Interactions of surfactant proteins A and D with Saccharomyces cerevisiae and Aspergillus fumigatus

Abstract

Surfactant proteins A (SP-A) and D (SP-D) are members of the collectin family of calcium-dependent lectins and are important pulmonary host defense molecules. Human SP-A and SP-D and rat SP-D bind to Aspergillus fumigatus conidia, but the ligand remains unidentified. To identify a fungal ligand for SP-A and/or SP-D, we examined the interactions of the proteins with Saccharomyces cerevisiae. SP-D but not SP-A bound yeast cells, and EDTA inhibited the binding. SP-D also aggregated yeast cells and isolated yeast cell walls. Treating yeast cells to remove cell wall mannoprotein did not reduce SP-D binding, and SP-D failed to aggregate chitin. However, SP-D aggregated yeast glucan before and after treatment with a beta(1-->3)-glucanase, suggesting a specific interaction between the collectin and beta(1-->6)-glucan. In support of this idea, SP-D-induced yeast aggregation was strongly inhibited by pustulan [a beta(1-->6)-linked glucose homopolymer] but was not inhibited by laminarin [a beta(1-->3)-linked glucose homopolymer]. Additionally, pustulan but not laminarin strongly inhibited SP-D binding to A. fumigatus. The pustulan concentration for 50% inhibition of SP-D binding to A. fumigatus is 1.0 +/- 0.3 microM glucose equivalents. Finally, SP-D showed reduced binding to the beta(1-->6)-glucan-deficient kre6 yeast mutant. Taken together, these observations demonstrate that beta(1-->6)-glucan is an important fungal ligand for SP-D and that glycosidic bond patterns alone can determine if an extended carbohydrate polymer is recognized by SP-D.

FIG. 1

SP-A and SP-D binding to S. cerevisiae. Aliquots of 4 × 10⁶ yeast cells were incubated with 20 μg of SP-A or SP-D per ml for 1 h at 25°C, followed by washing and similar incubations with primary and secondary antibodies. To determine background fluorescence, samples that contained cells and primary and secondary antibodies but not SP-A or SP-D were also included. For inhibition studies, the proteins were preincubated with EDTA for 15 min at 25°C, and the EDTA-surfactant protein mixture was then added to the cells. Binding was detected by flow cytometry and normalized to the mean fluorescence intensity of recombinant human SP-D (taken as 100% binding). Data represent the average ± standard error of three independent experiments (∗, P < 0.05 compared to binding without EDTA).

FIG. 2

S. cerevisiae aggregation by SP-D. Yeast cells were suspended in calcium-containing buffer with or without 10 mM EDTA at room temperature. After 5 min, recombinant human SP-D was added to a final concentration of 5 μg/ml. Buffer was added to the negative control sample. The A₇₀₀ of the suspensions was monitored every minute for 2 h after protein addition. For the graphs shown, the starting A₇₀₀ for all samples was normalized to the buffer control for ease of interpretation. The graph shows representative data from duplicate experiments.

FIG. 3

S. cerevisiae cell wall aggregation by SP-D. Yeast cell walls were suspended in calcium-containing buffer with or without 10 mM EDTA at room temperature. After 5 min, recombinant human SP-D was added to a final concentration of 5 μg/ml. Buffer was added to the negative control sample. The A₇₀₀ of the suspensions was monitored every minute for 2 h after protein addition. For the graphs shown, the starting A₇₀₀ for all samples was normalized to the buffer control for ease of interpretation. The graph shows representative data from duplicate experiments.

FIG. 4

Glucan aggregation by SP-D. Glucan powder was suspended in calcium-containing buffer with or without 10 mM EDTA at room temperature. After 5 min, recombinant human SP-D was added to a final concentration of 10 μg/ml. Buffer was added to the negative control sample. The A₇₀₀ of the suspensions was monitored every minute for 18 min after protein addition. For the graphs shown, the starting A₇₀₀ for all samples was normalized to the buffer control for ease of interpretation. The graph shows representative data from duplicate experiments.

FIG. 5

Aggregation of zymolyase-treated yeast glucan by SP-D. Insoluble material remaining after zymolyase digestion of yeast glucan was suspended in calcium-containing buffer with or without 10 mM EDTA at room temperature. After 5 min, recombinant human SP-D was added to a final concentrations of 10 μg/ml. Buffer was added to the negative control sample. The A₇₀₀ of the suspensions was monitored every minute for 2 h after protein addition. For the graphs shown, the starting A₇₀₀ for all samples was normalized to the buffer control for ease of interpretation. The graph shows representative data from duplicate experiments.

FIG. 6

Inhibition of SP-D-induced yeast aggregation. Yeast cells were suspended in calcium-containing buffer with or without carbohydrate inhibitor at room temperature. After 5 min, recombinant human SP-D was added to all samples except the negative control (final SP-D concentration was 5 μg/ml). Buffer was added to the negative control sample. The A₇₀₀ of the suspensions was monitored every minute for 2 h after protein addition. For the graphs shown, the starting A₇₀₀ for all samples was normalized to the buffer control for ease of interpretation. Maltose, pustulan, and laminarin concentrations are reported as glucose equivalents (Glc eq.). The graph shows representative data from duplicate experiments.

FIG. 7

Inhibition of SP-D binding to A. fumigatus. Aliquots of 2 × 10⁶ A. fumigatus conidia were incubated with 20 μg of recombinant human SP-D per ml for 1 h at 25°C, followed by washing and similar incubations with primary and secondary antibodies. To determine background fluorescence, samples that contained conidia and primary and secondary antibodies but not SP-D were also included. For inhibition studies, the proteins were preincubated with the indicated carbohydrate for 15 min at 25°C, and the carbohydrate-surfactant protein mixture was then added to the conidia. Binding was detected by flow cytometry and normalized to the mean fluorescence intensity of recombinant human SP-D without carbohydrate (taken as 100% binding). The graph shows representative data from three experiments for each inhibitor.

FIG. 8

SP-D binding to kre6 mutant S. cerevisiae. Aliquots of 4 × 10⁶ yeast cells were incubated with 20 μg of recombinant human SP-D per ml for 1 h at 25°C, followed by washing and similar incubations with primary and secondary antibodies. Binding was detected by flow cytometry and normalized to the mean fluorescence intensity of the untreated control (taken as 100% binding). Data represent the average ± standard error of three independent experiments (∗, P < 0.05 compared to wild-type parent cells).

FIG. 9

Glucosyl polysaccharides laminarin and pustulan. The carbon atom numbering schemes for each are indicated.