Mutated Leguminous Lectin Containing a Heparin-Binding like Motif in a Carbohydrate-Binding Loop Specifically Binds to Heparin

Abstract

We previously introduced random mutations in the sugar-binding loops of a leguminous lectin and screened the resulting mutated lectins for novel specificities using cell surface display. Screening of a mutated peanut agglutinin (PNA), revealed a mutated PNA with a distinct preference for heparin. Glycan microarray analyses using the mutated lectin fused to the Fc region of human immunoglobulin, revealed that a particular sulfated glycosaminoglycan (GAG), heparin, had the highest binding affinity for mutated PNA among 97 glycans tested, although wild-type PNA showed affinity towards Galβ1-3GalNAc and similar galactosylated glycans. Further analyses of binding specificity using an enzyme-linked immunoadsorbent assay demonstrated that the mutated PNA specifically binds to heparin, and weakly to de-2-O-sulfated heparin, but not to other GAG chains including de-6-O-sulfated and de-N-sulfated heparins. The mutated PNA had six amino acid substitutions within the eight amino acid-long sugar-binding loop. In this loop, the heparin-binding like motif comprised three arginine residues at positions 124, 128, and 129, and a histidine at position 125 was present. Substitution of each arginine or histidine residue to alanine reduced heparin-binding ability, indicating that all of these basic amino acid residues contributed to heparin binding. Inhibition assay demonstrated that heparin and dextran sulfate strongly inhibited mutated PNA binding to heparin in dose-dependent manner. The mutated PNA could distinguish between CHO cells and proteoglycan-deficient mutant cells. This is the first report establishing a novel leguminous lectin that preferentially binds to highly sulfated heparin and may provide novel GAG-binding probes to distinguish between heterogeneous GAG repeating units.

Fig 1. Tertiary structure of PNA and amino acid substitutions in mutated PNA clone H.

(A) Schematic illustration of the three-dimensional structure of PNA (PDB entry 2DVA). Ribbon representation of the main chain of PNA (green). Loop C consisting of the sugar-binding site is shown in red. Calcium and manganese ions are indicated by balls colored in cyan and magenta, respectively, and the sugar ligand (Galβ1-3GalNAc) complexed with PNA is shown as yellow sticks. (B) Amino acid substitutions of mutated PNA clone H (mPNA(H)) compared with wild-type PNA. The sugar-binding site of PNA consists of four loops (A-D) with six amino acid substitutions in loop C of mPNA(H).

Fig 2. SDS-PAGE of the purified mutated PNA clone H-IgG Fc fusion protein and its Ala-substituted variants.

mPNA(H)-Fc and Ala-substituted mPNA(H)-Fc at positions Arg124, His125, Arg128, or Arg129, respectively, were expressed, purified, and analyzed by electrophoresis on a 10% polyacrylamide gel under reduced conditions.

Fig 3. Glycan microarray analysis of wild-type and mPNA(H)-Fc.

Oligosaccharides and glycoproteins used in this experiment (97 in total) are shown in S1 Fig. The data are represented as the mean ± SD of n = 3 independent spots.

Fig 4. Binding of different glycosaminoglycan-conjugated BSAs to (A) mPNA(H)-Fc and IgG Fc (mock), and (B) mPNA(H)-Fc Ala-substituted at positions Arg124, His125, Arg128, or Arg129.

The data are represented as the mean ± SD of n = 3 independent wells. HP, heparin; 2-DS-HP, 2-O-desulfated heparin; 6-DS-HP, 6-O-desulfated heparin; N-DS-HP, 2-N-desulfated heparin; N-DS-Ac-HP, 2-N-desulfated, acetylated heparin; CSA, chondroitin sulfate A; CSB, chondroitin sulfate B; CSC, chondroitin sulfate C; HA, hyaluronic acid; HS, heparan sulfate; CH, chondroitin.

Fig 5. Binding inhibition assay and binding of PNA-Fc and mPNA(H)-Fc to CHO or its proteoglycan-deficient mutants using flow cytometry.

(A) Binding inhibition assay using heparin (white rectangle), chondroitin sulfate-A (red triangle), chondroitin sulfate-B (green rectangle) and GAG-unrelated heparin analogs, dextran sulfate (yellow circle), deoxyribonucleic acid (black circle), or phytic acid (blue square). The mutated PNA-Fc was preincubated with the indicated concentration of an inhibitor for 2 h at 4°C and then measured the binding of mutated PNA-Fc to heparin-coupled BSA was performed as described in Materials and Methods section. The binding of mPNA(H)-Fc in the absence of competitor was to be 100% (blue triangle). (B) Cells stained with wild-type PNA-Fc (left) or mPNA(H)-Fc (right) were analyzed by flow cytometry and the binding of mPNA(H)-Fc to the cells was monitored by measuring the mean fluorescent intensity (MFI). The data are represented as the mean ± SD of n = 3 independent experiments. * p<0.05, ** p<0.01 (one-way factorial analysis of variance).