Molecular Basis for Recognition of the Cancer Glycobiomarker, LacdiNAc (GalNAc[β1→4]GlcNAc), by Wisteria floribunda Agglutinin.
Aberrant glycosylation and the overexpression of specific carbohydrate epitopes is a hallmark of many cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy and diagnostics. Wisteria floribunda agglutinin (WFA) is a legume lectin that recognizes terminal N-acetylgalactosaminides with high affinity. WFA preferentially binds the disaccharide LacdiNAc (β-d-GalNAc-[1→4]-d-GlcNAc), which is associated with tumor malignancy in leukemia, prostate, pancreatic, ovarian, and liver cancers and has shown promise in cancer glycobiomarker detection. The mechanism of specificity for WFA recognition of LacdiNAc is not fully understood. To address this problem, we have determined affinities and structure of WFA in complex with GalNAc and LacdiNAc. Affinities toward Gal, GalNAc, and LacdiNAc were measured via surface plasmon resonance, yielding KDvalues of 4.67 × 10-4m, 9.24 × 10-5m, and 5.45 × 10-6m, respectively. Structures of WFA in complex with LacdiNAc and GalNAc have been determined to 1.80-2.32 Å resolution. These high resolution structures revealed a hydrophobic groove complementary to the GalNAc and, to a minor extent, to the back-face of the GlcNAc sugar ring. Remarkably, the contribution of this small hydrophobic surface significantly increases the observed affinity for LacdiNAc over GalNAc. Tandem MS sequencing confirmed the presence of two isolectin forms in commercially available WFA differing only in the identities of two amino acids. Finally, the WFA carbohydrate binding site is similar to a homologous lectin isolated from Vatairea macrocarpa in complex with GalNAc, which, unlike WFA, binds not only αGalNAc but also terminal Ser/Thr O-linked αGalNAc (Tn antigen).
From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 3P6, Canada and.
Omid
O
Haji-Ghassemi
Human Health Therapeutics, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada.
Michel
M
Gilbert
From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 3P6, Canada and.
Jenifer
J
Spence
Human Health Therapeutics, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada.
Melissa J
MJ
Schur
From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 3P6, Canada and.
Matthew J
MJ
Parker
From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 3P6, Canada and.
Meredith L
ML
Jenkins
From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 3P6, Canada and.
John E
JE
Burke
Human Health Therapeutics, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada.
Henk
H
van Faassen
Human Health Therapeutics, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada.
N Martin
NM
Young
From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 3P6, Canada and svevans@uvic.ca.
Stephen V
SV
Evans
D014408:Biomarkers, Tumor / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006801:Humans; D007785:Lactose / Q000031:analogs & derivatives* / Q000737:chemistry; D037121:Plant Lectins / Q000737:chemistry*; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D032447:Wisteria / Q000737:chemistry*
D014408:Biomarkers, Tumor; D037121:Plant Lectins; C093701:N-acetylgalactosaminyl-1-4-N-acetylglucosamine; D007785:Lactose
agglutinin; biomarker; cancer; carbohydrate; carbohydrate-binding protein; glycobiomarker; lectin; structural biology; x-ray crystallography