Immobilized Marasmius oreades agglutinin: use for binding and isolation of glycoproteins containing the xenotransplantation or human type B epitopes.

The type B-specific lectin from the mushroom Marasmius oreades was immobilized onto Sepharose 4B. The immobilized lectin bound murine laminin and bovine thyroglobulin, glycoproteins that contain the Galalpha1,3Galbeta1,4GlcNAc epitope. This epitope is responsible for hyperacute rejection of xenotransplants from lower mammals to humans, Old World monkeys, or apes. The immobilized lectin also bound a fraction of serum proteins from type B human serum but little or none from type A or O(H) serum. The major protein bound from human B serum was a portion of the alpha2-macroglobulin. Treatment of this fraction with N-glycosidase F resulted in decreased molecular weight of bands associated with alpha2-macroglobulin and loss of their M. oreades lectin reactivity, whereas on treatment with coffee bean alpha-galactosidase, this bound fraction also lost reactivity with M. oreades lectin but became reactive with Ulex europaeus I lectin, suggesting the presence of L-fucosyl-alpha1,2-terminated structures. The presence of blood group epitopes on alpha2-macroglobulin has been detected previously by immunological methods, but this is the first isolation and characterization of the specifically glycosylated fraction of this serum protein. The immobilized lectin also bound a number of proteins from pig, rabbit, and rat serum that were distinct in electrophoretic mobility from the human B-serum components and presumably contain the xenotransplantation epitope among their glycan structures. This report further demonstrates the utility of immobilized lectins in isolating and characterizing glycan structures of naturally occurring glycoproteins.

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 fern Phlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of approximately 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.

Differential expression of an alpha-galactosyl-containing trisaccharide on high- and low-malignant murine sarcoma cells: identification and regulation.

Past studies have shown that carbohydrate residues reactive with the Griffonia simplicifolia isolectin B4 (GS I-B4) are present on the surface of highly-malignant murine sarcoma cells but are lacking or expressed in much lower amounts on the surface of low-malignant cells isolated from the same parent tumors (Am J Pathol 111: 27; J Nat Cancer Inst 71: 1281). In the present study it is shown that an antibody which recognizes the trisaccharide Galalpha1-3Galbeta1-4GlcNAc- is reactive with the highly-malignant cells but is non-reactive with the low-malignant cells. Further studies show that the high-malignant cells not only bind GS 1-B4 but also bind Evonymus europaea lectin (which like GS I-B4 recognizes terminal galactose in alpha1-3 linkage) and Erythina crystagalli lectin (which recognizes sub-terminal galactose in the beta1-4 linkage--e.g., Galbeta1-4GlcNAc). In contrast, the low malignant cells bind Erythina crystagalli lectin as efficiently as the high malignant cells but do not bind (or bind much smaller amounts of) either GS I-B4 or Evonymus europaea lectin. The present studies also show that there is no significant difference between high- and low-malignant cells in expression of alpha-galactosidase activity. In contrast, the high-malignant cells express high levels of alpha-galactosyl transferase activity while this enzyme is virtually undetectable in low-malignant cells. Taken together, these studies indicate that differential expression of a single monosaccharide residue distinguishes high- and low-malignant murine sarcoma cells. These studies also identify a mechanism to account for surface carbohydrate differences between the high- and low-malignant cells.