A combination of biochemical and proteomic analyses reveals Bx-LEC-1 as an antigenic target for the monoclonal antibody 3-2A7-2H5-D9-F10 specific to the pine wood nematode

Abstract

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

Fig. 1.

Monoclonal antibody 3-2A7-2H5-D9-F10 (MAb-D9-F10) recognized only Bursaphelenchus xylophilus. A, Even though similar amounts of proteins from Bursaphelenchus xylophilus, B. mucronatus, B. doui and B. thailandae were loaded, MAb-D9-F10 recognized only one band from B. xylophilus. B, To assess the specificity of MAb-D9-F10, various samples including B. xylophilus, B. mucronatus, Drosophila melanogaster, human cervix carcinoma cells, mouse nerve cord, and mouse brain were tested with MAb-D9-F10. MAb-D9-F10 recognized only one band in B. xylophilus, even though similar or greater amounts of other proteins were loaded. For loading controls, α-tubulin was used.

Fig. 2.

A single spot was detected by MAb-D9-F10 when total proteins of B. xylophilus were separated by two-dimensional electrophoresis. Two identical two-dimensional electrophoresis (2-DE) gels were generated using total proteins from B. xylophilus. One 2-DE gel was processed with Coomassie Brilliant Blue staining to reveal protein spots. Another gel was processed with immunoblotting analysis using MAb-D9-F10. Only one spot very close to pI 3 was detected with MAb-D9-F10. As a reference, total proteins from B. xylophilus were loaded together with protein molecular weight markers.

Fig. 3.

Antigenic target proteins were precipitated together with MAb-D9-F10. To identify the antigenic target of MAb-D9-F10, total proteins of B. xylophilus and proteins precipitated with MAb-D9-F10 were separated and immunoblotted with MAb-D9-F10. One single band corresponding to the antigenic target was detected.

Fig. 4.

Separation and isolation of fractions containing antigenic targets for MAb-D9-F10 clone. To further characterize and isolate antigenic targets, B. xylophilus protein extracts were fractionated by anion exchange chromatography. In total, 101 fractions were recovered and tested by performing Western blot analysis with MAb-D9-F10. Fraction numbers 27 to 43 contained an antigenic target for MAb-D9-F10. The arrow indicates the MAb-D9-F10-specific bands. The line indicates the increasing linear gradient of NaCl. The y axis indicates relative protein concentration.

Fig. 5.

Localization of the LEC-1 in B. xylophilus. A, No MAb-D9-F10-positive immunoreactivities were observed in B. mucronatus. B, MAb-D9-F10-positive immunoreactivities were found in neighboring areas of the median bulb. C, The Nomarski differential interference contrast image of the same nematode at the same magnification revealed the median bulb (arrow) and the esophageal gland. D, The MAb-D9-F10 immunoreactivities at the anterior body of B. xylophilus, magnified.

Fig. 6.

Multiple alignment of deduced amino acid sequences of the Bx-LEC-1 and other evolutionarily conserved LECs. Two nonidentical GBDs connected by a short linker peptide sequence were boxed. Dots represent amino acids identical to those of the Bx-LEC-1. Background grayscale indicates the degree of amino acid residue conservation among 11 GaLECtins. White indicates 100% conservation, whereas black represents 0%. Asterisks (*) identify the eight highly conserved residues (H, S/N, R, V, N, W, E, and R) that have been shown to play key roles in the binding of sugars.

Fig. 7.

Three-dimensional homology models of two galactose-binding lectin domains in the Bx-LEC-1. Ribbon models of the N- and C- terminal galactose-binding lectin domains (GBD1, A; GBD2, B) are predicted together with space-filled models of N-acetyllactosamine (Lac-NAc). The four-stranded (F1–F4) and six-stranded (S1–S6) β-sheets are indicated by the letter-number code. The face and back of β-sheet structures are colored blue and aqua, respectively. Insets show side views of GBD1 (A1) and GBD2 (A2), clearly showing the anti-parallel β sheets of a β-sandwich fold. Amino acid residues forming the Lac-NAc binding sites of GBD1 (C) and GBD2 (D) are shown by rod model (green color) and labeled. Red colors are rod models of the amino acid residues of mouse galectin-9 (C) and mouse galectin-4 (D). The first residue number always belongs to the Bx-LEC-1, and the numbers and amino acid residues in parentheses belong to mouse galectin-9 (C) and mouse galectin-4 (D). Rod models of Lac-NAc are yellow.

Fig. 8.

Sensograms and K_Ds of tested carbohydrates for the immobilized Bx-LEC-1. Various concentrations of d-(+)-raffinose (A), N-acetyllactosamine (B), and l-(+)-rhamnose (C) solutions were introduced on the Bx-LEC-1-immobilized surface for 120 s at a flow rate 20 μl/min. The response unit was indicated as subtraction of the reference protein obtained on the immobilized surface from the values obtained on the Bx-LEC-1 immobilized surface.