The Xenograft Antigen Bound to Griffonia simplicifolia Lectin 1-B4

The purification and unique carbohydrate binding properties, including blood group B-specific agglutination and preferential binding to Gal␣1,3Gal-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 fulllength 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 polysaccharidedegrading 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 Gal␣1,3Gal and Gal␣1,3Gal␤1,4GlcNAc. 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.Specificity varies greatly among carbohydrate-binding proteins. Whereas some lectins broadly recognize all oligosaccharides containing particular terminal sugars, others show increasing affinity for specific di-and trisaccharides. Fewer still show almost no reactivity with a given sugar monomer yet bind strongly and specifically to particular oligosaccharides (1). As described in the accompanying paper (2), the Marasmius oreades agglutinin (MOA) 1 binds to Gal␣1,3Gal-containing sugars and falls into this last category.The Gal␣1,3Gal epitope has received considerable attention, stemming from its presence in the glycoproteins of most mammals and its conspicuous absence in humans, apes, and Old World monkeys (3). This absence is attributable to lack of the specific ␣1,3-galactosyltransferase because of frameshift mutations in its gene (4). The resulting immunogenicity of the Gal␣1,3Gal epitope is a significant barrier to xenotransplantation (5). Despite the importance of Gal␣1,3Gal epitope recognition, MOA is currently the only lectin known to have exclusive specificity for this disaccharide (2).Few proteins have been shown to bind with any specificity to Gal␣1,3Gal. While Clostridium difficile toxin A and antibodies recognizing the ␣-galactosyl epitope both bind well to some Gal␣1,3Gal-containing oligosaccharides (6), the size and species of origin of MOA suggest that it is fundamentally dissimilar to these proteins. On these grounds, the blood group Bspecific Griffonia simplicifolia I-B 4 isolectin is perhaps more appropriate for comparison (7). A recent x...

supporting

confidence: 51%

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

Kruger¹,

Winter²,

Simonson-Leff³

et al. 2002

“…the ␣-D-galactosyl group) (27,28). This concept of a restricted site was substantially verified by the recently completed x-ray crystallographic structure of the B 4 isolectin complexed with Gal␣1,3Gal in which it is shown that only the nonreducing ␣-galactosyl group makes contact with the lectin (29). Moreover, Kirkeby and Moe (30), using an enzyme-linked lectin assay, showed that the ␣-galactosyl group and the ␣1,2-, ␣1,3-, and ␣1,4-galactobiosyl groups as well as the linear B-trisaccharide (Gal␣1,3Gal␤1,4GlcNAc) linked to human serum albumin were very similar in their binding affinity to GS I-B 4 .…”

Section: Discussionmentioning

confidence: 70%

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

Winter¹,

Mostafapour²,

Goldstein³

2002

A blood group B-specific lectin from the mushroom Marasmius 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 Gal␣1,3Gal (but not for the isomeric ␣1,2-, ␣1,4-, and ␣1,6-disaccharides); Gal␣1,3Gal␤1,4GlcNAc; 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 Gal␣1,3Gal␤1,4GlcNAc 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.Blood group-specific lectins have served as serological reagents for typing blood for over 65 years (1)(2)(3). Among the useful reagents are Dolichos biflorus lectin for human type A 1 erythrocytes (4, 5), the B 4 Griffonia simplicifolia isolectin for type B erythrocytes (6), and the Ulex europeus I (7, 8) and eel (Anguilla anguilla) serum agglutinins for type O(H) erythrocytes (1, 2). Of all the blood group-specific lectins studied, group B-specific lectins are probably fewest in number.Presently, the fruiting bodies of mushrooms are being studied as sources for lectins with novel carbohydrate binding activity. Several surveys have been published that classify these proteins based on their ability to agglutinate human (and rabbit) erythrocytes according to blood type (9 -12).Crude extracts of the mushroom Marasmius oreades (Tricholomataceae) were first reported to exhibit human type B erythrocyte agglutinating activity in 1951 (9, 13). A lectin from this source was isolated by affinity chromatography on a matrix of ␣-galactosyl-polyacrylamide gel by Horejsi and Kocourek (14). It was reported to agglutinate type B erythrocytes six times more avidly than type A erythrocytes and was stated to be a heterodimeric protein of 50 kDa, with subunits of 33 and 23 kDa. No mono-or oligosaccharides (25 mM) tested were found to inhibit agglutination of human type B erythrocytes.We have undertaken a more detailed investigation of this potentially important B-specific lectin with interesting results. Quantitative precipitation studies using a highly purified preparation with A, B, and O(H) blood group substances show absolute specificity for B versus A substance, with slight Obinding activity. Binding stud...

“…Consequently, both galactose units are relatively tightly bound compared with the GlcNAc unit at the reducing end of the sugar. In contrast, the Griffonia simplicifolia isolectin GS I-B4 forms a deep pocket favoring the recognition of terminal galactose and thus broader specificity (60). The fact that the MOA accepts the fucosylated blood group antigen B, whereas the M86 antibody does not, is the best evidence for their different binding pocket structures.…”

Section: Discussionmentioning

confidence: 91%

Close-up of the Immunogenic α1,3-Galactose Epitope as Defined by a Monoclonal Chimeric Immunoglobulin E and Human Serum Using Saturation Transfer Difference (STD) NMR

Plum

Michel

Wallach

et al. 2011

Background: Antibody binding to xenobiotic ␣-Gal structures mediates anaphylaxis. Results: Humanized IgE antibodies exhibit recognition footprints similar to serum immunoglobulins but have no capability of effector cell activation. Conclusion: Recognition of the ␣-Gal epitope is based on the terminal disaccharide, but interaction does not imply cellular activation. Significance: These data provide the first insights into the recognition of carbohydrate IgE epitopes.