Odorranalectin is a small peptide lectin with potential for drug delivery and targeting

Abstract

Background: Lectins are sugar-binding proteins that specifically recognize sugar complexes. Based on the specificity of protein-sugar interactions, different lectins could be used as carrier molecules to target drugs specifically to different cells which express different glycan arrays. In spite of lectin's interesting biological potential for drug targeting and delivery, a potential disadvantage of natural lectins may be large size molecules that results in immunogenicity and toxicity. Smaller peptides which can mimic the function of lectins are promising candidates for drug targeting.

Principal findings: Small peptide with lectin-like behavior was screened from amphibian skin secretions and its structure and function were studied by NMR, NMR-titration, SPR and mutant analysis. A lectin-like peptide named odorranalectin was identified from skin secretions of Odorrana grahami. It was composed of 17 aa with a sequence of YASPKCFRYPNGVLACT. L-fucose could specifically inhibit the haemagglutination induced by odorranalectin. (125)I-odorranalectin was stable in mice plasma. In experimental mouse models, odorranalectin was proved to mainly conjugate to liver, spleen and lung after i.v. administration. Odorranalectin showed extremely low toxicity and immunogenicity in mice. The small size and single disulfide bridge of odorranalectin make it easy to manipulate for developing as a drug targeting system. The cyclic peptide of odorranalectin disclosed by solution NMR study adopts a beta-turn conformation stabilized by one intramolecular disulfide bond between Cys6-Cys16 and three hydrogen bonds between Phe7-Ala15, Tyr9-Val13, Tyr9-Gly12. Residues K5, C6, F7, C16 and T17 consist of the binding site of L-fucose on odorranalectin determined by NMR titration and mutant analysis. The structure of odorranalectin in bound form is more stable than in free form.

Conclusion: These findings identify the smallest lectin so far, and show the application potential of odorranalectin for drug delivery and targeting. It also disclosed a new strategy of amphibian anti-infection.

Figure 1. cDNA sequence of odorranalectin, (A) cDNA nucleotide sequence and deduced amino acid sequence of odorranalectin precursor.

The amino sequence of mature odorranalectin is boxed. The stop codon is indicated by a star (*); (B) The precursor of odorranalectin (LEC) shares a similar signal peptide with the antimicrobial peptide Nigrocin-OG20 (AMP) isolated from O. grahami . Both of the mature peptides are preceded by a Lys-Arg motif, which is a typical processing site for endoproteolytic cleavage. The signal peptide is given in italics. The mature peptide is boxed. Conserved amino acid residues are shaded.

Figure 2. Hemagglutinating and microbe-agglutinating activities of odorranalectin.

odorranalectin could agglutinate rabbit erythrocytes. In control (A) wells no lectins were added; in sample (B) wells 4 hemagglutination doses of odorranalectin were added; 4 hemagglutination doses of odorranalectin could also agglutinate microorganisms as illustrated in (C), untreated E.coli DH5α; (D), odorranalectin treated E.coli DH5α; (E), untreated C. albicans; (F), odorranalectin treated C. albicans; (G), untreated S. aureus; (H), odorranalectin treated S. aureus.

Figure 3. Sensor-grams showing the interaction between immobilized odorranalectin and glycoproteins or sugar with different concentrations as indicated.

(A), fetuin; (B), bovine submaxillary mucin (BSM); (C), porcine stomach mucin (PSM); (D), L-fucose; RU, resonance units; MIC is the minimum inhibitory concentrations required for inhibition of four hemagglutination doses of odorranalectin. sucrose, D(+)-cellobiose, D(+)-melezitose monohydrate, inulin, D(+)-raffinose pentahydrate, D(+)-galacturonic acid, lactose, D-glucose,D-ribose, D-trehalose, isopropy-β-D-thiogalactoside, 2-nitrophenyl-β-D-galactopyranoside, dulcitol, L-sorbose,D-sorbose, D-froctose, L(+)-rhamnose monohydrate, D(+)-xylose, D(+)-galactose, D(+)-arabinose, N-acetylgalactosamine, N-acetylglucosamine N-acetylneuraminic acid, D-sorbitol, inositol, adonitol, D-mannose, N-acetyl-D-mannosamine, 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside, N-glycolylneuraminc acid, D(+)-maltose monohydrate, and heparin did not inhibit at all at concentrations up to 200 mM. Association and dissociation rate constants (ka and kd) were calculated by using BIA evaluation 4.0 software (Biacore AB, Sweden). The affinity constant (K_D) was calculated from the ka and kd. For the calculation of rate constants, samples were appropriately diluted in 10 mM HEPES-buffered saline containing 3 mM EDTA and 0.005% surfactant P-20, pH 7.4 (HBS-EP) at various concentrations.

Figure 4. Titration of L-fucose to odorranalectin.

(A) Superposition of the ¹H-¹⁵N HSQC spectra of odorranalectin free and in the complex with L-fucose. The molar ratios of odorranalectin to L-fucose were 1∶0 (red), 1∶0.25 (orange), 1∶0.5 (blue), 1∶0.75 (purple), 1∶2 (pink), 1∶3 (cyan), respectively. (B) Each of five isolated peaks was split into two peaks upon titration with L-fucose which was indicative of interaction between odorranalectin and L-fucose. (C) Five residues consist of the L-fucose binding site on odorranalectin, which were displayed with side-chains in the structure of odorranalectin. Atoms C, O, N, H, S were shown in black, red, blue, light gray and yellow, respectively.

Figure 5. Radiocounting-time curves of blood and different tissues after per oral (Rhombus), intravenous (Rectangular) and intranasal (Triangle) administration into mice (n = 3), and the stability of ¹²⁵I-odorranalectin in plasma.

Figure 6. The solution structure of odorranalectin.

(A) Superposition of the 20 lowest-energy conformers calculated with NOE-derived ¹H-¹H distance restraints and J-coupling restraints. Backbone, side-chains of residues 4–16 and side-chains of residues 1–3, 4 were shown in blue, red and green, respectively. (B) The mean structure calculated from the 20 lowest-energy structures which highlighted three hydrogen bonds (green broken lines) and one disulfide bonds (black solid line). (C) Electrostatic surface of odorranalectin which took the same orientation as that in the Panel A. Positively charged region and negatively charged region were shown in blue and red, respectively. The electrostatic surface was calculated and colored using the program MOLMOL .