Harry C. Winter

A Lectin Isolated from Bananas Is a Potent Inhibitor of HIV Replication

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 enzyme-linked immunosorbent 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. Furthermore, 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.

Phylogenetic and specificity studies of two-domain GNA-related lectins: Generation of multispecificity through domain duplication and divergent evolution

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.

The Mushroom Marasmius oreades Lectin Is a Blood Group Type B Agglutinin That Recognizes the Galα1,3Gal and Galα1,3Galβ1,4GlcNAc Porcine Xenotransplantation Epitopes with High Affinity

A blood group B-specific lectin from the mushroomMarasmius oreades (MOA) was investigated with respect to its molecular structure and carbohydrate binding properties. SDS-PAGE mass spectrometric analysis showed it to consist of an intact (H; 33 kDa) and truncated (L; 23 kDa) subunit in addition to a small polypeptide (P; 10 kDa). Isolation in the presence of EDTA produced only the H subunits, indicating that the latter two are formed by metalloprotease cleavage of the intact H subunit. Tryptic digestion of the H, L, and P polypeptide chains followed by mass spectral analysis supports this view. The lectin strongly precipitated blood group type B substance, was nonreactive with type A substance, and reacted weakly with type H substance. Carbohydrate binding studies reveal a high affinity for Galα1,3Gal (but not for the isomeric α1,2-, α1,4-, and α1,6-disaccharides); Galα1,3Galβ1,4GlcNAc; and the type B branched trisaccharide. MOA also reacts strongly with murine laminin from the Engelbreth-Holm-Swarm sarcoma and bovine thyroglobulin, both of which contain multiple Galα1,3Galβ1,4GlcNAc end groups. This linear B trisaccharide is a component of porcine tissues and organs, preventing their transplantation into humans. MOA also shares carbohydrate recognition of this trisaccharide with toxin A elaborated by Clostridium difficile.

Cloning, expression in Escherichia coli and characterization of the recombinant Neu5Acα2,6Galβ1,4GlcNAc-specific high-affinity lectin and its mutants from the mushroom Polyporus squamosus

Lectin from the mushroom Polyporus squamosus (PSL) has a unique carbohydrate-binding specificity for sialylated glycoconjugates containing Neu5Acalpha2,6Galbeta1,4Glc/GlcNAc trisaccharide sequences of asparagine-linked glycoproteins. In the present study, we elucidate the molecular basis for its binding specificity as well as establish a consistent source of this useful lectin using a bacterial expression system. cDNA cloning revealed that PSL contains a ricin B chain-like (QXW)(3) domain at its N-terminus that is composed of three homologous subdomains (alpha, beta and gamma). A recombinant lectin was expressed in Escherichia coli as a fully active, soluble form. It agglutinated rabbit erythrocytes and showed the highest affinity for Neu5Acalpha2,6Galbeta1,4GlcNAc, but not for the sialyl alpha2,3-linked isomer. We also investigated the structure-function relationship of PSL. A monomeric C-terminal deletion mutant lacking 40% of the lectins molecular mass retained sugar-binding activity, indicating that the carbohydrate-binding sites are situated in the N-terminal portion of the lectin, whereas the C-terminal portion probably functions in oligomerization and structural stabilization. Mutant constructs that have single amino acid substitutions in the putative sugar-binding sites, based on sequence alignment with the ricin B-chain, indicate that the beta and gamma subdomains are most probably sugar-binding sites. The recombinantly expressed lectin will be a valuable reagent for the detection of the Neu5Acalpha2,6Galbeta1,4GlcNAc sequence of asparagine-linked glycans.

Glutamate uptake system in the presynaptic vesicle: Glutamic acid analogs as inhibitors and alternate substrates

A variety of naturally occurring amino acids, their isomers, and synthetic analogs were tested for their ability to inhibit uptake of [3H]glutamate into presynaptic vesicles from bovine cerebral cortex. Strongest inhibition (Ki<1mM) was observed fortrans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) anderythro-4-methyl-L-glutamic acid (MGlu), while 4-methylene-L-glutamic acid (MeGlu) was only moderately inhibitory (Ki=∼3mM), indicating that the synaptic vesicle glutamate translocator has higher affinity forrans-ACPD and MGlu than for glutamate. A few other amino acids, e.g., 4-hydroxyglutamic acid, S-carboxyethyl cysteine, and 5-fluorotryptophan, were slightly inhibitory; alll- anddl-isomers of protein amino acids and longer chain acidic amino acids were without measurable inhibition. Potassium tetrathionate and S-sulfocysteine exhibited strong to moderate noncompetitive or irreversible inhibition. Inhibition by t-ACPD, MGlu, or MeGlu was competitive with glutamic acid. Each of these competitive inhibitors was also taken up by the vesicle preparation in an ATP-dependent manner, as indicated by their being recovered unchanged from filtered vesicles. Similar results were obtained with reconstituted vesicles, while glutamate uptake by partially purified rat synaptosomes was inhibited only by MGlu. These results indicate that the glutamate translocator of presynaptic vesicles has stringent structural requirements distinct from those of the plasma membrane translocator and the metabotropic type of postsynaptic glutamate receptor. They further suggest possible structural requirements of pharmacologically significant compounds that can substitute for glutamic acid in the presynaptic side of glutamatergic synapses, thus serving to moderate or control glutamate excitation and associated excitotoxic effects in these neurons.

Cloning, Expression, and Characterization of the Galα1,3Gal High Affinity Lectin from the Mushroom Marasmius oreades

The purification and unique carbohydrate binding properties, including blood group B-specific agglutination and preferential binding to Galα1,3Gal-containing sugar epitopes, of theMarasmius oreades agglutinin (MOA) are reported in an accompanying paper (Winter, H. C., Mostafapour, K., and Goldstein, I. J. (2002) J. Biol. Chem. 277, 14996–15001). Here we describe the cloning, characterization, and expression of MOA. MOA was digested with trypsin and endoproteinase Asp-N, and the peptide fragments were purified by high performance liquid chromatography. Amino acid sequence data were obtained for eight peptides. Using oligonucleotides deduced from the peptide sequences for a reverse transcriptase-PCR, a 41-base pair cDNA was obtained. The 41-base pair fragment allowed the generation a full-length cDNA using 5′ and 3′ rapid amplification of cDNA ends. MOA cDNA encodes a protein of 293 amino acids that contains a ricin domain. These carbohydrate binding domains were first described in subunits of bacterial toxins and are also commonly found in polysaccharide-degrading enzymes. Whereas these proteins are known to display a variety of sugar binding specificities, none to date are known to share MOAs high affinity for Galα1,3Gal and Galα1,3Galβ1,4GlcNAc. Recombinantly expressed and purified MOA retains the specificity and affinity observed with the native protein. This study provides the basis for analyzing the underlying cause for the unusual binding specificity of MOA.

Structural Characterization of a Lectin from the Mushroom Marasmius oreades in Complex with the Blood Group B Trisaccharide and Calcium

MOA (Marasmius oreades agglutinin), a lectin isolated from fruiting bodies of the mushroom M. oreades, specifically binds nonreducing terminal Galalpha(1,3)Gal carbohydrates, such as that which 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 altered the conformation of the C-terminal domain by opening up the cleft containing a putative catalytic site.

Purification, Characterization, Molecular Cloning, and Expression of Novel Members of Jacalin-related Lectins from Rhizomes of the True Fern Phlebodium aureum (L) J. Smith (Polypodiaceae)

A lectin was purified from rhizomes of the fernPhlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of ∼15 kDa associated by noncovalent bonds. From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5′- and 3′-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues. The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins. PALa contained peptide sequences exactly matching those found in the isolated lectin. PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography. Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase. The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry. Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins. The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.

Xanthosoma sagittifolium Tubers Contain a Lectin with Two Different Types of Carbohydrate-binding Sites

An unusual lectin possessing two distinctly different types of carbohydrate-combining sites was purified from tubers of Xanthosoma sagittifolium L. by consecutive passage through two affinity columns, i.e.asialofetuin-Sepharose and invertase-Sepharose. SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and gel filtration chromatography of the purified lectin showed that theX. sagittifolium lectin is a heterotetrameric protein composed of four 12-kDa subunits (α2β2) linked by noncovalent bonds. The results obtained by quantitative precipitation and hapten inhibition assays revealed that the lectin has two different types of carbohydrate-combining sites: one type for oligomannoses, which preferentially binds to a cluster of nonreducing terminal α1,3-linked mannosyl residues, and the other type for complex N-linked carbohydrates, which best accommodates a non-sialylated, triantennary oligosaccharide withN-acetyllactosamine (i.e. Galβ1,4GlcNAc-) or lacto-N-biose (i.e. Galβ1,3GlcNAc-) groups at its three nonreducing termini.

Marasmius oreades agglutinin (MOA) is a chimerolectin with proteolytic activity

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 MOAs proteolytic activity for mushroom defense against pathogens is discussed.