Figure 2. LYSMD3 is a chitin and β-glucan binding protein.
(A) Direct binding of recombinant (r) LYSMD3 to immobilized carbohydrates or bovine serum albumin (BSA), determined by ELISA. The plate was coated with chitin DP5, DP7, LPS-EB, or BSA. An increasing amount ectodomain of LYSMD3 was added, and the amount bound was detected with the LYSMD3 antibody. See also Figure S1A.
(B) Direct binding of rLYSMD3 to chitin beads. A mixture of purified ectodomain of LYSMD3 and BSA was incubated with chitin magnetic beads. LYSMD3 bound chitin, while BSA remained in the soluble fraction (Unbound). Samples were assayed by SDS-PAGE and Coomassie blue stain.
(C) rLYSMD3 (150 ng) was incubated with chitin beads in the presence of chitin DP6 (3 mM). Bound LYSMD3 was detected by western using anti-LYSMD3. rLYSMD3 was run in the first two lanes. Western bands were quantified by densitometry using VisionWorks 9. Binding was normalized to the value without carbohydrate as 100%.
(D) rLYSMD3 and GFP were incubated (2.5 μg per sample) with suspensions of 1 × 107 heat-killed C. albicans SC5314 (HKCα). Proteins bound to fungal cell wall were detected by immunoblotting with anti-LYSMD3 or anti-GFP antibody. rLYSMD3 and GFP were run in the first two lanes.
(E) rLYSMD3, TLR2, TLR4 and GFP were incubated (2.5 μg per sample) with suspensions of 5 × 106 live Alternaria alternata (Alt) spores. Proteins bound to fungal cell wall were detected by immunoblot with anti-LYSMD3, anti-TLR2, anti-TLR4, or anti-His tag (GFP) antibody. rLYSMD3, TLR2, TLR4, and GFP were run in the first two lanes.
(F) Binding of rLYSMD3 to immobilized β-glucans or BSA by ELISA. See also Figures S1B-S1D.
(G) 100 ng of rLYSMD3 ectodomain was incubated with heat-killed C. albicans SC5314 (HKCα) or A. alternata spores in the presence of soluble WGP, laminarin, or chitin DP6 (2 mg/mL). Bound LYSMD3 was detected by western and quantified by densitometry. Binding was normalized to the value without carbohydrate as 100%.
Data are from one representative of three independent experiments (mean and SD of three samples per group in A and F).