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Title:  Isolation, characterization, cloning and use of a mushroom lectin

United States Patent:  6,958,321

Issued:  October 25, 2005

Inventors:  Goldstein; Irwin J. (Ann Arbor, MI); Winter; Harry C. (Ann Arbor, MI); Kruger; Robert P. (Ann Arbor, MI)

Assignee:  The Regents of the University of Michigan (Ann Arbor, MI)

Appl. No.:  137077

Filed:  May 2, 2002

Abstract

The isolation, characterization, cloning and expression of the lectin (agglutinin) from Marasmius oreades (MOA) is described. MOA displays unique carbohydrate binding properties, including blood group B-specific agglutination and preferential binding to Ga1α1,3Ga1-containing sugar epitopes, including but not limited to Ga1α1,3Ga1β1,4G1cNAc. MOA is contemplated as an affinity reagent, a therapeutic in the treatment of antibiotic-induced diarrhea and the field of xenotransplantation. MOA may also serve as a diagnostic reagent, e.g. for malaria.

SUMMARY OF THE INVENTION

In some embodiments, a method is contemplated, comprising: a) providing: (i) M. oreades mushrooms, and (ii) an extraction buffer comprising a protease inhibitor cocktail and ethylenediaminetetraacetic acid; b) preparing an extract of said M. oreades mushrooms in said extraction buffer; and c) treating said extract under conditions such that a lectin preparation is produced. In some embodiments, said preparing step b) is carried out under an atmosphere of argon. In some embodiments, said treating step c) comprises column chromatography. In some embodiments, said column chromatography comprises a melibiose-Sepharose affinity column.

In some embodiments, an additional purification step d) passing said lectin preparation through an affinity column to produce a purified lectin is contemplated. In some embodiments, said affinity column of step d) comprises G1α1,3 [L-Fucα1,2]Ga1βO(CH2)nCONH. In some embodiments, said lectin produced by passing said lectin preparation through an affinity column has high binding affinity for G1α1,3Ga1. In some embodiments, the lectin preparation is substantially free of proteolytic fragments of said lectin. In some embodiments, said binding affinity is determined by a method selected from the group consisting of isothermal titration calorimetry, blood cell agglutination and carbohydrate precipitation. Binding affinity of the lectin preparation and the purified lectin may be determined by a method selected from the group consisting of isothermal titration calorimetry, blood cell agglutination and carbohydrate precipitation. In some embodiments, a composition comprising the purified lectin is contemplated.

In some embodiments, a method is contemplated, comprising: a) providing: (i) a composition comprising labeled M. oreades agglutinin, and (ii) a sample obtained from a subject; and b) contacting said sample with said labeled M. oreades agglutinin under conditions such that a glycoconjugate in said sample is detected. It is not intended that the method be limited to any particular method of preparation of said M. oreades agglutinin for labeling and use in the method. A variety of M. oreades agglutinin preparations are contemplated. In some embodiments, recombinantly produced M. oreades agglutinin is contemplated. In some embodiments, biochemically purified M. oreades agglutinin is contemplated. In some embodiments, said biochemically purified M. oreades agglutinin is substantially free of proteolytic fragments, including fragments of approximately 23 kDa and approximately 10 kDa. In some embodiments, a biochemically prepared M. oreades agglutination preparation comprising proteolytic fragments is contemplated. In some embodiments, said subject has one or more symptoms of malaria. In some embodiments, said sample is a human blood sample. In some embodiments, said human blood sample comprises Plasmodium falciparum merozoites. In some embodiments, said labeled M. oreades agglutinin comprises a fluorescent label. In some embodiments, said fluorescent label is fluorescein.

In some embodiments, a composition comprising immobilized M. oreades agglutinin is contemplated. It is not intended that the composition be limited to any particular method of preparation of said M. oreades agglutinin for immobilization. A variety of M. oreades agglutinin preparations are contemplated. In some embodiments, recombinantly produced M. oreades agglutinin is contemplated. In some embodiments, biochemically purified M. oreades agglutinin is contemplated. In some embodiments, said biochemically purified M. oreades agglutinin is substantially free of proteolytic fragments, including fragments of approximately 23 kDa and approximately 10 kDa. In some embodiments, a biochemically prepared M. oreades agglutination preparation comprising proteolytic fragments is contemplated. In some embodiments, said immobilized M. oreades agglutinin is immobilized on Sepharose 4B. In some embodiments, said Sepharose 4B-immobilized M. oreades agglutinin is contained in a column.

In some embodiments, a method is contemplated, comprising: a) providing: i) an immobilized-M. oreades agglutinin affinity column, ii) a sample suspected of comprising a saccharide selected from the group consisting of G1α1,3Ga1, G1α1,3Ga1β1,4G1cNAc and G1α1,3 [L-Fucα1,2]Ga1; b) contacting said sample with said affinity column under conditions such that said saccharide is bound in a complex; and c) treating said complex under conditions such that said saccharide is released from said complex. It is not intended that the method be limited to any particular method of preparation of said M. oreades agglutinin for immobilization and use in the affinity column of the method. A variety of M. oreades agglutinin preparations are contemplated. In some embodiments, recombinantly produced M. oreades agglutinin is contemplated. In some embodiments, biochemically purified M. oreades agglutinin is contemplated. In some embodiments, said biochemically purified M. oreades agglutinin is substantially free of proteolytic fragments, including fragments of approximately 23 kDa and approximately 10 kDa. In some embodiments, a biochemically prepared M. oreades agglutination preparation comprising proteolytic fragments is contemplated. In some embodiments, said sample is a serum sample.

In some embodiments, a composition comprising an isolated nucleic acid having the sequence of SEQ ID NO: 1 is contemplated. In some embodiments, a vector comprising the isolated nucleic acid having the sequence of SEQ ID NO: 1 is contemplated. In some embodiments, said vector is an expression vector. In some embodiments, said nucleic acid sequence is operably linked to an inducible promoted in said expression vector. In some embodiments, a cell comprising an expression vector comprising a nucleic acid having the sequence of SEQ ID NO: 1 is contemplated. In some embodiments, said cell is a bacterial cell. In some embodiments, said bacterial cell further comprises the protein encoded by the nucleic acid having the sequence of SEQ ID NO: 1. In some embodiments, a composition comprising purified DNA having the nucleotide sequence of SEQ ID NO: 1 is contemplated. In some embodiments, RNA transcribed from said DNA is contemplated. In some embodiments, protein translated from said RNA is contemplated. In some embodiments, antibodies produced against said protein are contemplated. In some embodiments, transgenic animals comprising the DNA having the nucleotide sequence of SEQ ID NO: 1 are contemplated. In some embodiments, a vector comprising DNA having the nucleotide sequence of SEQ ID NO: 1 is contemplated. In some embodiments, said vector is an expression vector. In some embodiments, cells comprising said expression vector are contemplated. In some embodiments, said cells are selected from the group consisting of bacterial cells, yeast ells, insect cells and mammalian cells. In a preferred embodiment, said cells are bacterial cells. In some embodiments, a composition comprising an isolated polypeptide having the sequence of SEQ ID NO: 2 is contemplated. In some embodiments, a composition comprising an isolated polypeptide having the sequence of SEQ ID NO: 3 is contemplated.

Specificity varies greatly among carbohydrate binding proteins. While some lectins broadly recognize all oligosaccharides containing particular terminal sugars, others show increasing affinity for specific di- and tri-saccharides. Fewer still show almost no reactivity with a given sugar monomer, yet bind strongly and specifically to particular oligosaccharides. The present invention contemplates a protein, MOA, which binds to G1α1,3Ga1-containing sugars with specificity. In a preferred embodiment, the protein is isolated from a mushroom, such as M. oreades (which can be purchased from American Mushroom Hunter Corp. (Middle Town, N.J.).

In one embodiment, the protein is purified biochemically. In a preferred embodiment, a protease inhibitor cocktail (e.g. product P8215) is included during the extraction procedure at the level of at least 1 ml/L extract buffer in place of phenylmethylsulfonyl fluoride, eliminating Ca++ and including at least 1.25 mM EDTA in all buffers. Ideally, the extraction and ammonium sulfate precipitations are performed under an atmosphere of argon. Such a procedure minimizes proteolysis and yields a protein monomer (whereas proteolysis can create a dimer or trimer).

In a preferred embodiment, the protein is recombinant and expressed in a transfected host cell. A variety of host cells are contemplated including but not limited to bacterial cells, insect cells and mammalian cells. On the other hand, the protein can also be prepared without host cells using a coupled in vitro transcription/translation kit (available commercially from Promega, Madison Wis.).

In one embodiment, the present invention contemplates the protein having the amino acid sequence shown in "SEQ ID NO:2 shown in FIG. 1A". In another embodiment, the present invention contemplates the protein having the amino acid sequence shown in "SEQ ID NO: 3 shown in FIG. 2". On the other hand, proteins having conservative amino acid substitutions in this sequence are also contemplated. For example, one skilled in the art understand that certain amino acids can be readily substituted with no significant loss in activity (e.g. isoleucine for leucine, etc.). One skilled in the art will understand that some sequences may be polymorphic and polymorphisms in the amino acid sequence shown in FIG. 1A are contemplated. One such polymorphic sequence is shown in FIG. 2 [SEQ ID NO:3].

Whether prepared biochemically or recombinantly, the present invention contemplates labeling the protein (e.g. with a radiolabel, fluorescent label, etc.). In one embodiment, fluorescein-labeled MOA protein is provided. In one embodiment, fluorescein-labeled MOA is used in a method comprising; a) providing (i) labeled MOA and (ii) a blood smear, and (b) contacting said blood smear with said labeled MOA; and (c) examining said contacted smear for the presence of labeled MOA. In one embodiment, said blood smear is from a subject exhibiting one or more symptoms of malaria. In one embodiment, said blood smear is from a subject suspected of having malaria. In one embodiment, said presence of said labeled MOA in said examining step (c) is diagnostic of the presence of Plasmodium falciparum merozoites. In another embodiment, the present invention contemplates immobilizing the protein (e.g. on a resin for affinity purification of carbohydrate containing molecules, on beads for binding to cells, etc.). In one embodiment, said immobilization is on Sepharose B beads. In one embodiment, said immobilized MOA protein is used as an affinity reagent for isolation of carbohydrate containing molecules. In one embodiment, said carbohydrate containing molecules comprise a Gala 1,3Ga1 epitope. In one embodiment, affinity purification of a carbohydrate epitope from Plasmodium falciparum merozoites is contemplated.

The present invention contemplates the protein (or portions thereof) encoded by the nucleic acid sequence shown in FIG. 3. This nucleic acid sequence can be readily inserted in a variety of vectors for expression. Therefore, the present invention contemplates vectors comprising the nucleic acid sequence shown in "SEQ ID NO: 1 shown in FIG. 3" as well as host cells containing such a vector and the product of such host cells.

The present invention contemplates methods wherein the biochemically produced protein monomer or the recombinant protein are used for binding. It is not intended that the method of biochemically producing the protein be limiting. In some embodiments, biochemical methods are used to produce a M. oreades agglutinin preparation comprising proteolytic fragments of approximately 23 kDa and 10 kDa. In some embodiments, biochemical methods are used to purify the M. oreades agglutinin such that the preparation is substantially free of proteolytic fragments (including fragments of approximately 23 kDa and 10 kDa). Binding can be used for blood testing. In one embodiment, said blood testing is to detect the presence of Plasmodium falciparum merozoites. On the other hand, binding can be done to mask an immunogenic epitope on a tissue for transplantation. Furthermore, binding can be used to detect a microorganism (e.g. binding to the plasmodium of malaria) or cancer cell. Finally, binding can be used to cause disease (e.g. administration of the protein to cause a kidney pathology).

In some embodiments, the present invention provides a novel lectin (i.e. agglutinin) isolated from Marasmius oreades (hereinafter "MOA"). The lectin, MOA, has a high binding affinity for Gala 1,3Ga1 end groups and is specific for the α 1,3-linkage. MOA does not bind the isomeric a 1,4 and a 1,6-disaccharides or individual sugar residues. In preferred embodiments, intact MOA is isolated as polypeptide of approximately 33 kDa. Ideally, the 33 kDa protein is purified so as to be substantially free of proteolytic fragments.

In some embodiments, the present invention also provides methods for using MOA. It can be used as a research tool to detect the presence of and characterize di- and tri-saccharides having at least one Gala 1,3Ga1 unit present. An immobilized form of MOA can be used to isolate glycoconjugates, polysaccharides or oligosaccharides or to resolve mixtures containing molecules having at least one Gala 1,3Ga1 unit present. Therapeutic methods are also provided. MOA can be used to block transplant rejection of porcine tissue in humans. Porcine tissues and organs contain the Ga1α1,3Ga1β1,4G1cNAc epitope which is not found in humans and is involved in tissue rejection. Binding of MOA to this epitope would block recognition by the immune system and thereby block transplant rejection. The present invention further provides methods for blocking the action of Toxin A from Clostridium difficile which is responsible for antibiotic-induced diarrhea. Both MOA and Toxin A recognize the same trisaccharide epitope, Ga1α1,3Ga1↑1,4G1cNAc. A patient taking antibiotics or suffering from diarrhea caused by Toxin A can be treated with a therapeutically effective amount of MOA. MOA would then compete with Toxin A to bind the target cells, preventing Toxin A from causing any symptoms.

DESCRIPTION OF THE INVENTION

In several embodiments, the present invention contemplates a purified lectin, MOA, with specificity for Ga1α1,3Ga1-containing epitopes. In some embodiments, the MOA is purified from the fairy-ring mushroom Marasmius oreades. In some embodiments, MOA is expressed recombinantly, for example in bacteria. In some embodiments, the nucleic acid sequence encoding MOA is contemplated. In other embodiments, MOA is contemplated for use in a variety of applications, including but not limited to affinity purification, diagnostics, and therapeutics, including, for example, use in the field of xenotransplantation. The description that follows is divided into the following sections: I. Overview of the Purification of MOA, II. Binding Specificity of MOA, III. Cloning and Recombinant Expression of MOA, IV. Methods of Using MOA and IV. Therapeutic Formulations.

I. Overview of the Purification of MOA

In some embodiments, MOA is purified from M. oreades mushrooms. The mushroom may be harvested from outdoor plots, or may be ordered from commercial or gourmet food sources, for example from American Mushroom Hunter Corp. (Middle Town, NJ). Fresh mushroom tissue is homogenized and then strained and centrifuge (see Examples below). A series of chromatographic affinity columns is then used to purify the MOA lectin. In initial experiments, carried out in the presence of phenylmethylsulfonyl fluoride (PMSF), three peptides were detected by SDS-PAGE mass spectrometric analysis: one with a Mr of 33 KDa, one with a Mr of 23 kDa and one with a Mr of 10 kDa. A lectin from M. oreades having subunits of 33 kDa and 23 kDa was reported previously [Horejsl et al. Biochim. Biophys. Acta 538:299 (1978)].

Subsequent analysis (see Examples below), including analysis of tryptic fragments, isolation in the presence of EDTA and recombinant expression showed that the intact MOA lectin is a single peptide of approximately 33 kDa. While not to be limited to any particular mechanism, and with the understanding that knowledge of the underlying mechanism is not required for the practice of the present invention, it is believed that the originally identified 23 kDa and 10 kDa peptides are formed by metalloprotease cleavage of the intact, 33 kDa peptide.

In the presently preferred embodiments for isolation of MOA from mushrooms (see Example 2 below), the extraction procedure is carried out (i) in the presence of a protease inhibitor cocktail (including but not limited to product P8215, from Sigma), rather than the PMSF (i.e. the protease inhibitor cocktail replaces the PMSF), (ii) Ca2+ is eliminated, (iii) EDTA is included in all buffers and (iv) the extraction and initial ammonium sulfate precipitations are carried out under an atmosphere of argon. Again, while to be limited to any particular mechanism, it is believed that these conditions reduce or eliminate proteolysis and possibly oxidation of the MOA lectin.

Based on results of size exclusion chromatography (see Examples below) carried out using native MOA lectin isolated using the preferred methods (which include use of a protease inhibitor cocktail, the elimination of Ca2+, the presence of EDTA and an atmosphere of argon for the initial extraction and precipitation steps) it is believed that native MOA exists as a homodimer of the 33 kDa polypeptide.

In some embodiments, a composition comprising an isolated MOA polypeptide of approximately 33 kDa is contemplated, wherein said composition is substantially free of polypeptides of approximately 23 kDa and approximately 10 kDa (ie. fragment, of the 33 kDa polypeptide). In some embodiments, it is contemplated that said composition will have undetectable levels of 23 kDa and 10 kDa polypeptides, as detected by Coomassie blue staining of SDS-PAGE gels (i.e. said composition is substantially free of polypeptides of 23 kDa and 10 kDa). While other detection is methods (e.g. silver staining of SDS-PAGE gels) may detect small amounts of 23 kDa and 10 kda polypeptides, the inability to visually detect these polypeptides by Coomassie Blue staining of a purified MOA preparation is sufficient to classify such preparations as "substantially free" of the 23 kDa and 10 kDa polypeptides. In some embodiments, said composition is prepared from Marasmius oreades mushrooms. In some embodiments, said composition comprising said isolated MOA polypeptide, wherein said polypeptide is substantially free of polypeptides of 23 kDa and 10 kDa is contemplated as being used in a variety of applications, including but not limited to affinity purification, xenotransplantation, and diagnostics (e.g. detection of the presence of Plasmodium falciparum merozoites in a blood sample).

II. Binding Specificity of the MOA Lectin

While the MOA used in various individual experiments (see Examples below) may have been either isolated from M. oreades (as described supra), or produced recombinantly (see below), it should be noted that the binding specificity of the lectin is the same, regardless of the source (i.e. recombinant or biochemically isolated). As noted above, in instances where the lectin is biochemically isolated from M. oreades, the preferred isolation procedure is carried out in Ca2+-free buffers, in the presence of a protease inhibitor cocktail and EDTA, and (for certain steps) under an argon atmosphere.

A. Blood Group Specificity

In some embodiments, blood group specificity of MOA is determined in hemagglutination assays (see Examples below). Briefly, formaldehyde-treated erythrocytes in V-well microtitre plates are used in a protocol as described in Mo et al. [J. Biol. Chem 275:10623 (2000)], herein incorporated by reference. Strong hemagglutination activity was detected for human blood group B (human B erythrocytes), but very little activity was detected for human type A or type O erythrocytes. Example 3, below, presents the data for agglutination activity against a variety of cell types. MOA readily agglutinated Ehrlich ascites tumor cells, which contain the same Ga1α1,3Ga1 di- and Ga1α1,3Ga1β1,4Ga1 tri-saccharides in their cell membrane [Eckhardt and Goldstein. Biochemistry 22: 5290 (1983)].

In other embodiments, blood group specificity of MOA is determined by quantitative precipitation assays. Quantitative microprecipitation assays with soluble cyst blood group substances and inhibition of precipitation by sugar haptens were performed as described in Mo et al., [J. Biol. Chem. 275:10623 (2000)]. As shown in Example 4 below, MOA reacts strongly with human blood type B substance, not at all with type A substance, and rather weakly with type H substance.

B. Sugar Ligand Binding

Sugar ligand binding to MOA was conducted using three approaches: (i) inhibition of Type B hemagglutination, (ii) hapten inhibition of MOA-type B substance precipitation and (iii) isothermal calorimetry (see examples below). All three methods gave approximately the same results, with the calorimetric data being the most precise.

Being a blood type B agglutinin, MOA was assayed primarily against D-galactosyl-terminated sugars and oligosaccharides. As shown in the examples below, lactose, N-acetyllactosamine and melibiose (Ga1α1,6G1c) were very poor ligands. Methyl α-galactopyranoside was similarly very poor. The blood group B disaccharide, Ga1α1,3Ga1, was an excellent ligand with Ka 6.0103 M-1 whereas the isomeric disaccharides Ga1α1,2Ga1, G1α1,4Ga1 and Ga1α1,6Ga1 bound poorly or not at all. Addition of a G1cNAc group to the reducing end of Ga1α1,3Ga1 to give Ga1α1,3Ga1β1,4G1cNAc increased the binding by approximately 50% to Ka 9.7103 M-1. Similarly, adding an L-fucosyl group to the disaccharide to afford the blood group B branched trisaccharide (Ga1α1,3[L-Fucα1,2]Ga1) enhanced its affinity to MOA 4-fold (Ka 3.6104 M-1). Finally, the trisaccharide L-Fucα1,2Ga1β1,4G1c (fucosyllactose), related to the blood group H trisaccharide, had a Ka of 548 M-1 by isothermal titration calorimetry. It appears that the L-fucosyl residue makes a significant contribution to the binding affinity of the essentially inactive lactose (Ka=185 M-1). While not limited to any particular mechanism, and with the understanding that knowledge of the underlying mechanism is not required for the practice of the invention, it is believed that recognition of the L-fucosyl moiety is the reason for limited agglutination of human O erythrocytes and a weak precipitin curve with blood group H substance.

Other precipitation reactions were carried out (see Examples below) and it was found that MOA reacts strongly with laminin (which has been shown to contain Ga1α1,3Ga1β1,4G1cNAc-end groups [Shibata et al. FEBS Letter 214:194 (1982); Knibbs et al. Biochemistry 28:6379 (1989)]. MOA also reacted strongly with bovine thyroglobulin, which has been shown to have the same determinants [Spiro and Bhoyroo J. Biol. Chem. 259:9858 (1984)]. No precipitin reaction was observed with pigeon ovalbumin, which is known to contain multiple G1α1,4Ga1 end groups [Suzuki et al. J. BioL. Chem. 276: 23230 (2001)], thus demonstrating the specificity of MOA for Gala 1,3Ga1 groups. MOA also failed to recognize the blood group type A disaccharide (Ga1NAc≢1,3Ga1), as no precipitation was observed with this disaccharide-polyacrylamide glycoconjugate.

Binding assays with labeled MOA are also contemplated. It is not intended that the nature of the label be limiting. A variety of labels are contemplated, including but not limited to radioactive labels (e.g. 35S, 14C, 125I, 3H and 131I), fluorescent labels (e.g. rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red), and various enzyme-substrate labels (see e.g. U.S. Pat. No. 4,275,149, herein incorporated by reference, and leciferase, luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatasae (AP), β-galactosidase, glucomaylase, lysozyme, sacharide oxidases (e.g. glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase and the like). Biotin is also contemplated as a label. In one embodiment, fluorescein-labeled MOA is contemplated for use in binding assays. In one embodiment, fluorescein-labeled MOA is used to stain porcine striated skeletal muscle (see, Example 7 below). Endothelial cells lining the capillaries are the significant structures stained by the lectin. In another embodiment, fluorescein-labeled MOA is used to stain Plasmodium falciparum merozoites in a human blood smear (see Example 7 below).

Further insight into the carbohydrate specificity of MOA was obtained by using immobilized MOA. Immobilization of MOA on a variety of supports is contemplated. In one embodiment, MOA is immobilized on a matrix that can be used in an affinity column (see below). In some embodiments, the matrix comprises Sepharose 4B beads. In some Embodiments, the MOA-conjugated Sepharose 4B beads are packed in a column for binding and affinity isolation and purification studies. Briefly, the MOA-Sepharose column is loaded with glycan, glycoconjugate or serum sample to be tested, then washed with an appropriate buffer (including but not limited to PBS). Bound components are eluted with a suitable elution buffer (including but not limited to 1,3-diaminopropane in NaCl), and the collected fractions are analyzed for the presence of protein or glycan (see Example 6 below). The fractions containing the bound components (eluted as described herein) can be pooled and concentrated.

In order to confirm the binding specificity of the MOA lectin, various glycans and glycoconjugates were applied to the MOA-Sepharose column. Laminin purified from the EHS sarcoma and bovine thyroglobulin (both of which contain multiple G1α1,3Ga1β1,4G1cNAc end groups [Knibbs et al. Biochemistry 28: 6379 (1989); Spiro and Bhoyroo. J. Biol. Chem. 259:9858 (1984)] bound tightly to the immobilized MOA (see Example below). Asialofetuin and degalactosylated bovine thyroglobulin passed through the column, not being recognized by MOA. Pigeon ovalbumin (which contains Ga1α1,4Ga1-terminated N-glycans [Suzuki et al. J. Biol. Chem 276: 23221 (2001)] also did not bind the MOA-Sepharose column. Blood group H substance, which has terminal Fucα1,2Ga1β1,4G1cNAc chains, also failed to bind MOA-Sepharose, although the trisaccharide Fucα1,2Ga1=1,4G1c exhibited low but measurable binding in solution (see above). Galactomannan, which has Ga1α1,6Man branches on a linear Manβ1,4Manβ backbone also failed to bind immobilized MOA. While not to be limited to any particular mechanism, and with the understanding that knowledge of the underlying mechanism is not required for the practice of the invention, it is believed that MOA exhibits trisaccharide specificity and requires a Ga1α1,3Ga1 linkage for binding. Interactions of the Sepharose-immobilized MOA with other substances are described below.

III. Cloning and Expression of Recombinant MOA

The present invention contemplates the recombinant expression of MOA and the resulting composition, the only known Ga1α1,3Ga1 specific lectin. This is also the first reported protein sequence from the fairy-ring mushroom Marasmius oreades. MOA appears to belong to the ricin protein superfamily because of the presence of a conserved carbohydrate binding domain originally identified in the B chain of ricin, one of the first studied hemagglutinins. Many of these proteins, like the ricin B chain itself, promote the internalization of disulfide-linked toxic protomers through their binding to glycosylated cell surface receptors. There is no evidence that MOA functions in this manner.

Structural analysis of ricin domains suggests that they are composed of three repeating subdomains that may have originated from an ancestral galactose-binding motif. Closer analysis of the three subdomains of MOA indicates strong conservation with the key residues in the 1α and 2γ subdomains of ricin and ebulin (FIG. 4). Structural determination of these proteins in the presence of sugar shows binding to these two subdomains. All of the MOA subdomains have the conserved QXW motif. In ricin the conserved tryptophan is necessary for hydrophobic packing of the core structure, while the glutamine coordinates the conserved aspartic acid that hydrogen bonds with the third and fourth oxygens of the galactosyl moiety. The asparagine prior to the QXW motif also hydrogen bonds with the 03 and 04 of the sugar. The corresponding histidine found in the MOA subdomains could function similarly. Additionally, there is a conserved hydrophobic position, occupied by tryptophan, tyrosine, or phenylalanine between the conserved aspartic acid and asparagine. This residue forms a stacking interaction with the sugar ring. In the MOA subdomains this position is occupied by a conserved tryptophan.

Because the essential features required for galactosyl binding are conserved in MOA, it is interesting that the specificity of ricin is very different from that of MOA. While MOA is specific for G1α1,3Ga1-containing sugars, ricin binds well with β-1,3 or β-1,4-linked galactose-terminated sugars. Like MOA, ricin shows higher affinity for larger, more complex saccharides than for simple sugars. The affinity constant for lactose binding to ricin is 10-fold greater than for galactose alone. Similarly, MOA binds Ga1α1,3Ga1 with an affinity constant 44-fold greater than that for Meα-Ga1. While the structure of ricin shows hydrogen bonding exclusively to the terminal sugar, it is clear that elements outside of the main binding pocket are important for determining the strength and specificity of binding.

Of particular interest in explaining the difference between MOA and other ricin domain proteins could be the loop region between the stacking hydrophobic and sugar binding asparagine/histidine. Unlike other subdomain segments, it does not model well onto ricin. This loop is longer in MOA than in ebulin and ricin by one to three residues, and appears structurally different with the absence of a conserved proline followings the hydrophobic stacking residue. While the present invention is not limited to any particular mechanism, this region could provide an additional hydrogen bonding interface specific for G1α1,3Ga1 containing sugars either through direct side-chain contact or water-mediated interactions and would be appropriately positioned to sterically block sugars not in the 1,3 orientation.

The cloning and expression of the recombinant MOA provides a route for understanding the structure and unique carbohydrate binding specificity of this novel lectin. Crystallographic structure determination of MOA in the presence of bound sugar should provide the explanation for the specific binding of G1α1,3Ga1.

In order to clone the MOA cDNA (see Example 8 below), 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 oligonucleotide sequences prepared based on the peptide sequences, RT-PCR was carried out on mRNA isolated from M. oreades and a 41 base pair cDNA fragment was obtained. The full-length cDNA was obtained using 5′ and 3′ rapid amplification of cDNA ends (RACE). MOA cDNA encodes a protein of 293 amino acids that contains a ricin domain. Recombinantly expressed and purified MOA retains the specificity and affinity observed with the native protein.

In one embodiment, the cDNA sequence encoding MOA [SEQ ID NO: 1] is contemplated. In one embodiment, a polymorphism in SEQ ID NO: 1 is specifically contemplated. In one embodiment, the amino acid sequence encoded by SEQ ID NO: 1 is contemplated (i.e. the MOA amino acid sequence; SEQ ID NO: 2; FIG. 1A). In one embodiment, a polymorphism in SEQ ID NO: 2 is specifically contemplated. In one embodiment, the polymorphism comprises an asparagine at position 200, rather than an aspartic acid. The polymorphic sequence comprising asparagine at position 200 [SEQ ID: NO: 3] is presented in FIG. 2. While not limited to any particular mechanism, it is believed that this polymorphism is unlikely to alter binding specificity as it lies outside the predicted ricin domain.

Ir some embodiments, a purified nucleic acid having the sequence of SEQ ID NO: 1, or portions thereof is contemplated. In some embodiments, a purified nucleic acid sequence having a sequence polymorphism in SEQ ID NO: 1 is contemplated. Said polymorphism may comprise between one and four nucleotide differences in comparison to SEQ ID NO: 1. In some embodiments, an isolated nucleic acid having the sequence of SEQ ID NO: 1, or portions thereof is contemplated for use as a probe or primer (for example, for use in hybridization or amplification applications). In some embodiments, an isolated nucleic acid sequence having a sequence that is the complement of SEQ ID NO: 1 is contemplated. In some embodiments, said complementary sequence, or portions thereof, is contemplated for use as a probe or primer (for example, for use in hybridization or amplification reactions). In some embodiments, said probe or primer sequences are labeled. In some embodiments, said label comprises a radioactive label (including but not limited to 32P, 33P and 35S), while in other embodiments, said label comprises biotin. In other embodiments, said label comprise; a fluorescent moiety, including but not limited to fluorescein, Texas Red and rhodamine. In some embodiments, said hybridization reactions comprise Northern analysis. In some embodiments, said amplification reactions comprise PCR and RT-PCR.

In some embodiments, a vector comprising SEQ ID NO: 1 or portions thereof is contemplated. In some embodiments, said vector is an expression vector. In some embodiments, said expression vector comprises (in operable combination) an inducible promoter including but not limited to an IPTG-inducible promoter, for expression of SEQ ID NO: 1 (i.e. said inducible promoter is operably linked to SEQ ID NO: 1). In some embodiments, said expression vector is introduced into a host cell. Thus, in some embodiments, a cell comprising an expression vector comprising SEQ ID NO: 1 is contemplated. In some embodiments, said host cell is selected from the group consisting, of a bacterial cell, a yeast cell, an insect cell and a mammalian cell. In some embodiments, said host cell expresses SEQ ID NO: 1 from said vector. Thus, in some embodiments, a host cell comprising recombinantly expressed MOA protein (e.g. SEQ ID NO: 2) is contemplated. In some embodiments, said recombinantly expressed MOA protein is purified from said host cell. In some embodiments, said purification comprises the steps of (a) providing a host cell expressing said recombinant MOA, (b) lysing said host cells to generate an extract; and (c) purifying said recombinant MOA from said extract. In some embodiments, said purification step (c) comprises column chromatography. In some embodiments, said host cell is a bacterial cell. In some embodiments, said expression of recombinant MOA in said host cell comprises expression from an expression vector comprising SEQ ID NO: 1.

In some embodiments, said recombinantly expressed MOA is purified. Host cells expressing recombinant MOA are lysed in the presence of an appropriate buffer (see Example below). The resulting extract is run through a French press and the insoluble fraction is removed by centrifugation. The soluble fraction is then adsorbed n a melibiose-Sepharose column followed by lactose elution, as described supra and in the Examples below for purification of MOA from M. oreades extracts.

IV. Uses of the MOA Lectin

It is not intended that the present invention be limited by the particular use of MOA. Binding of this novel lectin can be used for blood testing. This lectin is a Blood Group Type B agglutinin. It can be used in standard agglutination assays. On the other hand, the present invention contemplates using the labeled protein for cell binding.

In a preferred embodiment, binding can be done to mask an immunogenic epitope on a tissue for transplantation. More specifically, MOA recognizes the porcine xenotran plantation epitope with high affinity. The reactions to xenografted tissues include hyperacute rejection [the activation of the host complement system after binding of xenoreactive natural antibody (XNA), subsequent endothelial cell, platelet and coagulation system activations] and delayed xenograft rejection (cell-mediated adherence and activation mechanisms). Because MOA binds xenograft tissue epitopes, MOA may effectively block XNA binding and rejection. In one embodiment, porcine tissue to be transplanted is treated with MOA prior to transplantation. In another embodiment, porcine tissue is transplanted and the patient is administered MOA (or a portion thereof) to avoid rejection (e.g. intravenous administration, local administration, etc.)

In yet another embodiment, binding can be used to detect a microorganism (e.g. binding to Plasmodium falciparum merozoites) or a cancer cell. Finally, binding can be used to cause disease (e.g. administration of the protein to cause a kidney pathology).

In other embodiments, MOA is contemplated for use in the detection and preliminary characterization of glycoconjugates containing G1α1,3Ga1 di- and Ga1α1,3Ga1β1,4G1cNAc tri-saccharides on cell surfaces and solution. These epitopes have been found, for example, in the basement membranes of mice, rats and rabbits, the surface of 3T3 cells, calf thyroid plasma membranes, and the plasma membranes of Ehrlich ascites tumor cells. As noted above, porcine tissues and organs contain the G1α1,3Ga1β1,4G1cNAc epitope. By comparing the strength of binding of MOA to an unknown saccharide to known standards (including but not limited to G1α1,3Ga1, Ga1α1,3Ga1β1,4Ga1Nac and Ga1α1,3[Fucα1,2]Ga1) one of skill in the art can make a preliminary determination of the structure of the unknown saccharide.

In an alternative embodiment, an immobilized form of MOA can be used for isolating glycoconjugates, polysaccharides and oligosaccharides. Methods for immobilizing lectins on a solid support are well known to the skilled artisan, and a representative conjugation of MOA to Sepharose B is presented in Example 6 below. Immobilized MOA can also be used for resolving mixtures of saccharides having G1α1,3Ga1 end groups. Methods of using immobilized MOA include the steps of contacting the immobilized MOA with a solution containing the saccharide of interest, incubating the solution and the immobilized MOA under conditions to permit binding of the saccharide to the MOA, washing the immobilized MOA-saccharide conjugate to remove all unbound compounds from the solution, and finally, eluting the saccharide from the immobilized MOA. The immobilized MOA can be used in a column and the solution run through the column, or the immobilized MOA can be added directly to the solution. The saccharide can be eluted from the immobilized MOA by contacting the MOA-saccharide conjugate with a solution containing, for example, G1α1,3Ga1. The saccharide of interest will then be released from the immobilized MOA due to competition from the disaccharide. Alternatively, the saccharide of interest can be eluted from the immobilized MOA by changing the pH of the eluting solution as compared to the pH of the washing solution. While not limited to any particular mechanism, it is believed that as the pH of the eluting solution becomes basic or acidic, MOA begins to denature, releasing the bound saccharide of interest.

In other embodiments, the immobilized MOA can be used in the affinity purification of serum components from human and animal serum. In one embodiment, serum samples from humans and a variety of animals can be applied to a column comprising Sepharose B-immobilized MOA. Following incubation and washing, bound serum components can be eluted. In one embodiment, α2-macroglobulin is eluted from serum from subjects with type B blood (see Example 6 below). Affinity isolation of α2-macroglobulin by immobilized MOA confirms the presence of blood group B epitopes on α2-macroglobulin from the serum of type B subjects.

In some embodiments, affinity isolation of other glycoconjugates is contemplated. In one embodiment, isolation of the Plasmodium falciparum merozoite glycoconjugate to which MOA binds is contemplated. Merozoites can be disrupted to produce an extract, using methods well known to one of skill in the art. For example, in one embodiment, merozoites can be disrupted by detergent, while in another embodiment, merozoites can be disrupted by sonication. It is not intended that the purification be limited to any particular mechanism of disruption. In some embodiments, the extract produced by disruption of the merozoites is subjected to centrifugation to remove insoluble material. The clarified supernatant comprising the merozoite glycoconjugate is then applied to a column comprising Sepharose B-conjugated MOA. Subsequent incubation, washing and elution steps are carried out as describe above and in the examples below. In some embodiments, the affinity isolated and purified P. falciparum merozoite glycoconjugate can be used in a variety of applications, including but not limited to vaccination against malaria (i.e. a composition comprising the affinity isolated and purified P. falciparum merozoite glycoconjugate can be administered to a subject under conditions such that protection against malaria is provided).

As one of skill in the art will appreciate, Sepharose B-immobilized MOA affinity columns can be used for the affinity isolation and purification of a variety of glycoconjugates, including but not limited to a Plasmodium falciparum merozoite glycoconjugate, glycoproteins from the serum of human and animals, including but not limited to α2-macroglobulin from the serum of type B human subjects and bovine thyroglobulin, as well as a variety of other glycoconjugates, including but not limited to laminin, glycoconjugates from basement membranes of mice, rats and rabbits, the surface of 3T3 cells, calf thyroid plasma membranes, the plasma membranes of Ehrlich ascites tumor cells and porcine tissue and organs.

In another embodiment, methods are provided for blocking transplant rejection of porcine tissues and organs transplanted into humans. Porcine tissues and organs comprise the Ga1α1,3Ga1β1,4G1cNAc epitope, the so-called Galili trisaccharide, which prevents their use for transplantation into humans. This epitope is present in most cells of nonprimate mammals and new world monkeys but not in humans, apes, or old world monkeys. The methods contemplated in certain embodiments of the present invention include administering a composition comprising a therapeutically effective amount of MOA to a patient receiving porcine tissue and or a porcine organ. The composition may be administered before, during and/or after transplantation to block transplant rejection by the patient. While not to be limited to any particular mechanism, it is believed that MOA binds the Galili trisaccharide, thereby blocking recognition of the trisaccharide by the patient's immune system. In an alternative embodiment, the porcine tissue or organ is contacted with MOA prior to transplantation into the patient. Said contacting is under conditions such that MOA binds the Galili trisaccharide on the porcine tissue or organ. In some embodiments, MOA is contacted with the porcine tissue or organ prior to transplantation into a human patient, and the patient is also administered a composition comprising MOA. Administration of a composition comprising MOA to a patient receiving a transplant comprising a porcine tissue or organ contacted with MOA may occur prior to, during and/or after transplantation. In some embodiments, said administration of a composition comprising MOA results in reduced rejection of the transplant than is (or has been) observed in the absence of MOA treatment. "Reduced rejection" may comprise one or more of the following: longer survival of the transplanted tissue or organ in the human subject, reduced severity of hyperacute rejection, reduced XNA binding the transplanted tissue and reduced activation of platelet and coagulation system mechanisms.

In a further embodiment, therapeutic methods are provided for treating a patient having Clostridium difficile Toxin A-induced diarrhea, commonly experienced by patients taking antibiotics, with a composition comprising a therapeutically effective amount of MOA. C. difficile Toxin A has been shown to bind to membrane receptors on epithelial cells of the human large intestine and then be internalized by endocytosis. Toxin A has been shown to bind the receptor through the Ga1α1,3Ga1β1,4G1cNAc trisaccharide epitope. Thus, administration of a composition comprising MOA to a subject either experiencing antibiotic-induced diarrhea, or at risk for antibiotic-induced diarrhea (i.e. a subject who is about to begin a course of antibiotic treatment), is expected to be of benefit. While not to be limited to any particular mechanism, it is believed that MOA competes with Toxin A for binding to the Ga1α1,3Ga1β1,4G1cNAc trisaccharide epitope on the receptor, thereby reducing the amount of bound and internalized Toxin A. In some embodiments, administration of a composition comprising MOA (either prophylactically or therapeutically) reduces the duration of antibiotic-induced diarrhea. In some embodiments, administration of a composition comprising MOA (either prophylactically or therapeutically) reduces the severity of antibiotic-induced diarrhea (for example, the patient loses less fluid or has fewer episodes of diarrhea during the course of antibiotic treatment), while in other embodiments, the patient does not experience antibiotic-induced diarrhea.

V. Therapeutic Formulations

As used herein, the terms "therapeutically effective amount" and a "therapeutically effective duration" preferably mean the total amount of each active component of the pharmaceutical composition and a duration of treatment that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions without undue adverse physiological effects or side effects. The term "therapeutically effective amount," when applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients, e.g., MOA, or portions of MOA, and other active ingredients, that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

The MOA lectin of the present invention may thus be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also comprise (in addition to MOA and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).

In practicing embodiments comprising methods of treatment or use of the present invention, a therapeutically effective amount of MOA of the present invention (or a composition comprising MOA or a portion of MOA) is administered to a patient having a condition to be treated, e.g. porcine tissue transplantation (including but not limited to a porcine cardiac valve replacement). MOA may be administered in accordance with the method of the invention either alone or in combination, including in combination with other conventional therapies. For example, MOA can be used as part of a multidrug regime.

Administration of MOA used in the pharmaceutical composition or to practice the method embodiments of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection, transmucosal (including but not limited to intranasal, sublingual, rectal and buccal administration).

When a therapeutically effective amount of MOA is administered orally, the MOA will be in the form of a tablet, capsule, powder, solution or elixir. When administer ed in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% MOA, and preferably from about 25 to 90% MOA. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical compositions may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of MOA of the present invention and preferably from about 1 to 50% MOA.

When a therapeutically effective amount of MOA is administered by intravenous, cutaneous or subcutaneous injection, the MOA will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable MOA solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to MOA, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

The amount of MOA in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of MOA with which to treat each individual patient. Initially, the attending physician will administer low doses of MOA and observe the patient's response. Larger doses of MOA may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
 

Claim 1 of 6 Claims

1. A method of detecting a glycoconjugate, comprising:

a) providing:

(i) a composition comprising labeled M. oreades agglutinin, and

(ii) a sample obtained from a subject; and

b) contacting said sample with said labeled M. oreades agglutinin under conditions such that a glycoconjugate in said sample is detected.

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