Elucidating the glycan-binding specificity and structure of Cucumis melo agglutinin, a new R-type lectin.

Plant lectins have garnered attention for their roles as laboratory probes and potential therapeutics. Here, we report the discovery and characterization of Cucumis melo agglutinin (CMA1), a new R-type lectin from melon. Our findings reveal CMA1's unique glycan-binding profile, mechanistically explained by its 3D structure, augmenting our understanding of R-type lectins. We expressed CMA1 recombinantly and assessed its binding specificity using multiple glycan arrays, covering 1,046 unique sequences. This resulted in a complex binding profile, strongly preferring C2-substituted, beta-linked galactose (both GalNAc and Fuca1-2Gal), which we contrasted with the established R-type lectin Ricinus communis agglutinin 1 (RCA1). We also report binding of specific glycosaminoglycan subtypes and a general enhancement of binding by sulfation. Further validation using agglutination, thermal shift assays, and surface plasmon resonance confirmed and quantified this binding specificity in solution. Finally, we solved the high-resolution structure of the CMA1 N-terminal domain using X-ray crystallography, supporting our functional findings at the molecular level. Our study provides a comprehensive understanding of CMA1, laying the groundwork for further exploration of its biological and therapeutic potential.

Expression, purification and biochemical characterization of AtFUT1, a xyloglucan-specific fucosyltransferase from Arabidopsis thaliana.

Efforts to identify genes and characterize enzymes involved in the biosynthesis of plant cell wall polysaccharides have yet to produce and purify to homogeneity an active plant cell wall synthesizing enzyme suitable for structural studies. In Arabidopsis, the last step of xyloglucan (XG) biosynthesis is catalyzed by fucosyltransferase 1 (AtFUT1), which transfers l-fucose from GDP-ß-l-fucose to a specific galactose on the XG core. Here, we describe the production of a soluble form of AtFUT1 (HisΔ68-AtFUT1) and its purification to milligram quantities. An active form of AtFUT1 was produced in an insect cell culture medium, using a large-scale expression system, and purified in a two-step protocol. Characterization of purified HisΔ68-AtFUT1 revealed that the enzyme behaves as a non-covalent homodimer in solution. A bioluminescent transferase assay confirmed HisΔ68-AtFUT1 activity on its substrates, namely GDP-fucose and tamarind XG, with calculated Km values of 42 µM and 0.31 µM, respectively. Moreover, the length of the XG-derived acceptor quantitatively affected fucosyltransferase activity in a size-dependent manner. The affinity of HisΔ68-AtFUT1 for tamarind XG and GDP was determined using isothermal titration calorimetry (ITC). Interestingly, ITC data suggest that HisΔ68-AtFUT1 undergoes conformational changes in the presence of its first co-substrate (XG or GDP), which then confers greater affinity for the second co-substrate. The procedure described in this study can potentially be transferred to other enzymes involved in plant cell wall synthesis.

Structure of Arabidopsis thaliana FUT1 Reveals a Variant of the GT-B Class Fold and Provides Insight into Xyloglucan Fucosylation.

The plant cell wall is a complex and dynamic network made mostly of cellulose, hemicelluloses, and pectins. Xyloglucan, the major hemicellulosic component in Arabidopsis thaliana, is biosynthesized in the Golgi apparatus by a series of glycan synthases and glycosyltransferases before export to the wall. A better understanding of the xyloglucan biosynthetic machinery will give clues toward engineering plants with improved wall properties or designing novel xyloglucan-based biomaterials. The xyloglucan-specific α2-fucosyltransferase FUT1 catalyzes the transfer of fucose from GDP-fucose to terminal galactosyl residues on xyloglucan side chains. Here, we present crystal structures of Arabidopsis FUT1 in its apoform and in a ternary complex with GDP and a xylo-oligosaccharide acceptor (named XLLG). Although FUT1 is clearly a member of the large GT-B fold family, like other fucosyltransferases of known structures, it contains a variant of the GT-B fold. In particular, it includes an extra C-terminal region that is part of the acceptor binding site. Our crystal structures support previous findings that FUT1 behaves as a functional dimer. Mutational studies and structure comparison with other fucosyltransferases suggest that FUT1 uses a SN2-like reaction mechanism similar to that of protein-O-fucosyltransferase 2. Thus, our results provide new insights into the mechanism of xyloglucan fucosylation in the Golgi.

Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP-galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.

Revisiting the expression and purification of MGD1, the major galactolipid synthase in Arabidopsis to establish a novel standard for biochemical and structural studies.

Monogalactosyldiacylglycerol, the major lipid of plants and algal plastids, is synthesized by MGDG synthases (MGD). MGDs belong to the large glycosyltransferase family. They catalyze the transfer of a galactose residue from the donor UDP-Gal to a 1,2-sn-diacylglycerol acceptor. MGDs are monotopic proteins localized in the plastid envelope and, as such, they are difficult to purify. This study re-examined previous purification procedures and aimed to set up a standard protocol for expression and purification of recombinant MGD1, addressing problems frequently encountered with the purification of glycosyltransferases, particularly protein aggregation, and enabling crystallization for structural studies. Briefly, His-tagged versions of MGD1 were expressed in Escherichia coli and purified by a two-step procedure, including immobilized metal affinity chromatography and size-exclusion chromatography. We demonstrated that E. coli is an appropriate host cell to produce a soluble and active form of MGD1. We also investigated the effects of various buffers and additives used during the purification and concentration steps on the biochemical behavior of the enzyme. The protocol we developed typically yields milligram quantities of pure and homogenous protein material and proved suitable for crystallization and biochemical studies. We also revisited the conditions for activity tests and effects of known positive effectors of MGD1 such as phosphatidic acid and phosphatidylglycerol.

A lectin from Platypodium elegans with unusual specificity and affinity for asymmetric complex N-glycans.

Lectin activity with specificity for mannose and glucose has been detected in the seed of Platypodium elegans, a legume plant from the Dalbergieae tribe. The gene of Platypodium elegans lectin A has been cloned, and the resulting 261-amino acid protein belongs to the legume lectin family with similarity with Pterocarpus angolensis agglutinin from the same tribe. The recombinant lectin has been expressed in Escherichia coli and refolded from inclusion bodies. Analysis of specificity by glycan array evidenced a very unusual preference for complex type N-glycans with asymmetrical branches. A short branch consisting of one mannose residue is preferred on the 6-arm of the N-glycan, whereas extensions by GlcNAc, Gal, and NeuAc are favorable on the 3-arm. Affinities have been obtained by microcalorimetry using symmetrical and asymmetrical Asn-linked heptasaccharides prepared by the semi-synthetic method. Strong affinity with K(d) of 4.5 µm was obtained for both ligands. Crystal structures of Platypodium elegans lectin A complexed with branched trimannose and symmetrical complex-type Asn-linked heptasaccharide have been solved at 2.1 and 1.65 Å resolution, respectively. The lectin adopts the canonical dimeric organization of legume lectins. The trimannose bridges the binding sites of two neighboring dimers, resulting in the formation of infinite chains in the crystal. The Asn-linked heptasaccharide binds with the 6-arm in the primary binding site with extensive additional contacts on both arms. The GlcNAc on the 6-arm is bound in a constrained conformation that may rationalize the higher affinity observed on the glycan array for N-glycans with only a mannose on the 6-arm.

Biochemical and structural analysis of Helix pomatia agglutinin. A hexameric lectin with a novel fold.

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin found in the albumen gland of the roman snail. As a constituent of perivitelline fluid, HPA protects fertilized eggs from bacteria and is part of the innate immunity system of the snail. The peptide sequence deduced from gene cloning demonstrates that HPA belongs to a family of carbohydrate-binding proteins recently identified in several invertebrates. This domain is also present in discoidin from the slime mold Dictyostelium discoideum. Investigation of the lectin specificity was performed with the use of glycan arrays, demonstrating that several GalNAc-containing oligosaccharides are bound and rationalizing the use of this lectin as a cancer marker. Titration microcalorimetry performed on the interaction between HPA and GalNAc indicates an affinity in the 10(-4) M range with an enthalpy-driven binding mechanism. The crystal structure of HPA demonstrates the occurrence of a new beta-sandwich lectin fold. The hexameric quaternary state was never observed previously for a lectin. The high resolution structure complex of HPA with GalNAc characterizes a new carbohydrate binding site and rationalizes the observed preference for alphaGalNAc-containing oligosaccharides.

Beta-propeller crystal structure of Psathyrella velutina lectin: an integrin-like fungal protein interacting with monosaccharides and calcium.

The lectin from the mushroom Psathyrella velutina recognises specifically N-acetylglucosamine and N-acetylneuraminic acid containing glycans. The crystal structure of the 401 amino acid residue lectin shows that it adopts a very regular seven-bladed beta-propeller fold with the N-terminal region tucked into the central cavity around the pseudo 7-fold axis. In the complex with N-acetylglucosamine, six monosaccharides are bound in pockets located between two consecutive propeller blades. Due to the repeats shown by the sequence the binding sites are very similar. Five hydrogen bonds between the protein and the sugar hydroxyl and N-acetyl groups stabilize the complex, together with the hydrophobic interactions with a conserved tyrosine and histidine. The complex with N-acetylneuraminic acid shows molecular mimicry with the same hydrogen bond network, but with different orientations of the carbohydrate ring in the binding site. The beta-hairpin loops connecting the two inner beta-strands of each blade are metal binding sites and two to three calcium ions were located in the structure. The multispecificity and high multivalency of this mushroom lectin, combined with its similarity to the extracellular domain of an important class of cell adhesion molecules, integrins, are another example of the outstanding success of beta-propeller structures as molecular binding machines in nature.

Structure-function analysis of the human sialyltransferase ST3Gal I: role of n-glycosylation and a novel conserved sialylmotif.

All eukaryotic sialyltransferases have in common the presence in their catalytic domain of several conserved peptide regions (sialylmotifs L, S, and VS). Functional analysis of sialylmotifs L and S previously demonstrated their involvement in the binding of donor and acceptor substrates. The region comprised between the sialylmotifs S and VS contains a stretch of four highly conserved residues, with the following consensus sequence (H/y)Y(Y/F/W/h)(E/D/q/g). (Capital letters and lowercase letters indicate a strong or low occurrence of the amino acid, respectively.) The functional importance of these residues and of the conserved residues of motif VS (HX(4)E) was assessed using as a template the human ST3Gal I. Mutational analysis showed that residues His(299) and Tyr(300) of the new motif, and His(316) of the VS motif, are essential for activity since their substitution by alanine yielded inactive enzymes. Our results suggest that the invariant Tyr residue (Tyr(300)) plays an important conformational role mainly attributable to the aromatic ring. In contrast, the mutants W301F, E302Q, and E321Q retained significant enzyme activity (25-80% of the wild type). Kinetic analyses and CDP binding assays showed that none of the mutants tested had any significant effect in nucleotide donor binding. Instead the mutant proteins were affected in their binding to the acceptor and/or demonstrated lower catalytic efficiency. Although the human ST3Gal I has four N-glycan attachment sites in its catalytic domain that are potentially glycosylated, none of them was shown to be necessary for enzyme activity. However, N-glycosylation appears to contribute to the proper folding and trafficking of the enzyme.

Production of recombinant xenotransplantation antigen in Escherichia coli.

The synthesis of sufficient amounts of oligosaccharides is the bottleneck for the study of their biological function and their possible use as drug. As an alternative for chemical synthesis, we propose to use Escherichia coli as a "living factory." We have addressed the production of the Galp alpha(1-3)Galp beta(1-4)GlcNAc epitope, the major porcine antigen responsible for xenograft rejection. An E. coli strain was generated which simultaneously expresses NodC (to provide the chitin-pentaose acceptor), beta(1-4) galactosyltransferase LgtB, and bovine alpha(1-3) galactosyltransferase GstA. This strain produced 0.68 g/L of the heptasaccharide Galp alpha(1-3)Galp beta(1-4)(GlcNAc)(5), which harbours the xenoantigen at its non-reducing end, establishing the feasibility of this approach.

Isolectins I-A and I-B of Griffonia (Bandeiraea) simplicifolia. Crystal structure of metal-free GS I-B(4) and molecular basis for metal binding and monosaccharide specificity.

Seeds from the African legume shrub Griffonia simplicifolia contain several lectins. Among them the tetrameric lectin GS I-B(4) has strict specificity for terminal alpha Gal residues, whereas the closely related lectin GS I-A(4) can also bind to alpha GalNAc. These two lectins are commonly used as markers in histology or for research in xenotransplantation. To elucidate the basis for the fine difference in specificity, the amino acid sequences of both lectins have been determined and show 89% identity. The crystal structure of GS I-B(4), determined at 2.5-A resolution, reveals a new quaternary structure that has never been observed in other legume lectins. An unexpected loss of both Ca(2+) and Mn(2+) ions, which are necessary for carbohydrate binding in legume lectins, may be related to a particular amino acid sequence Pro-Glu-Pro in the metal binding loop. Comparison with demetallized concanavalin A reveals a different process for the loss of metal ions and for the subsequent loss of carbohydrate binding activity. The GS I-A x alpha GalNAc and GS I-B x alpha Gal complexes were constructed using homology modeling and docking approaches. The unusual presence of an aromatic amino acid at position 47 (Tyr in I-A and Trp in I-B) explains the strong preference for alpha-anomeric sugars in both isolectins. Alteration at one amino acid position, Ala(106) in I-A versus Glu(106) in I-B, is the basis for the observed specificities toward alpha GalNAc and alpha Gal.