Altering the carbohydrate-binding specificity of the legume lectin FRIL through structure-guided engineering

Domain: chemistry
Published: 2026-03-05
Editorial outcome: minor_revision

Abstract
FRIL is a legume lectin from the hyacinth bean that has broad-spectrum antiviral activity. A distinctive trait of FRIL among similar mannose/glucose-specific legume lectins is that FRIL shows specificity for complex type N-glycans. We postulate that an extended binding site on FRIL facilitates this ligand selectivity. Here, we show legume lectin carbohydrate recognition domain (CRD) loop B is the main determinant of complex versus high-mannose N-glycan specificity in FRIL and Concanavalin A (ConA), respectively. First, we find that the inactive precursors of recombinant FRIL (rFRIL) and proConA (rproConA) can be activated via deglycosylation. Secondly, the cryo-EM structures of inactive apo rFRIL, active FRIL in complex with Galβ1,4-(Fucα1,3-)GlcNAcβ1,2-Man tetrasaccharide, and active rFRIL in complex with Mannose GlcNAc (Man9) N-glycan are determined, and residues H102 and Y101 on loop B are identified as crucial for complex glycan recognition. Finally, we swapped loop B residues 101 and 102 alongside loop C residue 145 on FRIL to their structural equivalent on ConA, resulting in a FRIL mutant that binds exclusively to high mannose N-glycans. Taken together, we have established a process of activating recombinant FRIL and related lectins through deglycosylation, and demonstrated the crucial role that loop B residues play in establishing oligosaccharide specificity.

Introduction
Legume lectins represent one of the largest families of lectins in nature, with over a hundred members displaying a wide range of glycan specificities. They are categorized as L-type lectins, which extend to other structurally-similar lectins found in species outside the Fabaceae family. An array of biomedical properties has been reported in legume lectins including anti-microbial, anti-cancer and immunomodulation1. Moreover, due to their ability to recognize core structural motifs on N-glycans that antibodies are less-equipped to distinguish, legume lectins remain power tools in the realm of glycan identification, glycoprotein purification, and characterization of cell surface glycoconjugates2. The legume lectin Flt3 Receptor Interacting Lectin (FRIL) from hyacinth bean (Lablab purpureus), also known as Dolichos lablab lectin, was found to preserve stem cells in suspension culture3, 4, mitigate tumor neoangiogenesis5, and possess anti-influenza, anti-HSV and anti-SARS-CoV-2 properties6, 7.

Legume lectins are produced as single-chained Nglycoprotein precursors and undergo extensive post-translational modifications to attain glycan-binding activity8, 9. In the case of the prototypical legume lectin ConA (Supplementary Fig. 1), a “circular permutation” process occurs where the glycosylated linker connecting α and β subunits of a pro-lectin is excised by the vacuole enzyme asparagine endopeptidase (AEP), and the N- and C-terminals of the two chains are then joined together in a transpeptidation event catalyzed by the same enzyme10. For FRIL (Fig. 1c, Supplementary Fig. 1), only one AEP cutting site exists on the glycosylated linker, and a single cut is made near the β subunit. The still-attached (and still-glycosylated) linker is then differentially digested by other carboxypeptidases in the vacuole, resulting in four variants of the α subunit3, 11. Unlike ConA circular permutation does not occur in FRIL, resulting in a “two-chain” lectin. These extensive post-translational modifications, manifested in an organelle only available to the plant cell, has been an obstacle to recombinant production and protein engineering of two-chain or circular permutated legume lectins.

Both FRIL and ConA belong to a group of D-mannose/D-glucose-specific legume lectins12, 13. The structures of FRIL and ConA in complex with a trimannose has been determined previously, and residues that make up FRIL and ConA’s monosaccharide binding site are largely conserved14, 15. This includes 6 residues distributed among four loops that make up FRIL and ConA’s CRD: Asp86 (FRIL numbering, ConA Asp208 due to circular permutation) on loop A, Gly103 and Gly104 (Con A Gly227 and Arg228) on loop B, Asn144 (ConA Asn14) on loop C, Gln229 and Asp230 (Con A Leu99 and Tyr100) on loop D. Furthermore, Tyr142 (Con A Tyr12) forms CH-pi stacking with mannose, and H102 (Con A Thr226) form water bridges with the O2 of mannose16. Despite similar monosaccharide preferences, FRIL and ConA’s binding affinity to N-glycans differ significantly. ConA binds strongly to oligomannose N-glycans while FRIL prefers complex N-glycans6, 17. This difference may be attributed to variations in their extended binding sites, which are residues outside the conserved monosaccharide binding site that stabilize larger oligosaccharides16. However, as far as we know no structure of ConA or FRIL binding to more complex oligosaccharides (such as N-glycans) have been resolved, and FRIL’s extended binding site remains unknown.

Changes in the binding specificity of lectins can have far-reaching consequences. For example, a fundamental basis of inter-species transmission of influenza virus are mutations on the viral attachment protein hemagglutinin (HA), which functions as a lectin interacting with host cell sialic acid glycans. Tremendous efforts have been made to understand the HA mutations needed to shift from avian-type to human-type glycan structures (terminal 2,3-linked sialic acid to 2,6-linked sialic acid, respectively)18, and lectin-glycan interactions on biologically relevant glycans in general19. Legume lectins present promising candidates to explore these interactions, as they consist of a large family of structurally homologous proteins with diverse glycan recognition including D-mannose/D-glucose, D-galactose/N-acetyl-D-galactosamine (GalNAc) and L-fucose16. However, progress has been hampered by difficulty in obtaining active recombinant legume lectins from two-chain legume lectins, and a lack of comprehensive knowledge of extended binding site interactions in these proteins.

We therefor set out to determine the structure of FRIL in complex with a tetrasaccharide representative of complex type N-glycans, and create a recombinant version of FRIL for mutagenesis analysis on the interacting residues identified in our structure. During the course of the alanine mutagenesis, we unexpectedly found two mutants that did not exhibit a loss of binding, but a loss of glycan specificity. Structural comparison between FRIL and ConA showed that the position these two mutants occupy on loop B were important in differentiating between different N-glycans. Taking this a step further, we then attempted to swap residues on loop B in order to switch glycan specificity. The overall goal is to gain structural insight into the diverse glycan recognition properties of legume lectins.

Results
Production of bioactive recombinant FRIL and recombinant proConA, and activation by deglycosylation. In order to explore the lectin-glycan interactions between FRIL and its ligand, we first attempt to overcome the challenge of producing recombinant two-chain and circular permutated legume lectins, which has been a major obstacle in the study of these agents for years9. Previously, we had struggled to express recombinant FRIL in Escherichia coli BL21 and Pichia pastoris, but resulted in poor expression levels and misfolding6. In this study, we expressed recombinant FRIL (rFRIL) residues 1-253, with a C-terminal His-tag and Brisbane HA signal peptide (Supplementary Data 1), in HEK 293-EBNA (293E) mammalian cell suspension culture. The 293E expression system yielded to approximately 4~5 mg of secreted glycosylated rFRIL protein per liter of culture medium after Ni-NTA purification. SEC-MALS (Supplementary Fig. 2g) suggests that our rFRIL proteins formed tetramers similar to native FRIL (nFRIL) purified from Lablab purpureus seeds, indicating proper folding. However, these recombinant lectins showed very little glycan binding activity in hemagglutination assay (glycosylated rFRIL 0.17 HAU/μg compared to nFRIL 2.67 HAU/μg, Supplementary Fig. 2j) or lectin binding ELISA with glycoprotein coatings (Fig. 1d, Supplementary Fig. 2i).

To find out why our glycosylated rFRIL has no activity, we employed cryo-electron microscopy (cryo-EM) to take a closer look at our expressed protein in its apo form, without the addition of carbohydrate ligands (Fig. 1a, Supplementary Fig. 3-4, Supplementary Table 1). The glycosylated rFRIL is revealed to be in a tetrameric structure with each monomer exhibiting the typical jellyroll motif of nFRIL and other legume lectins, indicating correct folding of the recombinant protein. But surprisingly, an extra density map was observed in the rFRIL’s CRD despite no ligand provided (Supplementary Fig. 4a). This density map allowed us to model three saccharides of GlcNAcβ1,2-Manα1,6-Man, in which a disaccharide GlcNAcβ1,2-Man is more confidently positioned with a clear density map (Supplementary Fig. 4a). This unexpected ligand observed in a rFRIL is most likely from the self-glycan on the inter-subunit linker, which further supports Sheldon et al’s20 hypothesis that the glycosylated linker between a legume lectin’s α and β subunits acts as a self-neutralizing ligand for the lectin’s CRD.

We attempted three strategies to release the inactive FRIL from its self-ligand: mutational removal of N-glycosites on linker, proteolytic digestion of linker, and enzymatic deglycosylation of linker N-glycans. In the first strategy N116/117Q, N123Q and N116/117/123Q (QQQ) mutants were created, but we observed decreased expression levels and significant retention of protein inside the cell, pointing to a lack of proper protein folding (Supplementary Fig. 2k). For proteolytic digestion we used trypsin to cleave the linker at Lys128 instead of naturally-occurring AEP cleavage at Asn113 or Asn116, but this failed to activate the lectin (Supplementary Fig. 2h-j). Finally, following enzymatic deglycosylation treatment with PNGase F (Fig. 1b, Supplementary Fig. 2h), we were able to obtain active rFRIL with similar hemagglutination (Supplementary Fig. 2j) and ELISA activity (Fig. 1d, Supplementary Fig. 2f) as native FRIL. This indicates that deglycosylation is responsible for FRIL activation.

After acquiring bioactive rFRIL, we wondered whether this process can be applied to other legume lectins. To test this out, we expressed recombinant proConcanavalin A (rproConA) in 293E mammalian cell suspension culture (Supplementary Fig. 2a). Expressed rproConA had comparable yield as rFRIL, in the 4~5 mg/L range, with very little initial activity. Deglycosylation via PNGase F treatment activated rproConA in a similar way as rFRIL (Supplementary Fig. 2a-c). However, without AEP-mediated circular permutation and stabilization of dimer-dimer interface contacts in mature ConA, the bioactive rproConA remained a dimer rather than a tetramer at neutral pH, exhibiting a lower molecular weight and lowered hemagglutination activity (Supplementary Fig. 2d-e). At acidic pH rproConA reverted to a tetramer, consistent with previous reports21. Nevertheless, we have established a reliable and consistent method for the production of two-chain and circular permutated legume lectins using a mammalian expression system followed by an enzymatic deglycosylation treatment, allowing further exploration into FRIL’s interactions with complex type N-glycans.

Identification of FRIL’s extended binding site residues. FRIL is reported to be a glucose/mannose lectin that binds to complex type N-glycans6, 12. Previously solved crystal structure of FRIL in complex with trimannose14 (M3M6M, 1QMO) only show density for a single mannose in the CRD with no extended binding site interactions, but oligosaccharides more representative of complex glycans have not been attempted. Therefore, we solved the X-ray crystal and cryo-EM structures of native FRIL complexed with a Galβ1,4-(Fucα1,3-)GlcNAcβ1,2-Man tetrasaccharide (Fig. 2a-b, Supplementary Table 1, 2), an antennary Lewis X branch that is found on complex N-glycans22. Overall, our X-ray crystal and cryo-EM structures are consistent with the previously solved tetrameric structure of native FRIL protein. In the crystal structure, there are sixteen monomers in one asymmetric unit, forming four tetramers (Supplementary Fig. 5 a,b). Each monomer contains a single bound ligand in an identical binding mode, with the Fo-Fc map showing an unambiguous density for each atom of the ligand (Supplementary Fig. 5 c-f). The crystal structure also showed a high consistency with the cryo-EM structure of the tetrasaccharide bound nFRIL at the resolution of 2.4 Å (Supplementary Fig. 3, Supplementary Table 1). The tetrasaccharide ligand interact with FRIL protein mainly via hydrogen bonds in all structures. The core mannose (Man) in the tetrasaccharide adopts a similar conformation with previous solved monosaccharide bound nFRIL’s structure14, involving previously-known interactions with monosaccharide binding residues Asp86 (Asp208 in ConA numbering), Asn144 (ConA Asn14), Gly104 (ConA Arg228), Tyr142 (ConA Tyr12), and backbone interactions with residues Gln229 (ConA Leu99) and Asp230 (ConA Tyr100) (Fig. 2a-b, Supplementary Fig. 6b). However, in our tetrasaccharide bound FRIL structure, additional extended binding site interactions with N-acetylglucosamine (GlcNAc) and fucose (Fuc) were revealed. The Oγ of Ser45 contacts to both the GlcNAc amide oxygen and the Fuc 2-OH; His102 forms hydrogen bonds with the Fuc 2-OH and GlcNAc 3-OH via its imidazole Nε2. His102 also forms hydrogen bonds with the GlcNAc 6-OH via its backbone carbonyl oxygen and amine nitrogen mediated by a water molecule. For galactose (Gal), its hydrophobic face (C3-C6) is packed with the aromatic ring of Tyr101 (Fig. 2a-b, Supplementary Fig. 6b).

Our tetrasaccharide bound structures suggest that all residues interacting with the β1,2-linked mannose in FRIL’s monosaccharide binding site fall within the four classical loops responsible for the architecture of legume lectin CRD (Asp86 on loop A, Gly103 and Gly104 on loop B, Tyr142, Tyr144 and Tyr147 on loop C, Gly228, Gln229 and Asp230 on loop D). Extended binding site interactions include another two residues (His102 and Tyr101) on loop B adjacent to the monosaccharide binding site, and Ser45 which falls outside the four classical loops (Fig. 2a-b, Supplementary Fig. 6a). To verify our structural observations, we performed alanine mutagenesis on Asp86, Gly103, Gly104, Tyr142, Asn144, Tyr147 and Asp230 of FRIL’s monosaccharide binding site, and Ser45, Tyr101, His102 of FRIL’s extended binding site (Supplementary Fig. 6c). These mutant’s N-glycan binding ability was analyzed by performing lectin binding ELISA with the glycoprotein A/California/07/2009 hemagglutinin (Cali09 HA), which contains both complex and high mannose N-glycans (Fig. 2c). Results show a loss in FRIL’s glycan binding ability when monosaccharide binding site residues Asp86, Asn144, Tyr147 and Tyr142 were replaced with alanine. G104A and D230Y mutants remain active due to those residues having backbone interactions with mannose (Fig. 2a). Of the residues that comprise FRIL’s extended binding site, S45A showed no binding on Cali09 HA ELISA, but interestingly, Y101A (EC 7.08 ng/ml) and H102A (EC 3.64 ng/ml) had slightly increased binding compared to wild type control (EC 9.16 ng/ml), despite presumably losing the Tyr101 CH-pi interaction with galactose and His102 hydrogen bonds with GlcNAc and fucose.

Structural-based alignment show that most other legume lectins have small hydrophilic residues in the Tyr101 His102 positions (ConA Ser225 Thr226) (Supplementary Fig. 6a). This raises the possibility that by replacing the bulky side chains of tyrosine and histidine with a smaller alanine, we had somehow altered rFRIL’s binding specificity instead of negating it. To test this hypothesis, we created five Cali09 HA proteins with different N-glycans, including a “fully glycosylated” (FG) HA with mostly complex type but also some high mannose N-glycans, an endoglycosidase H (EndoH) digested “complex-only” HA (CX) HA, a kifunensine-treated Man9-only (M9) HA, Mannose GlcNAc (M5) HA from GNT1-/- cells, and an HA where each glycosite has only one GlcNAc remaining, dubbed “mono-GlcNAc” HA (MG) (Supplementary Fig. 6d-e). These HAs were then coated onto plates for ELISA detection by Y101A and H102A mutants (Fig. 2e). As expected, rFRIL wild type (rFRIL-wt) displayed the strongest binding towards FG and CX-glycosylated HAs (EC 7.4 ng/ml and 16.7 ng/ml, respectively), with relatively low affinity towards M5 and M9 (EC 3218 ng/ml and 229.7 ng/ml, respectively). In contrast, Y101A and H102A displayed similarly strong binding towards FG, CX and M9 (EC of 6.8, 15.8 and 5.9 ng/ml in Y101A, 3.6, 7.1 and 3.4 ng/ml in H102A), indicating the lectin itself is active but binding specificity for complex glycans has been lost. This result is confirmed by glycan array (Fig. 2d, Supplementary Data 2), where Y101A and H102A showed strong preference towards high mannose type glycans. Therefore, we have identified residues in FRIL’s extended binding site responsible for the lectin’s interactions with complex type N-glycans. Two of these residues, Tyr101 and His102, appear crucial for FRIL’s glycan binding specificity and warrants further investigation.

Structure-guided conversion of rFRIL’s specificity towards oligomannose glycans. Due to the surprising loss of glycan binding specificity observed in rFRIL mutants Y101A and H102A, a more thorough exploration of FRIL’s extended binding site was performed. First, we examined the cryo-EM structure of rFRIL-wt in complex with a Man9 N-glycan (Fig. 3a, Supplementary Fig. 3, Supplementary Table 1). Density for the entire Man9 ligand could not be fully visualized in rFRIL-wt’s CRD, but extra clear density map at the ligand site with a resolution of 2.4 Å allowed us to build two α1,3 linked mannoses in (Manα1,3-Man, Supplementary Fig. 4b). Not seeing the entire oligomannose ligand in our cryo-EM structure is expected, as rFRIL-wt has comparably weak binding towards Man9 but strong binding to complex N-glycans (Fig. 2e). The two mannoses we see likely correspond to the α1,3 branching points on either D1 or D2 arm of Man9. The non-reducing end of the primary mannose binds to FRIL in an almost identical conformation as the mannose present in our rFRIL-wt-tetrasaccharide structure, suggesting that FRIL’s Glc/Man-recognizing monosaccharide binding site binds to the same mannose position on both complex type and high mannose N-glycans. With monosaccharide bind site recognizing the same sugar on both oligosaccharides, glycan specificity is likely determined by the extended binding site. Indeed, in contrast to our tetrasaccharide bound FRIL structures where GlcNAc, Gal and Fuc come into contact with loop B residues 101-104, the density map for Man9 complexed with rFRIL-wt show no further α1,2 mannose extensions of the D1/D2 branch beyond the core α1,3 linked mannose (Fig. 3a, Supplementary Fig. 4b-c). Taken together, this implies that loop B may play a role in determining oligosaccharide specificity.

A sequence alignment of FRIL loop B (101 ~104, ConA 223~228) with the loop B of other related legume lectins (Fig. 3b, Supplementary Fig. 6a) reveal that Tyr101-His102 on FRIL loop B is replaced by Ser223-Gly224-Ser225-X226, with X being a small side chain such as threonine (in ConA, high mannose specificity), glycine (in Dioclea grandiflora lectin DGL, high mannose specificity17), or alanine (in Platypodium elegans lectin PELa, asymmetrical complex type specificity23). From this alignment, we speculate that we can shift FRIL’s specificity away from complex N-glycans and towards high mannose N-glycans by conducting mutagenesis on loop B. First, we introduced single mutations H102T, Y101S and the insertion of a glycine and serine residue between 101 and 102 (101insGS102) to make the peptide chain longer. The binding affinity of these mutants were then analyzed by lectin binding ELISA with differently glycosylated HAs (FG, CX, M9 and M5) (Fig. 3c, Supplementary Fig. 7, Supplementary Data 3). Y101S and 101insGS102 showed increased binding towards M9 and M5 HAs without losing their affinity to CX HA, while H102T had decreased binding across the board. Next, we combined these three mutants to create 101insGS-H102T and Y101S-insGS-H102T mutants, which retained their binding towards M9 and M5 while experiencing a 5-fold decrease to their CX affinity (EC 5.04 ng/ml in Y101S to 25.59 ng/ml in Y101S-insGS-H102T). This puts our rFRIL mutants in the vicinity of rproConA, but still does not completely negate binding to CX glycans. Therefore, we introduced a P145T mutation (ConA Thr15) that in previous reports corresponds to ConA’s extended binding site in trimannoside complexes14, 15. Adding P145T to our triple mutant to create P145T Y101SinsGSH102T decreased binding to CX another 16-fold (EC 25.59 ng/ml to 398.1 ng/ml), while affinity towards Man9 was retained (EC 2.63 ng/ml to 3.19 ng/ml) (Fig. 3c-d, Supplementary Fig. 7, Supplementary Data 3). We re-named this final mutant, with a 24-fold decrease in binding to CX glycans and a 72-fold increase in Man9 glycan compared to rFRIL-wt, as oligomannose specific FRIL (FRIL-OMS). Bio-layer interferometry using FG, CX and M9 HAs as ligand confirms that FRIL-OMS has decreased response and larger Kd to CX HA compared to rFRIL-wt, and an increased response and smaller Kd to M9 HA (Fig. 3e, Supplementary Fig. 8a). Carbohydrate competition lectin binding ELISA also show that FRIL-OMS but not rFRIL-wt was inhibited by yeast mannan (IC 0.07 mg/ml), an oligomannose polysaccharide containing α1,3, α1,6 and α1,2-linked mannose (Supplementary Fig. 8b).

Consequently, we have engineered a FRIL mutant (FRIL-OMS) that specifically recognizes oligomannose rather than its native ligand of complex type N-glycan, by replacing FRIL residues responsible for complex type binding with their structural equivalents on related, high mannose binding lectins.

Structural and anti-viral activity of FRIL-OMS compared to rFRIL-wt. To confirm that FRIL-OMS preferentially binds to oligomannose and the biological implications that this entails, we first determined the cryo-EM structure of the mutant in complex with Man9 (Fig. 4a, Supplementary Fig. 3, Supplementary Table 1). We found that the mutation FRIL-OMS did not affect the protein tetramerization or the overall structure (RMSD ~ 0.28 Å from the wild type). A clear extra density was observed at the active site that allow us to model three mannose inside, Manα1,2-Man α1,3- Man (Supplementary Fig. 4d). Consistent with previous findings, the middle mannose in our FRIL-OMS structure has almost identical conformation with the core mannose in either the monosaccharide bound, oligomannose bound, or the tetrasaccharide bound FRIL wild type. The only structural difference observed between FRIL-OMS and wildtype is a displacement of ~ 5 Å in loop B at the mutation site (Fig. 4b, Fig. 5). The mutation of rFRIL-wt’s Tyr101-His102 (YH) to Ser101-Gly101-Ser101-Thr102 (SGST) in FRIL-OMS resulted in a longer loop B, closing off a “gap” in the outer rim of FRIL’s binding pocket that Galβ1,4-(Fucα1,3-)GlcNAcβ1,2-Man tetrasaccharide originally passed through and interacted with (Fig. 5). Instead, the extra density for glycans was observed downwards by the SGST bulge in the Man9-bound FRIL-OMS (Supplementary Fig. 4d). An extra α1,2 mannose was built in, showing interactions with Thr102, Ser45 and Gln229 (Fig. 4a, Supplementary Fig. 4d). Taken together, two original interactions between rFRIL-wt and the tetrasaccharide ligand were broken (His102 with fucose and GlcNAc, Tyr101 with galactose), and three new interactions between FRIL-OMS and its Man9 ligand were formed (Thr102, Ser45, Gln229 with the third α1,2 mannose). We were unable to resolve Man9 interactions with P145T on FRIL-OMS but extra density around that region could be seen (Supplementary Fig. 4d, left red arrow), possibly corresponding to the α1,6 arm of Man9.

Previous reports indicate FRIL possess anti-influenza activity6. Therefore, we were curious if FRIL-OMS’s antiviral spectrum differs from its rFRIL-wt progenitor. FG, CX, M9 and MG-decorated Cali09 influenza virus (X181) were prepared as previously described6, and subjected to lectin blotting and microneutralization assay. On lectin blot (Fig. 4c, Supplementary Fig. 9a-c) rFRIL-wt exhibited strong binding towards the HA protein on FG and CX viruses, but only weak binding to M9. In contrast, FRIL-OMS binds strongly to M9 HAs and had negligible binding to FG and CX. With the same FG and M9-decorated viruses, in a microneutralization assay (Fig. 4d) rFRIL-wt neutralizes FG but not M9 virus, while FRIL-OMS neutralizes M9 but not FG virus. The mutant H102A, which had previously shown a broadened glycan specificity including both complex type and mannose 9 glycans, was able to bind and neutralize both FG and M9 viruses, while rproConA also recognizes both FG and M9 but showed preference towards M9-decorated viruses (Supplementary Fig. 9c-d).

Finally, we compared the neutralization profiles of rFRIL-wt and FRIL-OMS against a panel of influenza viruses with differing density of oligomannose N-glycans on their HAs (Fig. 4e). The egg-grown Cali09-like (H1N1) vaccine strain X181 has 6 N-glycosylation sites per HA monomer, four of which are mostly complex type and two (104 and 304) mostly high mannose24, but lack glycans on top of the HA head. Egg-grown A/New Caledonia/20/1999 (NC99, H1N1 pre-pandemic) has 8 N-glycosylation sites, two of which (142 and 177) are on top of the HA head and are mostly oligomannose25. A/Victoria/361/2011 (Vic11, H3N2 strain) HA has 11 glycosylation sites, three of which (133, 165 and 285) being high mannose on the HA head, creating a “high-mannose patch”26. Results show that while rFRIL-wt has relatively similar neutralization titers across all three strains (IC Cali09 0.23 μg/ml or 2.02 nM, NC99 0.37 μg/ml or 3.25 nM, Vic11 0.78 μg/ml or 6.85 nM), FRIL-OMS performed the best against Vic11 (IC 4.10 μg/ml or 36 nM). This neutralization profile is in line with other known oligomannose binding antiviral lectins, such as Griffithsin27, H84T Banlec28 and Galanthus nivalis lectin (GNA)29 (Supplementary Fig. 9g). A concern we noticed previously6 was that ConA was cytotoxic to MDCK cells at higher concentrations, which shows up as false negatives in microneutralization assay. In our current study, neither rFRIL-wt nor FRIL-OMS caused cytotoxicity up to 500 μg/ml (Supplementary Fig. 9e). rproConA does show some cell death but not as severe as mature ConA, possibly due to being a dimer rather than tetramer.

In summary, a comparison between FRIL-OMS and rFRIL-wt structures (Fig. 5) reveal that in FRIL-OMS the removal of tyrosine and histidine residues from the 101 and 102 position breaks two interactions with galactose and fucose on our tetrasaccharide, and the elongation of loop B causes a clash between galactose and the binding site. In contrast to tetrasaccharide, the elongation of loop B on FRIL-OMS displaces Man9 downward, where an additional α1,2-linked mannose on the D1 branch of Man9 forms new interactions with the threonine present at the 102 position, and Ser45 which originally interacted with GlcNAc on the tetrasaccharide. The effects of this shifted glycan specificity can also be reflected in FRIL-OMS’s biological activity against influenza viruses, with FRIL-OMS showing a starkly different binding and neutralization profile than its wild type progenitor. These results show we have an engineered FRIL-OMS which completely shifts its binding specificity from Lewis X containing N-glycan to high-mannose type N-glycans.

Methods
Ethical statement
Chicken embryos used in this study were euthanized before 75% total incubation period (Day 16), and are therefore not subject to Academia Sinica Institutional Animal Care and Use Committee (IACUC) approval.

Biosafety considerations
Experiments using influenza viruses were approved by the Institutional Biosafety Committee of Academia Sinica (protocol no. BSF0420-00004402) and conducted by trained personnel in a Biosafety Level 2 laboratory.

Plasmid construction and protein expression
Codon-optimized FRIL, proConA and Cali09 HA sequences were synthesized by Genomics, Taiwan with a N-terminus Brisbane-HA signal peptide and C-terminus 6xHis tag. FRIL and Cali09 were cloned into a pTT expression plasmid (National Research Council of Canada) while proConA was cloned into pcDNA3.1 (Thermo Fisher). Primer sequences are provided as Supplementary Data 4. Plasmids were transiently transfected into early log-phase (1.6~2.2x106 cells/ml) HEK293 EBNA (ATCC CRL-10852) or HEK293S (ATCC CRL-3022 GnTI-, for M5 HA) cells with PEI (polyethylenimine, linear, MW25,000, Polysciences Inc.), in a ratio of 2 mg plasmid 4 mg PEI per 1 liter cell. For M9 and MG HA, kifunensine (Sigma Aldrich) is added to a final concentration of 1 mg/L. Transfected cells were cultured in a humidified incubator at 37°C with 8% CO2 with orbital shaking at 120 rpm for 5~6 days in Freestyle 293 medium (Thermo-Fisher Scientific) with 0.5% fetal bovine serum (Thermo-Fisher Scientific). After 5~6 days expression, supernatant is collected by centrifugation at 10543 rcf.

Recombinant proteins were purified with Ni-NTA (GE healthcare) beads. Beads were added to the harvested supernatant, and the mixture is incubated for 1 hour on an orbital shaker. The slurry is poured into an open column, and Ni-NTA beads are then washed twice with 10 column volumes of PBS, followed by 10 column volumes of 30 mM imidazole twice, 50 mM imidazole once, before the His-tagged proteins are eluted with 5 column volumes of 150 mM imidazole twice. The 150 mM imidazole eluate is buffer exchanged three times with PBS on a 50 kDa spin filter (Amicon).

For rFRIL and proConA, the eluate is then subjected to 4 hours 37°C PNGase F treatment at a ratio of 40 μg of PNGase F per 1 mg lectin. Some precipitation of the lectin is expected, especially for mutants. After PNGase F cleavage, the 36 kDa enzyme and our 100 kDa lectin are separated with a 50 kDa spin filter and 4~5 times washing. Do not use dextran or other polysaccharide-based size-exclusion chromatography during proConA and FRIL’s purification process.

For Cali09 HA, the PBS buffer-exchanged eluate undergoes a final polishing step by gel filtration. For MG and CX HA, endoglycosidase H cleavage happens before the polishing step, at a ratio of 1 mg HA to 100 μg Endo H for 1 hour in a 37°C waterbath. Afterwards, the HAs are loaded onto a Superdex 200 size exclusion column (GE healthcare), and the HA trimer peak is collected around 210 kDa.

Native FRIL purification from bean
Lablab purpureus beans were purchased from a traditional medicine store in Taiwan, and ground into fine powder using a blender (Blendtec). PBS was added to bean powder at a 10:1 ratio, and placed on an inverting mixer at 4°C overnight for protein extraction. Next day, the mixture is centrifuged at 10000g 25 min to remove bean powder. 20% w/w of ammonium sulfate is added to the supernatant and mixed at room temperature for 1 hour. The solution is centrifuged for 12000g 30 min, and pellet discarded. Another 40% w/w of ammonium sulfate is added to the supernatant for a total of 60% ammonium sulfate, and the solution is mixed at room temperature for 1 hour to precipitate nFRIL. After centrifugation at 12000g 30 min, the supernatant is discarded, and the pellet is re-suspended in PBS containing 1 mM CaCl2 and 1mM MgCl2.

1 g mannose beads (Sigma-aldrich) is added per 20 ml re-suspended pellet, and the mixture is incubated overnight at 4°C on an orbital shaker. The next day, the bead slurry is poured into an open column. The beads are then washed twice with 10 column volumes of PBS containing 1 mM CaCl2 and MgCl2. nFRIL is eluted from mannose beads with 200 mM of methyl-D-α-mannopyranoside (Sigma Aldrich), and the eluate is then dialyzed twice with PBS containing 1 mM CaCl2 and MgCl2 pH 8.

Ion exchange is used as the final polishing step for nFRIL purification. 1 ml of Unosphere Q beads (BioRad) is placed inside an open column, and the dialyzed nFRIL solution is passed through 3~4 times. nFRIL is found in the flowthrough while non-specific proteins bind to Unosphere Q beads. The flowthrough with nFRIL is then concentrated by a 50 kDa spin column (Amicon).

FRIL mutagenesis
Either KOD polymerase kit (Sigma-Aldrich) or Q5 Site-directed Mutagenesis Kit (New England Biolabs) were used to conduct mutagenesis, according to their respective protocols. Primers were synthesized by Genomics, Taiwan, and all mutants were sequenced for confirmation. Primer sequences are provided as Supplementary Data 4. Expression and purification of mutants followed the same protocol as rFRIL.

Influenza virus production and purification
10,000-fold diluted A/California/7/2009-like virus (X181, vaccine strain) was inoculated into the allantoic cavity of 9 day old fertilized chicken eggs (Animal Health Research Institute) at 100 μl/egg. The M9 and MG viruses were also injected with 0.2 mg/ml kifunensine. The injection hole was sealed with superglue (3M), and eggs were incubated for 48 hours in a 35°C incubator before being placed in 4°C for another 24 hours. Influenza viruses were harvested by collecting the allantoic fluid. The virus is then concentrated 10 to 20-fold using a 100 kDa spin filter. For MG viruses, endoglycosidase H was added to a final concentration of 20 μg/ml at this step, and allowed to cleave overnight at 4°C. Finally, the virus stocks are filtered through a 0.22 micron bottletop filter.

To purify virus particles for lectin blot, 25%, 30%, 40%, 50% and 55% (w/w) sucrose solution were prepared in PBS, and 5 ml of each solution was sequentially loaded onto a 25x89 centrifugal tube (Beckman Coulter), creating a 25~55% sucrose gradient. 10~14 ml of concentrated allantoic fluid is layered on top of the tube, and the tube is centrifuged at 20000 rpm (72100 rcf) for 15 hours (Optima L-90K, Beckman Coulter) on a SW-28 swing bucket rotor. The next day, virus particles were harvested in fractions of 1 ml starting from top of the tube, and hemagglutination assay on each fraction is conducted under a biosafety cabinet. Fractions with the highest hemagglutination titers are pooled and desucrosed by buffer exchange using a 100 kDa spin filter (Amicon).

Lectin binding ELISA and lectin blotting
In lectin binding ELISA, 2 μg/ml of HA, Spike or fetuin glycoprotein in PBS were coated onto an ELISA plate at 50 μl/well (0.1 μg/well) 4°C overnight. The next day, the ELISA plate is blocked by 10% bovine serum albumin (Bioman) at 100 μl/well for 1 hour room temperature. Blocking solution is removed, and rproConA, nFRIL, rFRIL or FRIL mutants are loaded onto the first well of the plate at 10 μg/ml 146 μl/well, and 100 μl dilution buffer (PBS with 1 mM CaCl2 and MgCl2 and 1% bovine serum albumin) loaded into subsequent wells. 0.5 log10 serial dilution is conducted by transferring 46 μl sample down to the last well. The plate is then incubated for 1 hour at room temperature. Afterwards, the lectins are removed, and the plate is washed twice with PBS-TN (PBS with 0.05% tween 20) containing 1 mM CaCl2 and MgCl2. 1:2500 rabbit polyclonal anti-FRIL or anti-ConA antibody (LTK laboratories) in PBS-T containing 1 mM CaCl2 and MgCl2 is applied to the plate at 100 μl/well. This primary antibody is incubated for 1 hour at room temperature. The plate is washed three times and 1:5000 anti-rabbit HRP (Jackson Immuno-Research) is applied to the plate at 100 μl/well. After another hour at room temperature, the plate is washed three times. 50 μl/well of KPL SureBlue (Seracare) substrate is added to each well, and the reaction is stopped with 50 μl/well of 1M H2SO4. OD is measured with Cytation5 (Biotek). Of note, calcium must be present in all buffers at each step beyond lectin application. Skim milk or any blocking solution that contain glycoproteins or sugars cannot be used.

In yeast mannan and methyl-D-α-mannopyranoside carbohydrate competition lectin binding ELISA, yeast mannan from Saccharomyces cerevisiae (Sigma Aldrich) was prepared at a concentration of 360 mg/ml in PBS (1 mM CaCl2, MgCl2), and methyl-D-α-mannopyranoside (Sigma Aldrich) was prepared at a concentration of 2M. These sugars were serially diluted on a U-bottomed 96-well plate, and an equal volume of 2 μg/ml rFRIL-wt, FRIL-OMS or rproConA were added to each well for a final concentration of 1 μg/ml lectin in all wells This mixture is incubated at room temperature for 15 mins before being transferred, well-to-well, to an ELISA plate coated with Cali09 HA glycoprotein and blocked by 10% BSA. The lectin-binding ELISA then proceeds as outlined in the previous section.

In lectin blotting, purified intact Cali09 virus particles adorned with different glycans were lysed using SDS loading dye (no DTT) and 100°C boiling for 10 minutes. The lysed viruses were then loaded onto a 4~20% gradient gel (BioRad). Viral proteins were transferred onto a PVDF membrane using the semi-dry method (Trans-Blot SD, BioRad), and blocking was performed using 10% bovine serum albumin overnight at 4°C on an orbital shaker. The next day, either rFRIL-wt, FRIL-OMS, or rproConA was added to the membrane at 2 μg/ml in PBS containing 1 mM CaCl2 and MgCl2. The lectins were then allowed to bind to glycoproteins on the lysed virus for one hour with orbital shaking. Afterwards, the lectins are removed, and the membrane is washed twice with PBS-T containing 1 mM CaCl2 and MgCl2. 1:2500 rabbit polyclonal anti-FRIL or anti-ConA antibody in PBS-T with 1 mM CaCl2 and MgCl2 is applied to the membrane. The primary antibody is incubated for 1 hour with orbital shaking at room temperature. Then, the plate is washed three times, and 1:5000 anti-rabbit HRP is applied to the membrane. After another hour of shaking at room temperature, the membrane is washed three times, and Clarity ECL substrate (BioRad) is applied. Chemiluminescence is visualized using iBright 1500 (Invitrogen). After visualization, the membrane is stripped using Restore Stripping Buffer (Thermo Scientific), and blocking is applied once more. 1:2500 rabbit polyclonal anti-NP antibody (LTK laboratories) is applied to the membrane, followed by 1:5000 anti-rabbit, and influenza NP protein is visualized as a loading control. Unprocessed and uncropped blots are available in source data files.

Microneutralization assay
TCID50 is first determined for the egg-grown third passage (P3) virus stocks of A/California/7/2009-like (X181), A/New Caledonia/20/1999-like (A/Taiwan/223/01) and A/Victoria/361/2011-like (IVR-165) influenza vaccine strains. 146 μl of the virus stock is diluted 1000-fold and loaded into the first well of a 96-well tissue culture plate, and 100 μl of virus dilution medium (DMEM with 25 mM HEPES, penicillin-streptomycin, 0.2% BSA) is loaded onto subsequent wells. Serially dilute the virus stock down to the 11th well by transferring 46 μl from the previous well to the next one (0.5 log10 dilution), for a 103 to 108 test. Plates were placed in 37°C 5% CO2 incubator for 1 hour to mimic microneutralization conditions, and 100 μl/well of 1.5x105 cells/ml MDCK cells (ATCC) in virus dilution medium is added. Incubate the plate for 18~20 hours in a 37°C 5% CO2 humidified incubator, or until CPE can be seen but before cell death and detachment. The plate is washed once with PBS, then fixed and permeated with ice cold 50% methanol 50% acetone. After drying the plate, 100 μl/well 10% skim milk 1% BSA in PBS is applied to the plate for 1 hour blocking. 100 μl/well 1:2500 rabbit polyclonal anti-NP antibody (LTK laboratories) in 10% blocking solution (1% skim milk 0.1% BSA in PBS with 0.05% tween 20) is added to plate, and allowed to incubate at room temperature for another hour. Afterwards, the plate is washed 3 times with PBS-T, and 100 μl/well 1:5000 anti-rabbit HRP (Jackson Immuno-Research) is added. After another hour of incubation, the plate is washed three times, and 50 μl/well of KPL SureBlue (Seracare) substrate is applied. Reaction is stopped with 50 μl/well of 1M H2SO4. OD is measured with Cytation5 (Biotek). TCID50/ml is calculated as the first serially-diluted concentration whose OD is above twice that of negative control wells.

For microneutralization assay, 73 μl of 50 μg/ml rFRIL-wt, FRIL-OMS, Griffithsin (NIH AIDS reagent program), H84T Banlec (kind gift from Dr Markovitz’s lab) and Galanthus nivalis agglutinin (GNA, Sigma Aldrich) were added to the first well (5 μg/ml for Griffithsin against NC99 Vic11, to keep its slope within the dilution range), and 50 μl/well of virus dilution medium is added to subsequent wells. 23 μl is sequentially diluted down from the first well in a 0.5 log10 serial dilution. 15 TCID50/50μl of virus is then added to each well. The plate is incubated at 37°C 5% CO2 1 hour for virus neutralization, then 100 μl/well of 1.5x105 cells/ml MDCK cells (ATCC) in virus dilution medium is added. Subsequent steps are the same as TCID50 test. Microneutralization % is calculated as {[(positive control – negative control) – (test well – negative control)]/(positive control – negative control)}x100%.

Glycan array analysis
All glycans used in the array analysis are prepared by the modular synthesis method42. Glycans were dissolved in printing buffer (300 mM phosphate buffer, 0.005% triton, pH 8.5) in 100 μM concentration. Microarrays were printed (BioDot; Cartesian Technologies) by a robotic pin (SMP3; TeleChem International) with the deposition of ∼0.6 nL of various solutions from 96-well plates onto NHS-coated glass slides (Nexterion H slide; SCHOTT North America). Printed slides were allowed to react in an atmosphere of 80% humidity for one hour followed by desiccation overnight. The prepared slides were stored in a humidity controlled dry box before the binding assay.

Glycan microarray slides with 169 different glycans were blocked for 1 hour with blocking buffer (Superblock, Thermo Fisher Scientific) and then washed with PBST (PBS buffer, 0.05% Tween 20).10 µg/ml His-rFRIL was applied onto the array and incubated at room temperature for 1 hour, and then washed with PBST to remove unbound rFRIL. Anti-His antibodies (Alexa Fluor® 647 anti-His, Biolegend) were applied (1:300 dilution, PBST+BSA) for another incubation at room temperature for 1 hour, and then washed with PBST. Microarray slides were spun dry prior to scanning with a GenePix 4300A reader (Molecular Devices, San Jose, California) at OD 635 nm and analyzed with GenePix Pro 7.0 software (Molecular Devices).

Hemagglutination assay
Turkey red blood cells (tRBC, Jianrong) were washed three times with PBS, and diluted to 0.5%. Add 50 μl PBS into each well of a V-bottomed 96 well plate, then add 50 μl of the lectin or virus sample to the first well for a total of 100 μl. Serially dilute 50 μl of the sample down to the last well, then add 50 μl of the 0.5% tRBC to each well (for a total of 100 μl). Allow the tRBC to settle for 30 min to 1 hour.

Biolayer interferometry
FG, CX and M9 HAs were labeled with biotin using N-hydroxysuccinimide (NHS) ester (EZ-link-LC-LC-Biotin, Thermo Scientific) at a 1-to-5 molar ratio 4°C overnight in PBS pH 7.4. Excess NHS-biotin were removed by size exclusion chromatography. Biolayer interferometry was performed on an Octet HTX system (Sartorius) using Octet SA Biosensors (Sartorius). 100 nM of biotinylated HA were loaded onto sensors for 300 seconds, followed by a baseline of 120 seconds, association 600 seconds and dissociation 600 seconds. A 2-fold serial dilution of various lectins starting from 200 nM were used as analyte (in PBS-T with 0.01% BSA, 1 mM CaCl2 and MgCl2), with single reference and no probe regeneration. Results were analyzed with Octet Data Analysis software (Sartorius) using a 2:1 heterogenous ligand model.

SEC-MALS
Size exclusion chromatography with multi-angle light scattering was performed by running the purified protein samples through an ENrich™ SEC 650 10x 300 column (BioRad) on an Akta FPLC (GE) connected to a three-angle light-scattering detector (mini-DAWN TREOS) and a refractive index detector (Optilab T-rEX. Wyatt Technology, Santa Barbara, California). Data analysis was done with ASTRA.

MTT cytotoxicity assay
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Invitrogen) was dissolved as a 12 mM solution in PBS. MDCK cells were grown to 80% confluence on a 96-well plate, and medium replaced with 100 µL/well of serially diluted lectins in culture medium (DMEM without FBS) for 20 hour incubation. 10 µL of MTT solution was added into each well before incubating for another 4 hours. 100 µL/well of MTT solvent (10 mM HCl, 10% SDS) was added, and the plate incubated overnight before absorbance at 570 nm was read to assess cell survival.

Crystallization, X-ray diffraction and structural refinement
Purified nFRIL was concentrated to 8 mg/mL and mixed with the tetrasaccharide at a final concentration of 10mM. The synthesis and validation of the Galβ1,4-(Fucα1,3-)GlcNAcβ1,2-Man Lewis X tetrasaccharide is shown in source data. Hanging-drop vapor diffusion crystallization was set at 20°C and a single crystal appeared on day 3 in the condition containing 0.2 M ammonium fluoride pH 6.2, 20% PEG 3350. Crystals were harvested without the addition of cryoprotectant and diffraction datasets were collected at National Synchrotron Radiation Research Center (NSRRC) beamline TPS05A, in the wavelength of 1.0000Å and temperature 100K. Data was processed in iMosflm43; Molecular replacement was performed by Phenix Phaser-MR44 using the monomer structure of PDB 1QMO14 as the searching ensemble. The 3D structure of Lewis X tetrasaccharide and all other figure-related glycans were encoded with CSDB glycoscience for building and optimizing in elBOW. The solved structure was further refined in phenix.refine44 (used NCS) with manual adjustments done in WinCoot45 until R/Rfree converged and all geometry statistics were satisfactory. Structural figures were prepared with PyMOL46 and ChimeraX UCSF47.

Negative stain transmission electron microscopy
Purified nFRIL or rFRIL protein was diluted to 20 μg/ml in PBS before being applied onto glow-discharged carbon-coated 400 mesh CF400-CU grids (Electron Microscopy Sciences) for 1 min at room temperature. Excessive protein solutions were blotted with filter paper and grids were washed 3 times in water prior to staining by 1% uranyl acetate for 60 seconds at room temperature. Data was collected using a FEI Tecnai G2 F20 S-TWIN electron microscope (Thermo Fisher Scientific) operating at 120 keV and a magnification of 29K that resulted in a pixel size of 3.13Å at the specimen plane. 3D reconstruction was processed by cisTEM48.

Cryo-EM sample preparation, data harvesting and processing and structural refinement
Purified nFRIL, rFRIL or FRIL-OMS lectins (0.06-0.08 mg/ml in 1/10 PBS buffer) were either mixed with the synthetic Galβ1,4-(Fucα1,3-)GlcNAcβ1,2-Man tetrasaccharide (10 mM), the Man9 GlcNAc2 (10 mM) oligosaccharide, or present in its apo state. The Man9 GlcNAc2 oligosaccharide is prepared by the modular synthesis method42. 3-4 μl of the sample was applied on to a graphene-oxide-coated Quantifoil R1.2/1.3 Cu 200 holey carbon grid mounted in a Mark IV Vitrobot (Thermo Fisher Scientific) equilibrated at 4°C with 100% humidity. Grids were blotted at force 0 for 3 seconds, waiting 10 seconds. Data was collected on a Titan Krios G3 at 300 kV (Thermo Fisher Scientific), equipped with a Gatan K3 detector and the Gif Quantum energy filter with 20 eV slit width. Movies were acquired in EPU (Thermo Fisher Scientific, v2.10) at two exposures per hole. Total electron dose was ~50 e-/A2 and fractionated into 50 frames. The movies were collected at the magnification of 105K (for apo rFRIL and nFRIL-tetrasaccharide) and 130K (for rFRIL-OMS-Man9 and FRIL-wt-Man9) in the defocus range from -1.2 to -2.0 μm. Movies were motion corrected using MotionCorr249. The micrographs were CTF-corrected, particle were picked and processed in relion 3.050 (for rFRIL apo and nFRIL+tetrasaccharide data sets) or CryoSPARC ver 451 (for nFRI-wt +Man9 and rFRIL-OMS +Man9 data sets). The D2 symmetry was applied, the resolution of the final volume determined by gold-standard Fourier shell correlation (FSC) cutoff at 0.143. PDB 1QMO14 was used for model fitting, and after the first cryo-EM structure was solved, the latter was used for model fitting on all subsequent datasets. The building of glycans and inter-subunit loops was done in Coot45. Geometry statistics were evaluated using Phenix real space refinement44.

Quantification and statistical analysis
All data were expressed as mean ± standard error unless otherwise specified. Curves were fitted by variable-slope nonlinear regression for EC50 or IC50 values using GraphPad Prism 9.0. Sequence alignment was performed using DALI52.

Biological materials availability
All biological materials produced in this study are available upon request to the corresponding author.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Liu et al., “Altering the Carbohydrate-Binding Specificity of the Legume Lectin FRIL Through Structure-Guided Engineering,” presents a well-executed study in which the glycan-binding specificity of FRIL is successfully redirected from complex-type to high-mannose-type N-glycans. The authors achieve this through rational loop mutagenesis based on structural insights. Furthermore, the study introduces a novel expression and activation protocol using mammalian cell systems followed by enzymatic deglycosylation. This dual approach represents a significant methodological and conceptual advance for the lectin and glycobiology fields.\nComments:\n1. Given that native FRIL is a double-chain lectin, the authors should comment on the potential impact of producing it as a single-chain polypeptide, as is the case for the recombinant form. It would be valuable for the authors to comment on whether this is expected to affect binding specificity or affinity.\n2. The manuscript convincingly shows that the lectin self glycan is responsible for its inactivation prior to cleavage. However, it is not clear for the reviewer the reason behind the recovery of the activity when the glycan is not removed completely, as it is the case for native FRIL. Additionally, the expected paucimannose glycan is expected to have a lower affinity for the lectin. Is it a matter of positioning and/or conformational flexibility of the glycan? Given the importance of the presented findings, the authors should expand on this.\n3. The authors provide a good rationale for the importance of most mutated residues. However, the high importance attributed to S45 and especially Y147 is surprising. As Y147 appears to be part of the canonical monosaccharide binding site, its specific role in modulating specificity in the extended binding site is not immediately obvious. The authors should include a more detailed explanation for the importance of these residues to better support the findings.\n4. The proposed importance of Y101 and H102 could be further substantiated through molecular docking simulations. Including such analyses would strengthen the conclusions drawn about the roles of these residues.", "reviewer_2": "This study by Liu et al presents a structure-guided engineering approach to alter the glycan-binding specificity of the legume lectin FRIL, a protein with broad-spectrum antiviral activity. The authors overcame longstanding barriers to producing bioactive FRIL by expressing recombinant protein in mammalian cells and activating it through enzymatic deglycosylation. Using cryo-EM and X-ray crystallography, they identified key residues in loop B (Y101, H102) as critical determinants of FRIL's preference for complex-type N-glycans. By replacing these and adjacent loop C residues with counterparts from the high-mannose-specific lectin ConA, they created a FRIL mutant (FRIL-OMS) that selectively binds high-mannose N-glycans. Structural analysis confirmed the altered binding mode, while functional assays showed that FRIL-OMS binds and neutralizes influenza strains rich in oligomannose, contrasting the specificity of wild-type FRIL. These findings establish a framework for reprogramming lectin specificity through rational loop engineering, with broad implications for antiviral design and glycan-targeting technologies.\nThe biochemical and structural data presented in the study are of high quality. The manuscript is logically organized and generally well written, though it may benefit from editing by a native English speaker. The figures are also of high quality.\nWhile the manuscript makes a meaningful and technically impressive contribution to the fields of glycoscience, lectin biology, and antiviral lectin engineering, its specialized focus on legume lectin structure-function relationships and glycan specificity reprogramming may limit its accessibility to a broader readership.", "reviewer_3": "In this manuscript titled “Altering the Carbohydrate-Binding Specificity of the Legume Lectin FRIL Through Structure-Guided Engineering” by Yo-Ming Liu et al. The authors present a comprehensive study demonstrating how structure-guided mutations can be used to alter the glycan binding specificity of a legume lectin, FRIL. Through binding assays, Cryo-EM studies, and molecular modeling, they successfully demonstrated how deglycosylation of their recombinant lectin, achieved by site-directed mutagenesis (Y101A, H102A, P145T), enables a specificity shift from complex N-glycans towards high-mannose glycans, while retaining structural integrity and binding activity. The study is well-executed, with all data and experiments presented clearly and logically, and the conclusions are well-supported by combinations of relevant structural, biochemical, and virological assays. There are several questions in the manuscript listed below that should be addressed before its publication.\n1. Binding avidities or dissociation constants along with Hill coefficients were not presented (pages 6,7, and 9; lines 205-235, 299-304) for both wild-type FRIL and its mutants. Providing these would enable a more quantitative comparison of their binding properties.\n2. How would the formation of a mutant that only has the P145T mutation without alterations to the other residues in Loop B affect binding? Where would this mutant stack in comparison to the others?\n3. How would this self-neutralizing ligand, as stated (page 11, line 372), operate in nature? Similarly, what would happen if you attempted Ramis et al (page 11, line 374) approach and attempted to actuate pH to cause protein activation?\n4. Did any mutation occur in which all binding was subsequently negated? (page 6, line 217) If no such mutant was found, would the possibility of other important residues exist in the full structure of the lectin?", "reviewer_5": "The manuscript by Liu and colleagues described how to switch the specificity of plant lectins such as FRIL from complex glycan towards oligomannosides for improved antiviral activity. They altered and swapped amino acids mainly in the loops B and C affecting ligand recognition in plant lectins. First of all, they also develop a protocol to produce those plant lectins in mammalian cells in inactive form and activate them thanks to the deglycosylation of their linker with PNGase F. They provide a thorough functional and structural characterization of the rationally designed mutants using a large panel and techniques: Elisa, lectin blotting, MALS, glycan arrays, neutralization assays as well as structurally to explain the impact of the mutations at the atomic level by CryoEM and X-ray crystallography. The used of SNFG symbol and coloring is greatly appreciated.\nThis very comprehensive work has been carefully done and provides an interesting approach to pave the way to create lectins with improved properties such as antiviral activities but also new molecular tools that could be used in some areas of biotechnology or even for biomedical applications. Whilst this study is of great significance with all claim supported by the results, there are a number of issues to be clarified and concerns addressed to further improve the manuscript for publication in Nature communications listed below.\n-The Summary is too long and should be shortened to the 200 words recommended. It should not contain any references. Some sentences are redundant.\n-The references should be updated to comply to Nat com style. Name, Initial. Title, abbreviated journal, volume, pages, year. The journal names should be abbreviated, some volume and pages number are missing as in ref 4,24. Reference 5 for a book chapter is incomplete. Organism name should be in italics.\n-There should be no coma before and when it is in a list. It should be only when connecting two independent clauses.\n-Add a paragraph at the end of the introduction on the aims and means of your study.\n-Line 21: replace ‘ Here we show legume lectin’ by Here, we show that legume lectin.\n-Line 23: add respectively after (ConA).\n-Line 32: this sentence is not clear, some word seem to be missing, rewrite in a clearer way.\n-Line 53: This sentence should be placed at the end of the paragraph after the principle of two chain and cyclic permutation are explained. In that case, the paragraph could start as Plant lectins are produced….\n-Line 111: replace “The 293E expression system yielded approximately 4~5 mg secreted” by The 293E expression system yielded to approximately 4~5 mg of secreted.\n-Line 151: error in figure number.\n-Lines 153-157: exemplify this sentence. It is not clear of the point to be made by the authors. -From the figure there is no hemagglutination even after deglycosylation at neutral pH. What is the relation with the dimerization. If it is deglycosylation and not hemagglutinating, does that mean it is not a dimer. Has MALS been done at different pH after degkycosylation?\n-Line 172: The sentence “ In the crystal structure, the tetrasaccharide ligand is fitted into the cleft between symmetry copies in crystal packing” is misleading since as said just after all the 16 binding sites in the asymmetric unit are occupied with an identical binding mode. The binding does not involved interaction due to crystal packing so that should not be mentioned. The “between symmetry copies” implies an interface and that is not the case. There is only one binding site per protomer and the binding is independent of the crystal symmetry. Please modified to make it clearer.\n-Line 180: replace adopt by adopts\n-Line 185: the interaction with the main chain of Asp230 is not visible in Fig2 as well those with His102 main chain. Perhaps a slight change in the orientation will help to visualize them better. From the figure 2A, it seems the interaction of the amine of His102 is made with the ring oxygen of GlcNAc through a water and with the 6-OH and Tyr147 OH is interacting with GlcNAC 6-OH with an interaction at 2.76 angstrom as shown in extended figure 3B. So, the sentence line 191 “Tyr147 may also interact with the GlcNAc” is incorrect. The anomeric oxygen of Man is also missing in Fig2A. There should be consistency between figure 2A and extended 3B. One water is added in 3B which no interaction whilst the one water mediated interactions with the main chain of His 101 are not described.\n-Paragraph starting line195: it could be mentioned that apart Ser45, all other residues belong to the 4 loops responsible for the architecture of plant lectin binding site and for fine specificity.\n-Line 211: replace glucose with GlcNAc.\n-Line 234: how do the authors explained that the IC50 for CX are almost identical or better for the H102A and Y101A mutants compared to the wildtype with good IC50 and is comparable with Man9 but on the glycan array no complex glycans are recognized? The selectivity for complex glycan is indeed lost but they seem to be still good ligand in Elisa. Have the authors an explanation for that?\n-Lines 308-309: add that after show for better English and rewrite seen in loop B mutation site for better clarity.\n-Line 307, 681, 820: typo in tetrasaccharide Idem for the legend of C panel extended figure 7\n-Lines 333. Why mentioning extended figure 5 panel D since nothing is said on those results in the text for H102A and rproConA. Add a sentence about those results.\n-Line 399, perhaps mentioned that here you manage to get an active rProConA dimer which present the same activity than the permutated active ConA tetramer.\n-Line 813: Replace GluNAc by GlcNAc\n-SNFG coloring should be mentioned in the legend of all figures using it. Otherwise introduced it in figure 1 and refer to that legend for the other figures.\n-The numbering of the extended data figures should be according their appearance in the main text.\n-The resolution of figure 5 and extended panel B extended data 3 need to be improves. In the interaction figures, perhaps using black dashes will be more visible than the cyan one and those between the sugar atoms can be removed for clarity. The transparency used for some sugar is a bit too high. It give the impression that the sugar are bound behind the loop.\n-The structure with Man9 show distortion for one mannose and that should not be the case. There should be no distortion of N-glycan bound to lectin in particular at this resolution. Constrain should be applied to obtain the 4C1 chair. This would be misleading to leave those distorded mannose. The authors could use the privateer program to check and validate their sugar. An application is available online (https://privateer.york.ac.uk/) or more complete in CCP4i2. See ref Dialpuri et al, acta cryst 2023.\n-In the extended data table 2, the authors should add the CC1/2 and the nb of measured and unique reflections collected in the data collection section and Ramachandran values in the refinement one. Unit cell values with only two digits number are sufficient as in PDB validation report. In the area of synchrotron and pixel detector, it is surprising to see structure with a low completeness , here 83.5% and 74.3 in the outer shell. It takes a few minutes to collect now a full 360 ° and redundancy and completeness have their importance in particular for complexed structures and for error estimation. It is always better to have too much data than not enough. Is here a reason why incomplete data very collected or why merging. The authors should mention in the crystallization paragraph if the crystal were cryoprotected and is yes how as well has the type of detector used on the beamline. Have the NCS been used for refinement? From the validation report, It is clear that the constrains used for the refinement are too tight or the library was not the most appropriate. They are too many bond lengths and torsions and chiral etc, listed in the validation report. The authors have used automatic weighting in phenix as seen for the general rsmd bond length. This could be taken into account for future structures. This is also responsible for the long list of close contact for the protein. There are also many close contact with water molecules and have the authors verify the listed ASN and Gln side chains should be flipped?"}, "author_response": "Reviewer #1 Comments and Response\nThe manuscript by Liu et al., “Altering the Carbohydrate-Binding Specificity of the Legume Lectin FRIL Through Structure-Guided Engineering,” presents a well-executed study in which the glycan-binding specificity of FRIL is successfully redirected from complex-type to high-mannose-type N-glycans. The authors achieve this through rational loop mutagenesis based on structural insights. Furthermore, the study introduces a novel expression and activation protocol using mammalian cell systems followed by enzymatic deglycosylation. This dual approach represents a significant methodological and conceptual advance for the lectin and glycobiology fields.\nThank you for your kind words. We very much hope that the methodology we employed in this study can also be of use to others in this field.\n1. Given that native FRIL is a double-chain lectin, the authors should comment on the potential impact of producing it as a single-chain polypeptide, as is the case for the recombinant form. It would be valuable for the authors to comment on whether this is expected to affect binding specificity or affinity.\nResponse:\nThank you for bringing up this important point. Producing an active recombinant FRIL was the first obstacle we had to overcome in the current study, and activity was initially assessed using the fast and simple method of hemagglutination assay. As a result, a lot of methods we developed later on (like ELISA with differentially-glycosylated hemaggutinin) were never tested on natural FRIL, and we are also curious whether there may be any differences in binding specificity or affinity between nFRIL and our recombinant version.\nTo assess this, we first conducted lectin binding ELISA with complex-type HA and mannose 9 HA comparing native FRIL to our recombinant FRIL (Supplementary Fig. 2f). The overall binding specificity are quite similar, although natural FRIL does seem to have a slightly higher affinity to high mannose glycoproteins compared to recombinant FRIL at high concentrations.\nHowever, a concern we have comparing natural FRIL with recombinant FRIL using lectin binding ELISA is that the rabbit polyclonal anti-FRIL primary antibody we used was originally raised against native FRIL, and it is possible that at least some of the serum antibodies are directed against plant paucimannose on the glycosylated linker rather than the lectin itself. To verify our ELISA results, we also did bio-layer interferometry (BLI) using biotinylated FG, CX and M9 HAs as ligands and natural FRIL and deglycosylated recombinant FRIL as analyte (Supplementary Fig. 8a).\nNatural FRIL and recombinant FRIL also had roughly similar binding on BLI, with natural FRIL perhaps slightly better.\nWe had done glycan array on native FRIL in a previous publication (Liu et al 2020) and recombinant FRIL in the current manuscript (Fig. 2d). While the results are not directly comparable as in our previous study native FRIL was conjugated to a cy3 dye while in the current manuscript recombinant FRIL binding was detected with an anti-His antibody, the native and recombinant FRILs on glycan array seem to recognize the same glycans, namely hybrid or complex type glycans with a subterminal α1-3-linked fucose on one of its Gal-GlcNAc-Man branches.\nAside from specificity and affinity, one difference we observed while working on the deglycosylated recombinant FRIL is precipitation. Natural FRIL is incredibly stable, we had some samples that were kept at 4oC in buffer for over a year with no decrease in activity, and Daniell et al 2024 has kept FRIL in chewing gum at ambient temperature for up to 790 days. Our single-chain polypeptide recombinant FRIL is also very stable by protein standards, but we’ve noticed it tends to precipitate after being kept for more than 3 months at 4oC in buffer. This is similar to what others have observed in mature ConA.\nIn summary, we haven’t found notable differences in binding specificity between two-chain native and one-chain recombinant FRIL in lectin binding ELISA with differentially glycosylated HA (Supplementary Fig. 2f) or BLI (Supplementary Fig. 8a), the slight variation in binding affinity could perhaps be attributed to protein stability and storage conditions.\n2. The manuscript convincingly shows that the lectin self glycan is responsible for its inactivation prior to cleavage. However, it is not clear for the reviewer the reason behind the recovery of the activity when the glycan is not removed completely, as it is the case for native FRIL. Additionally, the expected paucimannose glycan is expected to have a lower affinity for the lectin. Is it a matter of positioning and/or conformational flexibility of the glycan? Given the importance of the presented findings, the authors should expand on this.\nResponse:\nWe agree with Reviewer #1. We also believe that in paucimannose N-glycans, the core trimannose without any extensions creates a poor ligand for FRIL. This would be consistent with other seed lectins, in that they evolved as defense-related proteins that show preference for foreign glycans over plant glycans (Van Damm et al 2008, Peumans et al 2000).\nWe therefore speculate that unlike proConA with its high mannose linker (Ramis et al 2001), pauci-mannose on FRIL act as a much weaker self-ligand, so that a single cut at linker position Asn113 with AEP is enough to activate native FRIL without needing complete deglycosylation. Unfortunately, our lab does not have a plant expression system that can produce recombinant FRIL adorned with plant paucimannose to verify our speculation, or to see if FRIL linker glycan acts as a self-ligand in plants.\nHowever, we can try to deglycosylate the native FRIL we purified from Lablab beans, to see if binding can be improved.\nWe first attempted to deglycosylate native FRIL by PNGase F digestion following the same conditions as recombinant FRIL, then by increasing the PNGase F concentration 2.5-fold (100 μg/mg) and increasing the digestion time to overnight (Supplementary Fig. 2h). None of the three conditions were able to deglycosylate native FRIL. This is perhaps due to the fact that plant pauci-mannose contains a α1-3-linked core fucose (Kumar et al 2014) that is resistant to PNGase F cleavage.\nAs the FRIL glycosylated linker is bracketed by an AEP cutting site on one end (Asn113) and a trypsin cutting site on the other (Lys128), we next tried to remove the glycosylated linker altogether with trypsinization, which would deglycosylate native FRIL in the process. As seen in Supplementary Fig. 2h lane 2, we were able to collapse the myriad of α bands on native FRIL into a single deglycosylated α. When this deglycosylated native FRIL was tested in a lectin binding ELISA (Supplementary Fig. 2i), it showed very slightly better binding than glycosylated natural FRIL, but probably not enough to be conclusive.\nIn summary, when the glycosylated linker was completely removed on native FRIL (Supplementary Fig. 2h), no further improvement was seen in FRIL binding (Supplementary Fig. 2i). So we speculate that pauci-mannose act as a weak self-ligand, and a single cut at linker position Asn113 with AEP is enough to activate native FRIL without needing complete deglycosylation. We have added a description in the discussion section of our manuscript to expand upon this.\n3. The authors provide a good rationale for the importance of most mutated residues. However, the high importance attributed to S45 and especially Y147 is surprising. As Y147 appears to be part of the canonical monosaccharide binding site, its specific role in modulating specificity in the extended binding site is not immediately obvious. The authors should include a more detailed explanation for the importance of these residues to better support the findings.\nResponse:\nThank you for your important comment.\nAfter concerns were raised by both Reviewer 1 and Reviewer 5 on the classification of Y147, we went back and refined the structure and re-analyzed the interactions using PDBePISA. Your statement is correct; Y147’s role in core mannose interactions are more crucial to overall glycan binding than its interactions with the extended site.\nThe Y147A alanine mutation results in a complete loss of FRIL's glycan binding activity. Because of Y147’s involvement in core mannose interactions, we could not further investigate other roles it may potentially play in determining binding specificity to complex type vs high mannose type glycans.\nFor S45, the location of S45 is distant from the core mannose interaction. It binds non-specifically to GlcNAc, fucose (in complex-type glycan, Fig. 2A), but also to an extended mannose (in high-mannose type glycan, Fig. 4A). Therefore, S45 can be classified as \"extended binding site residues\". However, S45 is seemingly not involved in determining glycan specificity, as it can bind to anything. S45 also forms a hydrogen bond with the core mannose mediated by water, when other glycans are not present (Fig. 3A). We believe this is why the mutation of S45A contributes to the instability of the core glycan binding, resulting in a loss of the whole glycan binding ability (Fig. 2C).\n4. The proposed importance of Y101 and H102 could be further substantiated through molecular docking simulations. Including such analyses would strengthen the conclusions drawn about the roles of these residues.\nResponse:\nThank you for the suggestion. We have tried molecular docking simulations using AlphaFOLD: Protenix (ByteDance AML AI4Science Team et al 2025) for Y101S and H102A, with the two ligands of Galβ1,4-(Fucα1,3-)GlcNAcβ1,2-Man (tetrasaccharide surrogate for complex type N-glycans), and Manα1,2-Manα1,3-Man (a trisaccharide found on high mannose type N-glycans). The ligand input files were derived from our EM structure coordinates.\nThe modelling criteria was to retain the majority of interactions with the core mannose residues. Docking with complex-type tetrasaccharide did not yield any acceptable models, but docking of the Y101S and H102A mutants with our high-mannose ligand produced acceptable conformations (Figures below).\nIn both Y101S and H102A mutants, no direct interactions were observed between these substituted residues and the third mannose. The interaction originally formed by Ser45 with GlcNAc and fucose in complex type ligand was replaced by contact with the third mannose in the high mannose ligand.\nMutations of Tyr101 or His102 of FRIL’s extended binding site on loop B to smaller side chains, like alanine and serine, did not introduce steric clashes or new interactions with either the complex-type or high-mannose ligand. This could explain the loss of binding specificity in our single residue mutants, which is consistent with our lectin binding ELISA results (Fig 3C).\nReviewer #2 Comments and Response\nThis study by Liu et al presents a structure-guided engineering approach to alter the glycan-binding specificity of the legume lectin FRIL, a protein with broad-spectrum antiviral activity. The authors overcame longstanding barriers to producing bioactive FRIL by expressing recombinant protein in mammalian cells and activating it through enzymatic deglycosylation. Using cryo-EM and X-ray crystallography, they identified key residues in loop B (Y101, H102) as critical determinants of FRIL's preference for complex-type N-glycans. By replacing these and adjacent loop C residues with counterparts from the high-mannose-specific lectin ConA, they created a FRIL mutant (FRIL-OMS) that selectively binds high-mannose N-glycans. Structural analysis confirmed the altered binding mode, while functional assays showed that FRIL-OMS binds and neutralizes influenza strains rich in oligomannose, contrasting the specificity of wild-type FRIL. These findings establish a framework for reprogramming lectin specificity through rational loop engineering, with broad implications for antiviral design and glycan-targeting technologies.\nThe biochemical and structural data presented in the study are of high quality. The manuscript is logically organized and generally well written, though it may benefit from editing by a native English speaker. The figures are also of high quality.\nWhile the manuscript makes a meaningful and technically impressive contribution to the fields of glycoscience, lectin biology, and antiviral lectin engineering, its specialized focus on legume lectin structure-function relationships and glycan specificity reprogramming may limit its accessibility to a broader readership.\nResponse:\nThank you for your kind words. This project has been a 5-year journey with numerous obstacles, some false leads, and many setbacks. At times we have also wondered whether it was all worth it. It is our sincerest hope that the techniques and conclusions gleaned from this study can have wider applicability to not only legume lectins but other L-type lectins in general, and perhaps in this way appeal to a broader readership.\nWe have gone back and re-written certain parts of the abstract and discussion sections to make things clearer.\nReviewer #3 Comments and Response\nIn this manuscript titled “Altering the Carbohydrate-Binding Specificity of the Legume Lectin FRIL Through Structure-Guided Engineering” by Yo-Ming Liu et al. The authors present a comprehensive study demonstrating how structure-guided mutations can be used to alter the glycan binding specificity of a legume lectin, FRIL. Through binding assays, Cryo-EM studies, and molecular modeling, they successfully demonstrated how deglycosylation of their recombinant lectin, achieved by site-directed mutagenesis (Y101A, H102A, P145T), enables a specificity shift from complex N-glycans towards high-mannose glycans, while retaining structural integrity and binding activity. The study is well-executed, with all data and experiments presented clearly and logically, and the conclusions are well-supported by combinations of relevant structural, biochemical, and virological assays. There are several questions in the manuscript listed below that should be addressed before its publication.\nThank you for your kind words. The questions raised by Reviewer #3 are very interesting, and we will attempt to address them to the best of our abilities.\n1. Binding avidities or dissociation constants along with Hill coefficients were not presented (pages 6,7, and 9; lines 205-235, 299-304) for both wild-type FRIL and its mutants. Providing these would enable a more quantitative comparison of their binding properties.\nResponse:\nThank you for the comment. Before our first manuscript submission, there had been internal discussion over whether we should do bio-layer interferometry (BLI) to determine dissociation constants for our mutants, thank you for giving us the impetus to finally do it.\nFor our BLI assay, we used streptavidin probes, biotin-conjugated FG, CX and M9 hemagglutinin as ligand, and nFRIL, rFRIL-wt, and three mutants as analyte. For the mutants, FRIL-OMS was selected for its oligomannose specificity, Y101S was chosen for its broad binding to both high mannose and complex type N-glycans, and G103A was used as control due to its lack of binding on ELISA and location on loop B near Tyr101 and His102. A 2:1 heterogenous ligand model was used to calculate dissociation constants due to FG having both complex type and high mannose glycans, and CX endoH cleavage leaving a single GlcNAc at previously high mannose positions (Supplementary Fig. 6d). The results are shown in Fig. 3e and supplementary Fig. 8a, and broadly speaking they do seem to confirm our ELISA results, although the calculated K values are likely due to avidity rather than affinity.\nd\nFor a more quantitative comparison of our lectin binding ELISA, we have included Supplementary Table 4 with Hill slope and 95% confidence interval on all mutants.\nWe are also compelled to report that while checking the Hill slope and 95% CI, we noticed some of the EC values of mutants reported on Fig. 3c are slightly different, 50\nthough the difference is negligible. We believe this is likely due to those values having been calculated individually on an older version of Prism with the same data but different constraints, rather than the pooled data on Prism 9 that were used to plot our current graphs. Though the difference is small, we have re-plotted Fig. 3c with EC s under uniform constraints (EC >0) and made sure data analysis is consistent 50 50\nacross the board. The full list of changes made to Fig. 3c are: H102T-FG 52.21→52.23, H102T-CX 90.65→90.76, H102T-M9 271.2→272.0, H102T-M5 2645→2661, Y101S-FG 2.59→2.62, Y101S-CX 4.96→5.04, Y101S-M9 5.97→6.03, Y101S-M5 14.32→14.35, 101insGS102-FG 9.36→9.41, 101insGS102-CX 17.42→17.44, 101insGS102-M9 10.57→10.60, 101insGS102-M5 52.25→52.34, P145T-FG 12.02→12.07, P145T-CX 13.09→13.10, P145T-M9 324.1→324.8, P145T-M5 145.0→145.2, P145T H102A-FG 8.53→8.60, P145T H102A-CX 25.40→25.45, P145T H102A-M9 3.41→3.46, P145T H102A-M5 847.7→848.8, 101insGSH102T-FG 6.75→6.81, 101insGSH102T-CX 23.22→23.26, 101insGSH102T-M9 3.07→3.11, 101insGSH102T-M5 9.56→9.62, Y101SinsGSH102T-FG 6.65→6.71, Y101SinsGSH102T-CX 25.55→25.59, Y101SinsGSH102T-M9 2.59→2.63, Y101SinsGSH102T-M5 17.96→17.98, and H102A-FG 3.43→3.59. Graphs on Supplementary Fig. 7 are not affected, as they were plotted together in Prism 9. The authors apologize for this mistake, and thank Reviewer #3 for letting us catch it.\nIn summary, for a more quantitative comparison of binding properties, we have provided Hill slope and 95% CI for our ELISA data (Supplementary Table 4), and performed BLI on select mutants for dissociation constants (Fig. 3e, Supplementary Fig. 8a). We have also found slight issues with some EC s calculated in Fig. 3c, and 50\ncorrected them.\n2. How would the formation of a mutant that only has the P145T mutation without alterations to the other residues in Loop B affect binding? Where would this mutant stack in comparison to the others?\nResponse:\nWe thank Reviewer #3 for this interesting question. P145T is a curious mutant as it was not identified through our own structural data, but by previous structural studies done on FRIL and ConA (Hamelryck et al 2000, Naismith & Field, 1996). These studies suggested that ConA Thr15 (FRIL Pro145) played a role in trimannoside binding. Thus, after alanine mutagenesis, FRIL-P145T was among the first batch of single residue mutants we attempted while trying to shift FRIL glycan specificity.\nDisappointingly, as seen in Figure 3c and Supplementary Fig. 7, P145T mutation without alterations to the other residues in Loop B did not change the binding preference of FRIL towards oligomannose. However, due to several previous studies highlighting the importance of Thr15 (FRIL Pro145), this mutation remained in the back of our minds and was occasionally added into our then-ongoing mutagenesis on loop B residues. The FRIL P145T/H102A double mutant was able to retain FRIL-H012A single mutant’s binding to mannose 9 (Fig. 3c), but decreased H102A’s binding to complex type glycan 4-fold.\nBased on this result, when our final Loop B mutant (Y101SinsGSH102T) came out with good binding to mannose 9 but still showed an unacceptable amount of binding towards complex type N-glycans, we re-introduced P145T back into the mix, hoping that it can do to Y101SinsGSH102T what it did to H102A. Fortunately, P145T did exactly what we wanted it to do, decreasing Y101SinsGSH102T binding towards complex type glycan 8-fold without interfering with its mannose 9 binding.\nIn summary, P145T without alterations to other residues in Loop B did not change the glycan specificity or affinity of FRIL (Fig. 3c, Supplementary Fig. 7). However, when coupled with other mutants on loop B it consistently decreased FRIL recognition towards complex type glycans. Structurally, we observed density around the Pro145 region, but resolution was inadequate to determine how P145T presumably interacts with the α1-6 arm of the glycan.\n3. How would this self-neutralizing ligand, as stated (page 11, line 372), operate in nature? Similarly, what would happen if you attempted Ramis et al (page 11, line 374) approach and attempted to actuate pH to cause protein activation?\nResponse:\nWe thank Reviewer #3 for these two important questions. Previous reports have noted that for seed lectins involved in defense/storage, they are usually synthesized as inactive precursors that are only activated after sequestration into specialized organelles (Van Damm et al, 2008). In the case of FRIL and ConA, this would be the plant vacuole. We speculate that due to these lectin’s multivalency and the huge quantity being expressed in the maturing seed (studies have suggested that seed lectins can constitute up to 10% of the weight of the seed. Our own estimates based on native FRIL purification suggest that FRIL constitutes up to 4% dry weight of a Lablab bean), if the lectins were already active in the ER, Golgi or during transport, they would likely cross-link or otherwise interfere with other glycoproteins or glycans inside the cell. Therefore, it makes sense to produce them in an inactive form until they are transported to their destination.\nAfter the lectin is sequestered into the vacuole, which step actually activates the lectin is still (as far as we know) debatable, whether it’s removal of the glycan, removal of the linker, AEP-mediated circular permutation, or as Ramis et al’s results suggest, refolding of the ConA protein initiated by deglycosylation and the acidic environment of the vacuole.\nTo mimic Ramis et al’s approach of pH-mediated ConA activation, we performed lectin binding ELISA with PNGaseF-deglycosylated proConA under pH 7.4 and pH 5.5, and glycosylated proConA under pH 5.5 conditions. The pH is kept the same throughout the entire assay, and fetuin was chosen as the glycoprotein coating because we were concerned about influenza hemagglutinin conformational change under acidic conditions. Results show little difference in proConA binding between pH 7.4 and pH 5.5, while glycosylated proConA did not bind to fetuin at pH 5.5 (Supplementary Fig. 2c). This would seemingly leave deglycosylation as the crucial step in proConA activation, as linker cleavage and circular permutation did not happen in our rproConA, and acidification had minimal effect.\nHowever, Ramis et al assessed proConA activity not with ELISA, but binding on an ovalbumin column. Unexpectedly, we may have also encountered a similar phenomenon while conducting one of Reviewer #5’s experiments. In that experiment, we were trying to determine whether rproConA forms a tetramer under acidic pH with size exclusion chromatography followed by multi-angle light scattering (SEC-MALS, Supplementary Fig. 2e). Due to ConA’s known binding to dextran, we deliberately chose a size-exclusion column that was not dextran-based. However, the column was not new and it is very difficult to completely get rid of glycoproteins and other glycans retained within the beads. The deglycosylated rproConA at pH 7.4 came out with the calculated molecular weight of 55. 3 kDa, the expected size of a dimer albeit at a slightly higher retention volume than the molecular weight would suggest. The glycosylated rproConA pH 5.5 came out as 108.2 KDa, the expected size of a tetramer. However, the deglycosylated rproConA at pH 5.5 did not come out of our size exclusion column in any meaningful way at all, a possible explanation being that it was retained inside the column by affinity binding to glycans. This is somewhat confirmed when we loaded mature ConA pH 7.4 into the column, which also resulted in lectin retention. Our speculation for this phenomenon is that the pH-dependent dimer-tetramer equilibrium (as noted by Nonis et al 2021) is not important for ELISA binding, but the multi-dimensional orientation of a proConA tetramer’s four carbohydrate recognition domains mean that it is especially proficient at agglutination of multiple red blood cells (Supplementary Fig. 2d) or perhaps being retained on a column. This is only our speculation and probably beyond the scope of our current study, but it is possible that what Ramis et al observed was not entirely binding but also increased multivalency due to tetramerization.\nIn summary, we believe ConA and FRIL’s self-neutralizing ligand allows the lectins to be synthesized and transported as an inactive precursor before its sequestration in the vacuole. Our ELISA results (Supplementary Fig. 2c) indicate that acidic pH alone is not enough to activate glycosylated rproConA, and acidic pH does not enhance the glycan binding properties of PNGaseF-activated rproConA. However, acidic pH does affect the dimer-tetramer state of proConA (Supplementary Fig. 2e), and our speculation is that this may result in observable differences in how the lectin behaves in certain biological assays.\n4. Did any mutation occur in which all binding was subsequently negated? (page 6, line 217) If no such mutant was found, would the possibility of other important residues exist in the full structure of the lectin?\nResponse:\nThank you for the question. The original goal of our project was to determine the structure of FRIL in complex with complex type N-glycans, and created a bioactive recombinant version of FRIL for alanine mutagenesis on the oligosaccharide-interacting residues identified in our structure. It was only later, with the identification of two residues whose alanine mutants came with broadened glycan specificity (Y101A, H102A), that the project took a turn and went down a new and difficult path.\nOf the original alanine mutants we produced (Fig. 2c), D86A, G103A, Y142A, N144A, Y147A and S45A did not show any binding to the fully glycosylated hemagglutinin, which contains both high mannose and complex type glycans. To verify our ELISA results, we selected G103A for BLI analysis against FG, CX and M9 HAs (Supplementary Fig. 8a). We did not observe binding at the highest concentration used (200 nM). Also, although not shown in the presented data, our Y142A mutant has been used in a lot of unrelated experiments as a negative control due to its ease of production and lack of activity.\nHowever, our study has only highlighted residues that directly interact with an oligosaccharide ligand. Other residues on FRIL potentially play important roles in maintaining the structural stability of the carbohydrate recognition domain, especially conserved residues in the two metal binding sites such as Asp149 (Asp19 in ConA), His154 (His24 in ConA), Glu138 (Glu8 in ConA) or Asp140 (Asp10 in ConA). Residues involved in FRIL dimer-dimer contacts such as His181 may also be worth looking into in the future.\nIn summary, D86A, G103A, Y142A, N144A, Y147A and S45A did not show any binding (Fig. 2c, supplementary Fig. 8a).\nReviewer #5 Comments and Response\nIn the revised version of the manuscript by Liu and colleagues, the authors thoroughly answered to all the comments of the 5 reviewers. Their new implementations have greatly improved the manuscript and they amended the structures. They gave detailed answers to the main comments which was greatly appreciated. One little mistake was made in figure 2 panel b where M9 has been replaced by HM and this abbreviation has not been used in the text. It was fine in the previous version. I mentioned the distortion of mannose in the M9 structure which has been corrected but I forgot to mention this was also the case for the rFRIL-wt with self-ligand for the alpha 1-6linked mannose (9VBY) as it is visible in panel a from figure S4 (formally S7a). I am sorry about this oversight. It would be great if the authors could also correct it but this moiety is not in figure 1 in the main text and its does not make interaction as it is solvent exposed. It is not an essential part of the work but it would just avoid to have PDB coordinates with geometry error on the sugar and a misleading figure in supplemental material. Refinement of oligosaccharides could be complicated notably at low resolution.\nAll the main issues have been resolved and this work is of significant importance for glycosciences but also synthetic biology with the introduction of new methodology. The manuscript is now suitable for publication in Nature Communications.\nResponse:\nOnce again, we thank Reviewer #5 for their meticulous review of our manuscript. The M9 to HM reversion was a mistake, a previous version of our figures was used during revisions without checking for consistency, causing a now-disused abbreviation to resurface (“HM” stood for high mannose N-glycan, a designation we originally used for M9 but felt may cause confusion with M5, which is also high mannose). We have changed it back to M9.\nWe have also refined the conformation of the alpha 1-6 linked mannose in PDB: 9VBY, and the supplementary figure 4 was changed accordingly."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have provided convincing responses, and the revision satisfactorily addresses the raised questions.", "reviewer_2": "I have nothing further to add. I previously raised my concern about the suitability of this work for publication given its highly specialized scope. Otherwise, I consider it a solid study. The authors chose not to address my comments, so I do not have additional feedback at this stage.", "reviewer_3": "We find that the authors satisfactorily answered all our questions and comments. We thank the authors and the journal for this opportunity to review this document. In doing so we find there is no further reason to withhold the publication of this work.", "reviewer_5": "In the revised version of the manuscript by Liu and colleagues, the authors thoroughly answered to all the comments of the 5 reviewers. Their new implementations have greatly improved the manuscript and they amended the structures. They gave detailed answers to the main comments which was greatly appreciated. One little mistake was made in figure 2 panel b where M9 has been replaced by HM and this abbreviation has not been used in the text. It was fine in the previous version. I mentioned the distortion of mannose in the M9 structure which has been corrected but I forgot to mention this was also the case for the rFRIL-wt with self-ligand for the alpha 1-6linked mannose (9VBY) as it is visible in panel a from figure S4 (formally S7a). I am sorry about this oversight. It would be great if the authors could also correct it but this moiety is not in figure 1 in the main text and its does not make interaction as it is solvent exposed. It is not an essential part of the work but it would just avoid to have PDB coordinates with geometry error on the sugar and a misleading figure in supplemental material. Refinement of oligosaccharides could be complicated notably at low resolution.\nAll the main issues have been resolved and this work is of significant importance for glycosciences but also synthetic biology with the introduction of new methodology. The manuscript is now suitable for publication in Nature Communications."}, "author_response": "Reviewer #1 Comments and Response\nThe authors have provided convincing responses, and the revision satisfactorily addresses the raised questions.\nResponse:\nWe thank reviewer 1 for their comprehensive review of our manuscript, and the points they raised on the role of Y147 and S45.\nReviewer #2 Comments and Response\nI have nothing further to add. I previously raised my concern about the suitability of this work for publication given its highly specialized scope. Otherwise, I consider it a solid study. The authors chose not to address my comments, so I do not have additional feedback at this stage.\nResponse:\nWe thank reviewer 2 for their review of our manuscript, and the concerns they have raised. We understand reviewer 2’s point that lectin structure-function relationships and glycan specificity reprogramming may be considered too narrow in scope. We feel that in recent years there has been renewed interest in lectin-glycan interactions from diverse disciplines, and we hope that our study can appeal to a broad readership.\nReviewer #3 Comments and Response\nWe find that the authors satisfactorily answered all our questions and comments. We thank the authors and the journal for this opportunity to review this document. In doing so we find there is no further reason to withhold the publication of this work.\nResponse:\nWe also thank reviewer 3 for taking the time to read and thoroughly review our manuscript.\nReviewer #5 Comments and Response\nIn the revised version of the manuscript by Liu and colleagues, the authors thoroughly answered to all the comments of the 5 reviewers. Their new implementations have greatly improved the manuscript and they amended the structures. They gave detailed answers to the main comments which was greatly appreciated. One little mistake was made in figure 2 panel b where M9 has been replaced by HM and this abbreviation has not been used in the text. It was fine in the previous version. I mentioned the distortion of mannose in the M9 structure which has been corrected but I forgot to mention this was also the case for the rFRIL-wt with self-ligand for the alpha 1-6linked mannose (9VBY) as it is visible in panel a from figure S4 (formally S7a). I am sorry about this oversight. It would be great if the authors could also correct it but this moiety is not in figure 1 in the main text and its does not make interaction as it is solvent exposed. It is not an essential part of the work but it would just avoid to have PDB coordinates with geometry error on the sugar and a misleading figure in supplemental material. Refinement of oligosaccharides could be complicated notably at low resolution.\nAll the main issues have been resolved and this work is of significant importance for glycosciences but also synthetic biology with the introduction of new methodology. The manuscript is now suitable for publication in Nature Communications.\nResponse:\nOnce again, we thank Reviewer #5 for their meticulous review of our manuscript. The M9 to HM reversion was a mistake, a previous version of our figures was used during revisions without checking for consistency, causing a now-disused abbreviation to resurface (“HM” stood for high mannose N-glycan, a designation we originally used for M9 but felt may cause confusion with M5, which is also high mannose). We have changed it back to M9.\nWe have also refined the conformation of the alpha 1-6 linked mannose in PDB: 9VBY, and the supplementary figure 4 was changed accordingly."}]

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