title: Cloning, expression in Escherichia coli and characterization of the recombinant Neu5Acalpha2,6Galbeta1,4GlcNAc-specific high-affinity lectin and its mutants from the mushroom Polyporus squamosus.
abstract: Lectin from the mushroom Polyporus squamosus (PSL) has a unique carbohydrate-binding specificity for sialylated glycoconjugates containing Neu5Acalpha2,6Galbeta1,4Glc/GlcNAc trisaccharide sequences of asparagine-linked glycoproteins. In the present study, we elucidate the molecular basis for its binding specificity as well as establish a consistent source of this useful lectin using a bacterial expression system. cDNA cloning revealed that PSL contains a ricin B chain-like (QXW)(3) domain at its N-terminus that is composed of three homologous subdomains (alpha, beta and gamma). A recombinant lectin was expressed in Escherichia coli as a fully active, soluble form. It agglutinated rabbit erythrocytes and showed the highest affinity for Neu5Acalpha2,6Galbeta1,4GlcNAc, but not for the sialyl alpha2,3-linked isomer. We also investigated the structure-function relationship of PSL. A monomeric C-terminal deletion mutant lacking 40% of the lectin's molecular mass retained sugar-binding activity, indicating that the carbohydrate-binding sites are situated in the N-terminal portion of the lectin, whereas the C-terminal portion probably functions in oligomerization and structural stabilization. Mutant constructs that have single amino acid substitutions in the putative sugar-binding sites, based on sequence alignment with the ricin B-chain, indicate that the beta and gamma subdomains are most probably sugar-binding sites. The recombinantly expressed lectin will be a valuable reagent for the detection of the Neu5Acalpha2,6Galbeta1,4GlcNAc sequence of asparagine-linked glycans.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606, USA.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tateno
authors.forename: Harry C
authors.initials: HC
authors.lastname: Winter
authors.forename: Irwin J
authors.initials: IJ
authors.lastname: Goldstein
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001487:Basidiomycota / Q000235:genetics; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D050260:Carbohydrate Metabolism; D011232:Chemical Precipitation; D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006384:Hemagglutination; D006385:Hemagglutination Inhibition Tests / Q000379:methods; D006358:Hot Temperature; D007785:Lactose / Q000031:analogs & derivatives* / Q000378:metabolism*; D037102:Lectins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics*; D010455:Peptides / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods; D017384:Sequence Deletion / Q000235:genetics; D017186:Titrimetry / Q000379:methods
chemical_list: C426347:2,6-sialyllactose; D005656:Fungal Proteins; D037102:Lectins; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D007785:Lactose