Bivalent carbohydrate binding is required for biological activity of Clitocybe nebularis lectin (CNL), the N,N'-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacdiNAc)-specific lectin from basidiomycete C. nebularis.
Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N'-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency.
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia,. Electronic address: jure.pohleven@ijs.si.
Jure
J
Pohleven
Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute and Centre of Excellence CIPKeBiP, Jamova 39, SI-1000 Ljubljana, Slovenia.
Miha
M
Renko
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Špela
Š
Magister
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
David F
DF
Smith
Institute of Microbiology, Department of Biology, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland, and.
Markus
M
Künzler
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia,; Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia.
Borut
B
Štrukelj
Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute and Centre of Excellence CIPKeBiP, Jamova 39, SI-1000 Ljubljana, Slovenia.
Dušan
D
Turk
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia,; Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia.
Janko
J
Kos
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Jerica
J
Sabotič
D000017:ABO Blood-Group System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000363:Agaricales; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism; D018360:Crystallography, X-Ray; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D007785:Lactose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D012276:Ricin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity
D000017:ABO Blood-Group System; C568807:CNL protein, Clitocybe nebularis; D011994:Recombinant Proteins; C093701:N-acetylgalactosaminyl-1-4-N-acetylglucosamine; D012276:Ricin; D007785:Lactose