title: Cloning, expression, and characterization of the Galalpha 1,3Gal high affinity lectin from the mushroom Marasmius oreades.
abstract: The purification and unique carbohydrate binding properties, including blood group B-specific agglutination and preferential binding to Galalpha1,3Gal-containing sugar epitopes, of the Marasmius 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 MOA's high affinity for Galalpha1,3Gal and Galalpha1,3Galbeta1,4GlcNAc. 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.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Kruger
authors.forename: Harry C
authors.initials: HC
authors.lastname: Winter
authors.forename: Nancy
authors.initials: N
authors.lastname: Simonson-Leff
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Stuckey
authors.forename: Irwin J
authors.initials: IJ
authors.lastname: Goldstein
authors.forename: Jack E
authors.initials: JE
authors.lastname: Dixon
mesh_terms: D000363:Agaricales / Q000737:chemistry*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D037102:Lectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D037102:Lectins; D011994:Recombinant Proteins