In this work, we analyzed at high-resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the haemolytic protein LSLa (LSL(150)) from the mushroom Laetiporus sulphureus, and also provide functional in vitro evidences suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We firstly demonstrate that recombinant LSL(150) behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47 Å resolution, and also that of the [LSL(150):(lactose)β, γ)] binary complex at 1.67 Å resolution. This complex reveals two lactose molecules bound to the beta and gamma sites of LSL(150), respectively. Isothermal titration calorimetry indicates that LSL(150) binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL(150) exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL(150)(LSL(t)) to the N-terminal end of various recombinant proteins. All the designed LSL(150)-tagged fusion proteins were successfully produced at high yield and, furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL(150) as affinity tag. An optimized protocol for target protein purification was devised which involved removal of the LSL(150) tag through in-column cleavage of the fusion proteins with His(6)-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications.

The Marasmius oreades mushroom lectin (MOA) is well known for its exquisite binding specificity for blood group B antigens. In addition to its N-terminal carbohydrate-binding domain, MOA possesses a C-terminal domain with unknown function, which structurally resembles hydrolytic enzymes. Here we show that MOA indeed has catalytic activity. It is a calcium-dependent cysteine protease resembling papain-like cysteine proteases, with Cys215 being the catalytic nucleophile. The possible importance of MOA's proteolytic activity for mushroom defense against pathogens is discussed.

Glycan chains that terminate in sialic acid (Neu5Ac) are frequently the receptors targeted by pathogens for initial adhesion. Carbohydrate-binding proteins (lectins) with specificity for Neu5Ac are particularly useful in the detection and isolation of sialylated glycoconjugates, such as those associated with pathogen adhesion as well as those characteristic of several diseases including cancer. Structural studies of lectins are essential in order to understand the origin of their specificity, which is particularly important when employing such reagents as diagnostic tools. Here, we report a crystallographic and molecular dynamics (MD) analysis of a lectin from Polyporus squamosus (PSL) that is specific for glycans terminating with the sequence Neu5Acα2-6Galβ. Because of its importance as a histological reagent, the PSL structure was solved (to 1.7 Å) in complex with a trisaccharide, whose sequence (Neu5Acα2-6Galβ1-4GlcNAc) is exploited by influenza A hemagglutinin for viral adhesion to human tissue. The structural data illuminate the origin of the high specificity of PSL for the Neu5Acα2-6Gal sequence. Theoretical binding free energies derived from the MD data confirm the key interactions identified crystallographically and provide additional insight into the relative contributions from each amino acid, as well as estimates of the importance of entropic and enthalpic contributions to binding.

The parasitic mushroom Laetiporus sulphureus produces a family of lectins (LSL's) sharing 80-90% sequence identity that possesses a low but significant sequence similarity to the bacterial pore-forming toxins mosquitocidal toxin Mtx-2 from Bacillus sphaericus and a toxin from Clostridium septicum. The crystal structure of one member of the L. sulphureus lectins family (LSLa) reveals unexpected structural similarities to the 1-pore-forming toxins from the aerolysin family, namely, aerolysin from the Gram-negative bacterium Aeromonas hydrophila, epsilon-toxin from Clostridium perfringens and parasporin from B. thuringiensis. This similarity presumably indicates that the hemolytic activity of LSLa proceeds through a molecular mechanism that involves the formation of oligomeric transmembrane beta-barrels. Comparison of the crystal structures of the above mentioned proteins reveals common pore-forming modules, which are then distributed both in bacteria and fungi. Currently, it can be stated that the above three dimensional structures have been key in revealing structural similarities that were elusive at the sequence level. A potential corollary from this is that structural studies aimed at determining high resolution structures of aerolysin-like pore-forming toxins, whose biological activity involves large conformational changes, are mandatory to define protein domains or structural motifs with membrane-binding properties.

BanLec is a jacalin-related lectin isolated from the fruit of bananas, Musa acuminata. This lectin binds to high-mannose carbohydrate structures, including those found on viruses containing glycosylated envelope proteins such as human immunodeficiency virus type-1 (HIV-1). Therefore, we hypothesized that BanLec might inhibit HIV-1 through binding of the glycosylated HIV-1 envelope protein, gp120. We determined that BanLec inhibits primary and laboratory-adapted HIV-1 isolates of different tropisms and subtypes. BanLec possesses potent anti-HIV activity, with IC50 values in the low nanomolar to picomolar range. The mechanism for BanLec-mediated antiviral activity was investigated by determining if this lectin can directly bind the HIV-1 envelope protein and block entry of the virus into the cell. An ELISA-based assay confirmed direct binding of BanLec to gp120, and indicated that BanLec can recognize the high mannose structures that are recognized by the monoclonal antibody 2G12. Further, BanLec is able to block HIV-1 cellular entry as indicated by temperature sensitive viral entry studies, and by the decreased levels of the strong-stop product of early reverse transcription seen in the presence of BanLec. Thus, our data indicate that BanLec inhibits HIV-1 infection by binding to the glycosylated viral envelope and blocking cellular entry. The relative anti-HIV activity of BanLec compared favorably to other anti-HIV lectins, such as snowdrop lectin and griffithsin, and to T-20 and maraviroc, two anti-HIV drugs currently in clinical use. Based on these results, BanLec is a potential component for an anti-viral microbicide that could be used to prevent the sexual transmission of HIV-1.

Evidence based on the quantitative precipitin method and hapten inhibition technique demonstrates that concanavalin A may interact with internal 2-O-linked alpha-D-mannopyranosyl residues as may occur in glycoproteins and polysaccharides.

MOA, a lectin isolated from the fruiting bodies of the mushroom Marasmius oreades, specifically binds non-reducing terminal Galalpha(1,3)Gal-carbohydrates, such as occurs in the xenotransplantation epitope Galalpha(1,3)Galbeta(1,4)GlcNAc and the branched blood group B determinant Galalpha(1,3)[Fucalpha(1,2)]Gal. Here, we present the crystal structure of MOA in complex with the blood group B trisaccharide solved at 1.8 A resolution. To our knowledge, this is the first blood group B specific structure reported in complex with a blood group B determinant. The carbohydrate ligand binds to all three binding sites of the N-terminal beta-trefoil domain. Also, in this work Ca(2+) was included in the crystals, and binding of Ca(2+) to the MOA homodimer alters the conformation of the C-terminal domain by opening up the cleft containing a putative catalytic site.

Changes to the glycan structures of proteins secreted by cancer cells are known to be functionally important and to have potential diagnostic value. However, an exploration of the population variation and prevalence of glycan alterations on specific proteins has been lacking due to limitations in conventional glycobiology methods. Here we report the use of a previously-developed antibody-lectin sandwich array method to characterize both the protein and glycan levels of specific mucins and CEA-related proteins captured from the sera of pancreatic cancer patients (n = 23) and control subjects (n = 23). The MUC16 protein was frequently elevated in the cancer patients (65% of the patients) but showed no glycan alterations, while the MUC1 and MUC5AC proteins were less frequently elevated (30% and 35%, respectively) and showed highly-prevalent (up to 65%) and distinct glycan alterations. The most frequent glycan elevations involved the TF antigen, fucose, and Lewis antigens. An unexpected increase in the exposure of alpha-linked mannose also was observed on MUC1 and MUC5ac, indicating possible N-glycan modifications. Because glycan alterations occurred independently from the protein levels, improved identification of the cancer samples was achieved using glycan measurements on specific proteins, relative to using the core protein measurements. The most significant elevation was the CA 19-9 antigen on MUC1, occurring in 19/23 (87%) of the cancer patients and 1/23 (4%) of the control subjects. This work gives insight into the prevalence and protein carriers of glycan alterations in pancreatic cancer and points to the potential of using glycan measurements on specific proteins for highly effective biomarkers.

Previous reports on the carbohydrate specificities of Amaranthus caudatus lectin (ACL) and peanut agglutinin (PNA, Arachis hypogea) indicated that they share the same specificity for Thomsen-Friedenreich (T(alpha), Galbeta1-3GalNAcalpha1-Ser/Thr) glycotope, but differ in monosaccharide binding - GalNAc>>Gal (inactive) for ACL; Gal>>GalNAc (weak) with respect to PNA. However, knowledge of the recognition factors of these lectins was restricted to several monosaccharides and T-related oligosaccharides. In this study, a wider range of interacting factors of ACL and PNA toward known mammalian structural units, natural polyvalent glycotopes and glycans were examined by enzyme-linked lectinosorbent and inhibition assays. The results indicate that the main recognition factors of ACL should be a combination of T glycotope and its polyvalent complexes. Although GalNAc was the only active monosaccharide in ACL, the contribution of its polyvalent form (poly GalNAcalpha1-Ser/Thr, Tn in asialo OSM) was limited. Human blood group precursor disaccharides Galbeta1-3/4GlcNAcbeta (I(beta)/II(beta)) were weak, while their clusters (multiantennary II(beta)) and polyvalent forms were active. The major recognition factors of PNA were a combination of alpha or beta anomers of T disaccharide and their polyvalent complexes. Although I(beta)/II(beta) were weak haptens, their polyvalent forms played a significant role in binding. From the 50% molar inhibition profile, the shape of the ACL combining site appears to be a cavity type and most complementary to a disaccharide of Galbeta1-3GalNAc (T), while the PNA binding domain is proposed to be Galbeta1-3GalNAcalpha or beta1- as the major combining site with an additional disaccharide as subsite (partial cavity type), and most complementary to a linear tetrasaccharide, Galbeta1-3GalNAcbeta1-4Galbeta1-4Glc (T(beta)1-4L, asialo GM(1) sequence). These results should help us understand the differential contributions of polyvalent ligands, glycotopes and subtopes of these lectins to binding, and make them useful tools to study glycosciences, glycomarkers and their biological functions.