Enhancing the Receptor Affinity of the Sialic Acid-binding Domain of Vibrio cholerae Sialidase through Multivalency

Abstract

Many glycoside hydrolases possess carbohydrate-binding modules (CBMs) that help target these enzymes to appropriate substrates and increase their catalytic efficiency. The Vibrio cholerae sialidase contains two CBMs, one of which is designated as a family CBM40 module and has been shown through structural and calorimetry studies to recognize the ␣-anomer of sialic acid with a K D of ϳ30 M at 37°C. The affinity of this V. cholerae CBM40 module for sialic acid is one of the highest reported for recognition of a monosaccharide by a CBM. As Nature often increases a weak substrate affinity through multivalency, we have explored the potential of developing reagents with an increased affinity for sialic acid receptors through linking CBM40 modules together. The V. cholerae CBM40 was subcloned and crystallized in the presence of sialyllactose confirming its ability to recognize sialic acid. Calorimetry revealed that this CBM40 demonstrated specificity to ␣(2,3)-, ␣(2,6)-, and ␣(2,8)-linked sialosides. Polypeptides containing up to four CBM40 modules in tandem were created to determine if an increase in affinity to sialic acid could be achieved through an avidity effect. Using SPR and a multivalent ␣(2,3)-sialyllactose ligand, we show that increasing the number of linked modules does increase the affinity for sialic acid. The four-CBM40 module protein has a 700-to 1500-fold increase in affinity compared with the single-CBM40 module. Varying the linker length of amino acids between each CBM40 module had little effect on the binding of these polypeptides. Finally, fluorescence-activated cell sorting analysis demonstrated that a green fluorescent protein fused to three CBM40 modules bound to subpopulations of human leukocytes. These studies lay the foundation for creating high affinity, multivalent CBMs that could have broad application in glycobiology.

Prevention of influenza by targeting host receptors using engineered proteins

“…1B). Multivalency was achieved either through tandem repeats (17) or by fusing one or two tandemly linked CBM domains to an oligomerization domain (TD) derived from Pseudomonas aeruginosa sialidase (Fig. 1C) (19),…”

“…1D and Table S2). Glycan array screening of VcCBM (17) and SpCBM (Fig. S1) showed that they recognize glycans containing terminal α-2,3-or α-2,6-linked sialic acids, and crystallographic analysis with bound sialyllactose reveals that only the terminal sialic acid interacts with each domain (18).…”

“…Numerous sialic acid-binding proteins are known, but most have low affinity for sialic acid (e.g., the HA monomer that has ∼2.5 mM affinity for its receptor but gains affinity by being present in high copy number on the virus surface) (16). We have shown previously that engineered multivalent polypeptides containing up to four tandemly linked copies of the sialic acid-recognizing carbohydrate-binding module (CBM) from Vibrio cholerae nanH sialidase display low (nanomolar) binding affinity compared with the 30-μM affinity of the single domain (17).…”

Sialic Acid-Binding Protein Sp 2CBMTD Protects Mice against Lethal Challenge with Emerging Influenza A (H7N9) Virus

“…For histopathological examination, an additional 2 mice from each group were sacrificed on days 0, 3, 6, and 9 p.i. Three weeks after the initial inoculation with A/Anhui/1/2013 (H7N9) influenza virus, the surviving mice received a second administration of Sp2CBMTD (10-g dose) once (day Ϫ7 or Ϫ3), twice (days Ϫ3 and Ϫ1), or three times (days Ϫ7, Ϫ3, and Ϫ1) prior to being reinfected with 20 were measured using a mouse MCYTOMAG-70K-PMX Milliplex premixed kit (Millipore), according to the manufacturer's instructions. For each cytokine, the standard curve ranged from 3.2 to 10,000 pg/ml.…”

“…By using structural information, Connaris, Crocker, and Taylor (20) and Connaris et al (21) recently developed multivalent proteins based on SA-recognizing carbohydrate-binding modules (mCBMs) derived from bacterial sialidases with high affinity to SA receptors (Vc2CBMTD and Sp2CBMTD) (20,21 (21). The biologics appear to stimulate inflammatory mediators that confer protective ability in mice (21).…”