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 KD values of 4.67 × 10-4 m, 9.24 × 10-5 m, and 5.45 × 10-6 m, 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).