Isolation, characterization, cloning and use of a mushroom lectin

#The invention relates to the field of carbohydrate binding proteins, and more specifically a protein which binds specifically to particular oligosaccharides associated with particular medical significance.

#The Ga1α1,3Ga1 epitope has received considerable attention stemming from its presence in glycoproteins of most mammals and its conspicuous absence in humans, apes, and Old World monkeys. The loss is attributable to frameshift mutations in α,3-galactosyltransferase and the resulting immunogenicity of the epitope is a significant barrier to xenotransplantation.

#There are a few proteins that bind the Ga1α,3Ga1 epitope. For example, antibodies which recognize the α-galactosyl epitope and Clostridium difficile toxin A both bind well to some Ga1α1,3Ga1-containing sugars. However, merely binding to this epitope is not useful if the same protein binds many other related epitopes.

#The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

#FIG. 1A shows a derived full-length MOA amino acid sequence [SEQ ID NO: 2]. Underlined regions denote position of the MOA peptides sequence during the cloning (Table 1; SEQ ID NOs: 9-16). The boxed region shares homology with the ricin B chain lectin domain. FIG. 1B shows an alignment of a portion of the MOA amino acid sequence [SEQ ID NO: 4] with an assortment of bacterial toxins and carbohydrate degrading enzymes [SEQ ID NOs: 5-8]. Three conserved QXW/F motifs (each identified with a line) are noted. Residues that are identical to the consensus sequence are boxed and shaded. Residues that are similar to the consensus are shaded. Numbers indicate the amino acid positions of the selected full-length sequences.

#FIG. 2 shows the predicted amino acid sequence of a polymorphic form of MOA [SEQ ID NO: 3]

#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)].