding of mucins to mItln2\nand hItln2 and convincingly confirm these interactions by different methods, including dot blot, fluorescence\nmicroscopy, and spectroscopic assays. However, how specific are the interactions of mItln2 with the different\nmucin isoforms? Are there any mucin isoforms that are not recognized by mItln2? Can the authors provide an\nexplanation for the differential binding of hItln2 (e.g., why MUC5B is not bound by hItln2)?\nResponse: We appreciate the reviewer's thoughtful question regarding mucin specificity. In our binding assays,\nwe found that mItln2 binds all major secreted mucins (MUC2, MUC5AC, MUC5B) relevant to intestinal and\nrespiratory tract, as well as porcine gastric mucus (mixture of different mucins). However, hItln2’s binding is\nmore restricted, with strong affinity for MUC2 and MUC5AC. This differential binding may reflect subtle\ndifferences in j patterns (density, modification, and accessibility) between mucin isoforms. Given that both\nintelectins bind β-D-galactopyranose, differences in galactose accessibility or the influence of adjacent glycans\nlikely contribute to selectivity. For example, porcine MUC2 displays glycans that are reported to be shorter and\nless extended than in MUC5AC and MUC5B (https://doi.org/10.1038/s41522-023-00378-4). We have expanded\nthis observation and explanation in the Results section (page 7).\n4. Supplementary Figure 6: To optimize hItln2 expression, the authors mutated some amino acid residues for an\nimproved solubility and reduced aggregation. Could these modifications have affected the binding properties of\nhItln2?\nResponse: We appreciate the reviewer raising this issue. We introduced conservative changes in hItln2 to\nreduce aggregation without changing carbohydrate binding. These mutations were guided by structural\nhomology models with hItln1 (>90% sequence similarity), where we mapped the hItln2 ligand binding site to\nthose determined in the hItln1’s crystal structure. We now provide more description for the changes we\nintroduced (detailed in Figure S7C):\n1. Installation of a glycosylation site in the N-terminal domain that is conserved in hItln1 and mItln2. The\nspecificities of these lectins are different though both have a glycosylation site, indicating glycosylation\n2\ndoes not have a role in binding. Introduction of an N-glycan in hItln2 improved protein expression and\nyields.\n2. Removal of an unpaired cysteine (Cys 311) on a loop in the carbohydrate recognition domain distal\nfrom the ligand binding site. Replacing this cysteine reduced the aggregation of hItln2 protein, but the\nresulting protein had glycan-binding activity.\nIndependent evidence that the binding we detect is relevant is from a recent report on human lectin arrays,\nreleased while this manuscript was in submission. An Itln2 engineered to be monomeric (all cysteines involved\nin interchain disulfide linkages and unpaired Cys 311 were replaced) to afford a mutant protein that binds\ngalactose (https://doi.org/10.1016/j.jbc.2024.107869).\nThe revised manuscript includes all the above information in the Results section (page 6).\n5. According to the Methods section, binding studies to microbial isolates were done with live bacteria, while\nlectin binding to pathogenic isolates was determined using formaldehyde-fixed bacteria. Did the fixation\nprocedure affect the binding behavior of the bacteria (did the authors compare Itln2 binding to live vs. fixed\nbacteria)?\nResponse: Previous studies have compared lectin binding to live versus formaldehyde-fixed bacteria and found\nthat fixation had minimal impact on lectin binding patterns, suggesting that the surface glycans are preserved\nduring fixation (https://doi.org/10.1074/jbc.RA120.012783). Biosafety limitations preclude running live\npathogens at our flow cytometry core facility to compare all directly. We observe binding, however, to fixed\npathogenic isolates and dose-dependent effects on live pathogenic isolates, including S. aureus and K.\npneumoniae, in our growth assay (done in BL2 confinement). These data, and the cited literature, indicate that\nformaldehyde fixation neither impairs nor enhances lectin binding. The revised manuscript includes this\ninformation in the Methods section (page 16).\n6. Titration of Itln2 for determination of antimicrobial activity: In some cases, the authors used different Itln2\nconcentrations for bacteria binding and killing assays, which renders comparisons of antimicrobial activities of\none Itln2 against different bacteria species and/or among mItln2 and hItln2 difficult. This should be made\nconsistent by titration of both lectins over a wider concentration range. For instance, in Figure 4B, it looks as if\nonly the highest concentration of 5 µM mItln2 is active against L. reuteri (i.e. no activity up to 2.5 µM),\nwhereas in a different assay for microbicidal activity, even the lowest concentration of 1 µM Itln2 exhibited a\nmaximum antimicrobial effect. Thus, Ki values should be measured to determine the effective antimicrobial\nconcentrations of mItln2 and hItln2.\nResponse: We appreciate the opportunity to clarify our experimental approach. The flow-based assays can\nidentify microbial \"binders\" but are not quantitative measures of cell killing. Using flow cytometry, we\nobserved that some, but not all, microbes showed changes in FSC vs SSC upon mItln2 treatment at 0.5 μM. We\nsubsequently evaluated a panel of binders and found similar dose-dependent phenotypes in these isolates\n(Figure 4B and Figure S5, as the reviewer noted). The flow studies revealed concentrations that induced\nchanges in cell size and scattering properties; they do not effectively measure antimicrobial activity. We\nemployed CFU assays and growth inhibition studies for quantitative assessment of antimicrobial effects. In\nFigures 4E, 4G, and 4H, we found that lower concentrations of mItln2 (0.5-1 μM) affect the growth of various\nisolates, including L. reuteri and K. pneumoniae. These data indicate that mItln2 treatment exerts antimicrobial\neffects at lower concentrations.\nWe appreciate the value of standardized MICs, but direct comparison of lectin-mediated antimicrobial\nactivity is difficult. Unlike traditional antibiotics that target specific cellular processes, lectins exert\nantimicrobial effects through glycan-binding interactions that can vary significantly between bacterial species\ndue to differences in surface glycan sequences, cell wall composition, and glycan accessibility. As established in\nthe antimicrobial susceptibility literature, MIC values are inherently specific to particular \"drug/bug\"\ncombinations and can vary even between strains of the same species. The observed variation in Itln2\neffectiveness between microbial isolates is expected and biologically meaningful. To address the reviewer's\n3\nconcern, we have replotted the K. pneumoniae data for both hItln2 and mItln2 to establish MIC values for the\nsame microbe using identical assay conditions for comparison (Figures S5M and S8J).\n7. The authors found that hItln2 retained binding to beta-galactopyranose and additional ligands at low pH and\nionic strength (Figure 7). Were similar experiments performed with mItln2 as well? EDTA addition did not\nabrogate the interaction of hItln2 with mucins and microbes at low pH and low salt concentrations, which is an\ninteresting but somewhat unexpected finding. The authors suggest an “alternate binding mode” (p.7) as a\npotential explanation. Does that mean that the orientation of the carbohydrate ligands in their interaction with\nItln2 is expected to be completely different under these conditions? Does such an altered binding mode have an\nimpact on the specificity of the recognized microbes (i.e., is bacterial recognition of hItln2 altered, as compared\nwith Supplementary Figure 7)?\nResponse: We did not test mItln2 at low pH and ionic strength because of its potent antimicrobial effects in\nneutral pH and physiological ionic strength at concentrations consistent with other antimicrobial proteins in the\nliterature (0.5-5 μM). Moreover, the isoelectric point of mItln2 is ~5.5, so low pH is likely to lead to protein\naggregation.\nWe agree that mucin and microbial recognition by hItln2 under altered conditions is a fascinating\nobservation and understanding its basis is a goal of future work. In the current study, we observed that hItln2\nretained binding to “mucin and microbial binders\" (Figures 7D, S8C, and S8E) and “galactopyranose and\n6SO3LacNAc over galactofuranose) at low pH and ionic strength. For mucin, there was still a stronger\ninteraction of hItln2 with MUC2 at low pH and ionic strength, with some weaker interaction with MUC5B and\nMUC5AC. Moreover, treatment of human fecal samples with hItln2 at both conditions yielded similar\npercentage of bound microbes (Figures 6A and 7B), therefore, the specificity of hItln2 for the recognized\nmucins and microbes is largely retained. Further analyses, using methods such as lectin-seq\n(https://doi.org/10.1126/sciadv.add8766) and glycan array studies of hItln2 under low pH and ionic strength\nconditions, are major undertakings that could shed further light into whether the specificity of hItln2 is\nexpanded in such conditions.\nReviewer #2 (Remarks to the Author):\nThis is a fine manuscript describing an animal lectin for which no activity could be assigned previously. The\nextensive study brings molecular characterization, specificity study with some measurement of affinity, and\nfunctional role against pathogen infection. The manuscript reads very well, but, presumably because of space\nlimitation, lacks some better description of the large amount of experimental data.\nMajor points\n1. The oligomeric state of the proteins is quickly discussed and some more description of experimental data\nwould be helpful. If mItln2 is composed of a mixture of different oligomers, does this mean that only part of the\nsample is correctly associated? what about the effect on activity? As for hItln2, it is stated that it occurs as a\ncovalent trimer, but supp. Fig 6e is more in evidence of a hexamer (Supp Fig 6d should be explicated). A Cys\ninvolved in oligomerization has been mutated, what is the effect on oligomerization?`\nResponse: We thank the reviewer for highlighting this aspect. We now provide a more detailed characterization\nof the oligomeric states of the lectins in the Results sections (pages 3, 6, and 7). MItln2 exists as a mixture of\ndimers, trimers, and higher-order oligomers as determined by DLS and crosslinking studies (Figures S2A and\nS2B). Attempts to isolate specific oligomerization states by size exclusion chromatography were unsuccessful\nbecause the lectin typically bound to standard matrices (Sepharose). Moreover, literature precedents for other\nlectins suggest that oligomerization is driven by multivalent glycan ligands and that multiple oligomeric forms\ncan retain activity (https://doi.org/10.1146/annurev-biochem-062917-012322). For example, MBL (a disulfide-\nlinked trimer) can form oligomers up to dodecamers that all bind mannose and recruit complement\n(https://doi.org/10.1016/j.imbio.2008.11.003). Similarly, RegIIIgamma functions can bind oligomerize upon\n4\nbinding its membrane ligands to form hexameric pores (https://doi.org/10.1038/nature12729). These data\nsuggest that mItln2 oligomers have functional activity, an interesting avenue for future studies.\nThe reviewer notes that Figure S6E (now Figure S7F) shows that hItln2 can form trimers and hexamers.\nThe N-terminal cysteines involved in hItln1 oligomerization are conserved in hItln2 and preserved in our study.\nWe observe stable covalent oligomers through intermolecular disulfide bonds. HItln2 has an additional unpaired\ncysteine near the C-terminus that led to aggregation issues, but the location of this cysteine is inconsistent with\na role in physiological oligomerization. Moreover, our observation of recombinant hItln2 forming trimers and\nhexamers is consistent with the oligomeric state reported for native hItln2 in human ileal tissue\n(https://doi.org/10.1096/fj.202101870R). That we observe the same oligomerization behavior as native protein\nis reassuring evidence that our recombinant protein behaves like the endogenous protein. This information is\ndescribed in our revised manuscript. Similarly, the DLS data for hItln2 (previously Figure S6D, now Figure\nS7E) shows hydrodynamic radius value of around 40 nm, suggesting the presence of higher order oligomers.\nThese data provide a qualitative measure of the oligomeric state, as DLS analysis assumes that the protein is\nglobular, is more biased for larger particles; thus, it cannot determine the oligomeric state of the protein. So, our\nestimation of the oligomeric states of hItln2 was mainly guided by the SDS-PAGE data. Lastly, we point to a\nrecent publication that emerged as we were submitting this manuscript suggesting that monomeric hItln2 can\nbind (https://doi.org/10.1074/jbc.RA120.012783). Together, the data indicate that oligomerization is beneficial\nfor avidity but not required for binding.\n2. The glycan array data are of high importance. They provide the basis of the lectin specificity that was\nunknown for intelectin2. Fig 2B should include identification (labeling) of the highly positive ligands. Some\nmore discussion about the observation would be of interest. In the mammalian array, results pointing to N-\nglycans and to O-glycans should be discussed: biantennary glycan with LacNAc repeats are excellent ligands,\nspecially those with 3 LacNAc repeats (540 and 567) and no binding is observed with only 2 repeats.\nTriantennary is even better. More numerous LacNAc repeats result in weaker binding. This could be discussed\nin term of lectin topology. Similarly, the binding of terminal sulfated Gal is not that simple: it is efficient only\nwhen bound to 6SGlcNAc or 6SGlc by b1-4 linkage. Figure 2C is rather misleading: the two disaccharides at\nthe bottom are not recognized by the lectin.\nResponse: We appreciate the reviewer's thoughtful advice regarding the analysis of the glycan array data. We\nadded labels to identify the highest-binding ligands in Figure 2B. Additionally, we expanded our discussion on\nthe structure-activity relationships (pages 3 and 4). We agree with the reviewer's observations about biantennary\nand triantennary N-glycans with LacNAc repeats being excellent ligands for mItln2 and have included\ncomments about lectin topology as possible determining factor influencing the interactions. We have corrected\nthe disaccharide structure in Figure 2C to reflect the glycan hits from the CFG microbial and mammalian arrays.\nHowever, we clarify that the two disaccharides [LacNAc disaccharide and 6SO3LacNAc (with sulfation on\ngalactose but not glucose)], listed previously at the bottom in the Figure 2C are recognized by mItln2 as\ndemonstrated by the ELLA and biolayer interferometry assays (Figures S3G-S2J). Additionally, an important\nlimitation in interpreting glycan array data is the lack of information on relative affinities or ranked binding\npreferences. The apparent differences in signal intensity may reflect differences in array density, presentation\ncontext, or other technical factors rather than true affinity differences. We therefore validated the glycan array\nresults through complementary biochemical approaches. We incorporated this more nuanced discussion of the\nglycan array results and their interpretation limitations into the main text, while emphasizing that these data\nprovide the foundation for our subsequent detailed characterization of mItln2 binding specificity (page 3).\n3. It is not clear why hItln2 was not assayed on the mammalian array.\nResponse: The hItln2 protein was tested on both mammalian and microbial glycan arrays by the CFG, but this\nrun appeared to fail as it had no binding on the former. We postulate there was a problem with the array or that\nthe protein was no longer active, as we have encountered this issue previously with different proteins. We since\ntested hItln2 on select mammalian glycans using alternative approaches (dot blot, biolayer interferometry, and\n5\nmicroscopy) and confirmed hItln2 and mItln2 have similar binding patterns. We added this clarification to the\nMethods section (page 15).\n4. The excel files provided in supplemental data are not easy to understand (except for CFG mammalian). There\nis no identification of the polysaccharides/microbes in the microbial analysis, and only code names for the\nRayBiotech data. This glycan array is never mentioned in the main text (Supp Fig 2b is cited with no explanation)\nand the strong discrepancy between SFG glycan array and RayBiotech is not discussed.\nResponse: We agree that presenting more of the glycan data would be helpful. We added comprehensive\nlegends and identifiers to all supplemental data files. We additionally included references for glycan structure\nidentifications for the microbial array data. We have also amended the main text to discuss the RayBiotech array\nresults in the main text, as noted in our response to Reviewer #1 (page 3).\n5. 3D-models are important for comparing intelectin-2 (mouse and human) with intelectin-1. In the present\nversion, one model is obtained with Swissmodel and the other with Alpha-Fold3 without justification. The same\nprocedure (either one or the other) should be used for both. The two methods also allows for modeling\noligomeric state and it would be of interest to have a view of the modelled trimers. Alignment of sequences is\nprovided only for 2 sequences in Supp Fig1a. It would be useful to have a global alignment for mouse and\nhuman intelectins (1 and 2). Finally docking galactose would not be much work (for example with Autodock)\nand will rationalize the effect of sulfate (but of course could be kept for further study).\nResponse: We thank the reviewer for these suggestions. We have now generated all structural models using\nAlphaFold3 for consistency and have included predicted oligomeric assemblies for both proteins (Figures S2D\nand S7G). We have also included alignment of mouse and human Itln1 and Itln2 (Figure S1B). We agree that\ndocking studies would be informative; however, the current models are not especially adept at modeling\ncalcium-dependent interactions. Structural studies are underway with these lectins, and we hope to report a\nbinding mode in our future studies.\nReviewer #3 (Remarks to the Author):\nThis manuscript provides important new insights into mucosal immunity by characterizing the intelectin-2\n(Itln2) family in both mice and humans. Building on the authors' prior work on intelectin-1 (Itln1), the study\nreveals distinct and complementary roles for Itln2 in host defense at mucosal surfaces. These lectins are\nexpressed by epithelial cells in barrier tissues, and the current study focuses on their function in intestinal\nimmunity.\nThe authors show that mouse Itln2 expression is induced by type 2 cytokines and that the protein exhibits\nantimicrobial activity via direct bacterial killing and membrane disruption. In contrast, human ITLN2 primarily\nagglutinates bacteria. Both forms can cross-link mucins, suggesting a dual role in antimicrobial defense and\nenhancing mucosal barrier integrity. Importantly, the authors demonstrate that mItln2 binds terminal β-D-\ngalactopyranose, a specificity distinct from mItln1 and hITLN1. This divergence is supported by structural data.\nOverall, the data are quite robust and the conclusions are well-supported. The work makes a significant\ncontribution to our understanding of the diversity of antimicrobial proteins (AMPs) at epithelial surfaces. That\nsaid, I have a few specific questions and suggestions that could further strengthen this already strong\nmanuscript:\n1. The microarray data indicate that intelectins also bind to mammalian glycans. More discussion is warranted\non how carbohydrate structure enables discrimination between host and microbial targets. What prevents host\ncell targeting in vivo?\nResponse: Several factors could contribute to host vs. microbial discrimination by intelectin-2. Firstly, Itln2 is\nsecreted by Paneth and goblet cells into the mucus layer where it encounters high concentrations of secreted\nmucins (which it cross-links) and resident or invading microbes. This locale gives Itln2 limited access to host\n6\nepithelial cell surfaces. Additionally, microbial surface glycans may present β-galactopyranose residues at a\nhigher density, thereby facilitating preferential recognition of intelectin-2 to microbes. We expanded the\ndiscussion to delineate these possibilities (page 9).\n2. Several known AMPs show preferential activity against log-phase bacteria. Has the antimicrobial activity of\nmouse Itln2 been tested across different bacterial growth phases?\nResponse: Our initial assays for Itln2 binding and activity against microbial isolates were conducted in log and\nstationary phases. We saw binding and antimicrobial effects in both growth phases. Given these results, we\nperformed assays at mid-log as this is the most standard format in the literature, and it allowed us to use a\nconsistent protocol between different microbial isolates.\n3. The liposome disruption assays use mammalian-type lipid compositions, yet these membranes are disrupted\nby mItln2. Can the authors clarify why they think that these proteins don’t show toxicity to mammalian cell\nmembranes?\nResponse: We thank the reviewer for this clarifying question. This apparent paradox likely reflects several\nfactors, including the complexity of cell membranes compared to liposomes. Our liposomes contain simplified\nlipid compositions, while mammalian cell membranes have complex asymmetric lipid distributions, variable\ncholesterol content, and associated proteins that may resist disruption. We additionally postulate that while\nligands present on mammalian cells may result in Itln2 binding, the differences in the location and distribution\nof mammalian glycans preclude Itln2 assemblies that lead to cell killing. For example, the Reg proteins are\nbelieved to bind abundant ligands on microbial cells and then assemble into hexameric pores that result in cell\nlysis (https://doi.org/10.1038/nature12729). If Itln2 operates through a similar mechanism, the lectin likely\nbinds to the most accessible galactose residues of the glycocalyx. In mammalian cells, these residues may be\ntoo far away from the membrane to assemble into a pore. Moreover, Itln2 is secreted in environments rich with\nmicrobes and mucins, and these components may compete for binding or create local concentration gradients\nthat favor microbial targeting over mammalian cell interaction. We have incorporated these discussions in the\nmain text (page 9).\n4. What is the estimated concentration of mouse Itln2 in the intestine or enteroids? This would help\ncontextualize the in vitro findings.\nResponse: There are no accurate measures of mItln2 concentration in the intestine or enteroids. Proteomic\nanalyses of the enteroids and mouse tissue would be informative for future work examining mItln2 function in\nvivo. Still, mItln2 has been reported to be one of the most abundant proteins in the BALB/c mouse jejunum\nfollowing nematode infections (https://doi.org/10.1002/pmic.200300658). Moreover, in murine organoid\ncultures, mItln1 transcripts are expressed abundantly at levels comparable to those found in tissue\n(https://doi.org/10.1073/pnas.2312453120), suggesting a correlation between enteroid and tissue expression.\nThus, we reason that mItln2 is abundantly expressed in the intestine and enteroids in the type-2 inflammatory\nconditions. Moreover, hItln2 is even more highly expressed than hItln1 in ileal tissue. Association of Itln2 with\nthe mucus layer would result in very high local concentrations that would be difficult to assess by analyzing\nsoluble lectin alone.\n5. The section on pH sensitivity could be clarified. While the authors note that low pH and low salt are linked to\ninflammation, gut epithelial surfaces also experience these conditions physiologically. Where are Itln2 proteins\nexpressed relative to such environments? Notably, their ability to function at pH 7.4 and 150 mM NaCl is\nunusual for AMPs and could be emphasized.\nResponse: We thank the reviewer for this comment. Under normal conditions, hItln2 is expressed by Paneth\ncells in the distal small intestine (highest expression is in the ileum). A healthy ileum, a site of nutrient\n7\nabsorption, tends to be neutral pH and have physiological salt concentration. In contrast, mItln2 is not expressed\nin healthy mice but by mucus-secreting cells in the intestines and lungs during type 2 inflammation. We now\nelaborate on the distinct abilities of these lectins to function at physiological pH and ionic strengths in the\nDiscussion sections (pages 8 and 9).\n6. How do intelectins complement or differ from other AMPs? In other words, how are they solving a distinct\n“evolutionary challenge”? Is it just that they are induced by type 2 cytokines? Their broad bacterial specificity\nwould suggest that they are not simply targeting a subset of bacteria that is “missed” by other AMPs.\nResponse: We thank the reviewer for this excellent question. Though more information on the microbial\nspecificity of other antimicrobial lectins and AMPs is needed to answer this question fully, intelectins represent\nan evolutionarily ancient solution to pathogen recognition that predates the adaptive immune system. Their high\nconservation across chordates suggests they fill a need for immediate, broad-spectrum antimicrobial activity\nwithout requiring the clonal selection that characterizes adaptive immunity. Moreover, in contrast to canonical\nAMPs that directly disrupt bacterial membranes through electrostatic interactions, intelectins function through\nmore specific targeting of distinct carbohydrate epitopes. Additionally, the family can participate in a range of\neffector functions, including immune cell recruitment, a property that also distinguishes them from classic\nAMPs. We found that Itln2 not only engages microbes but also mucins to establish different modes of defense.\nIn this way, by altering the mucosal environment, it can protect against microbes for which direct recognition is\nlacking. This more passive defense mechanism is advantageous in clearing infections in tissues where\ninflammatory collateral damage needs to be reduced. We believe these properties set the intelectins apart from\nAMPs in the mucosal environment. We highlighted these properties in the Discussion (page 9).\n7. Since type 2 cytokines promote goblet cell hyperplasia and mucus production, the co-induction of\nantimicrobial intelectins that also cross-link mucins is intriguing. This synergistic relationship could be\nhighlighted more explicitly in the discussion.\nResponse: We appreciate the reviewer’s advice. The dual functions of Itln2 suggest it evolved to address a\nspecific challenge in barrier immunity: the generation of antimicrobial barriers that are structurally robust and\nfunctionally active. This is particularly critical when tissues are undergoing active remodeling and repair during\ntype 2 inflammation. We have taken the reviewer’s advice to augment the discussion of this issue (page 9).\n8. Given the role of type 2 cytokines, are STAT6 binding motifs present in the promoters of the mItln2 and\nhITLN2 genes?\nResponse: We appreciate this reviewer suggestion. There is evidence that mItln2 is a STAT6-dependent gene\n(https://doi.org/10.1016/j.exppara.2007.02.015), which we now cite. Additionally, analysis of promoter\nsequences of mItln2 (Figure S1A) and hItln2 (Figure S7A) showed presence of STAT6 binding motifs.\nHowever, we note that the Th2-dependent upregulation of hItln2 (which is constitutively expressed) require\nfurther confirmation.\n9. Do N-linked glycosylation sites on the recombinant proteins affect their oligomerization state or binding\nactivity? Are any of the N-glycans capped with β-D-galactopyranose, potentially affecting specificity?\nResponse: mItln2 is naturally N-glycosylated and introducing an N-glycosylation site in hItln2 helped improve\nprotein expression and solubility. If any Itln2 self-association occurs, it would lead to competition between the\nprotein and other targets on the cell surface or mucins, but we observe robust binding to cells and mucins as\nwell as immobilized glycans. The observation that glycosylation of hItln2 improves its solubility also supports\nthat no competing self-interactions occur with the lectins, as these could lead to formation of large aggregates.\nReviewer #4 (Remarks to the Author):\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition for\nEarly Career Researchers who co-review manuscripts.\nWe thank this reviewer for their transparency and valuable comments, which improved our manuscript.\n9"}, {"round": 2, "reviewer_comments": {"reviewer_1": "In their manuscript entitled \"Intelectin-2 is a broad-spectrum antimicrobial lectin\", Dugan et al. report an in-depth functional\ncharacterization of murine and human intelectin-2 (Itln2). The authors provide evidence for an interaction of both mItln2 and\nhItln2 with different host mucin isoforms as well as a variety of gram-negative and gram-positive bacteria, thus suggesting a\nrole for Itln2 in microbial recognition. Moreover, they convincingly show direct antimicrobial effects of Itln2, thereby indicating\na relevance for Itln2 in mucosal defense. In summary, the manuscript presents novel mechanistic insights into the role of\nintelectins in host-microbe interactions.\nOverall, the manuscript contains a wealth of experimental data that allow for elucidating the glycan preferences of both\nmItln2 and hItln2. The present manuscript builds on previous studies that reported an upregulated expression of mouse Itln2\nduring nematode infection and postulated a role for Itln2 in inflammatory processes. By using an innovative combination of\nbiophysical techniques, glycan arrays, biochemistry, and microbiology methods, the authors demonstrate a dual role of Itln2\nfor host protection, i.e., via binding to host-derived mucins on the one hand and recognition and killing of bacteria on the\nother hand. The manuscript is well written and of interest to a wide readership.\nThe authors have convincingly addressed my concerns, either by providing additional data and/or discussion or by a\nreasonable rebuttal of the points that I raised. Thus, this already strong manuscript has been further strengthened during\nrevision.", "reviewer_2": "All my questions have been answered clearly. I have no other points to rise.", "reviewer_3": "The authors have done a good job of responding to our questions and concerns. I look forward to seeing this exciting story\npublished!"}, "author_response": ""}]

Decision letter