Additional file 7: of Description of the interaction between Candida albicans and macrophages by mixed and quantitative proteome analysis without isolation

Changed proteins of macrophage interacting with C. albicans (five proteins)

Protein sequence alignment between Sap1–3 and Sap7.

<p>Multiple-sequence alignments were performed with CLUSTAL W. The active site of Sap7 was predicted to be D244 and D464 by homology. The cleavage point of Sap7 was found in the Sap7-specific insertion sequence N422-N451, which did not exist in the sequences of Sap1–3.</p

<i>N</i>-glycosylation and fragmentation of Sap7 did not affect its insensitivity to pepstatin A.

<p>(A) Proteolytic activity of deglycosylated Sap7. The influence of <i>N</i>-glycosylation on its insensitivity to pepstatin A was evaluated by EndoH treatment. Deglycosylated Sap7 did not show any significant change in proteolytic activity and pepstatin A insensitivity. (B) SDS-PAGE analysis of Sap7Δ422–451 with or without EndoH treatment. The sum of the 52-kDa and 15-kDa wild type fragments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032513#pone-0032513-g001" target="_blank">Fig. 1A</a>) was almost equal to the 68-kDa fragment of Sap7Δ422–451, indicating that Sap7Δ422–451 existed in a non-fragmented form. M: marker. (C) Proteolytic activity of Sap7Δ422–451. Sap7Δ422–451 was insensitive to pepstatin A. Thus, there was no relationship between the fragmentation of Sap7 and pepstatin A insensitivity. The data represent the average of at least 3 independent experiments. Error bars are shown as ±S.E.M. n.s.; not significant by Tukey's test.</p

Additional file 5: of Description of the interaction between Candida albicans and macrophages by mixed and quantitative proteome analysis without isolation

Proteins identified in this study for C. albicans monoculture (976 proteins) and macrophage monocultre (1,357 proteins)

D244A and D464A mutants possess no proteolytic activity.

<p>(A) SDS-PAGE analysis with or without EndoH treatment. The D244A and D464A mutants prevented fragmentation, and lost the 15-kDa fragment found in the Sap7 wild type lane. M: marker. (B) Proteolytic activity of the mutants. The D244A and D464A mutants completely lost their proteolytic activity. This result suggests that D244 and D464 represent the active site of Sap7. Averages of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** <i>P</i><0.01 determined by Tukey's test after a significant one-way factorial ANOVA (<i>P</i><0.01).</p

Surface modeling of Sap7.

<p>The surface structure of WT Sap7 was calculated using SWISS-MODEL based on the crystal structure of Sap2 (1eag), and visualized using Pymol. M242 and T467 were located near the entrance to the active site. Red: D244 and D464, green: M242, blue: T467, pale yellow: fragment 1, orange: fragment 2, and pink: N422–N451.</p

M242 and T467 are important amino acids in restricting the accessibility of pepstatin A to the active site.

<p>(A) SDS-PAGE analysis of alanine substitution mutants with or without EndoH treatment. All mutants were successfully produced by <i>P. pastoris</i>, and a 15-kDa band was confirmed in all samples. M: marker. (B) Proteolytic activity with or without pepstatin A. After pepstatin A treatment, M242A showed some proteolytic activity, while T467A showed none. Relative activity of the T467A mutant was significantly stronger than that of wild type. Average of at least 3 independent experiments are plotted, and the error bars are shown as ±S.E.M. ** <i>P</i><0.01 determined by Tukey's test after a significant one-way factorial ANOVA (<i>P</i><0.01).</p

Biochemical characteristics of Sap7.

<p>(A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously <i>N</i>-glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of <i>P. pastoris</i> transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** <i>P</i><0.01 determined by the Tukey's test after a significant one-way factorial ANOVA (<i>P</i><0.01), n.s.; not significant.</p

Structure of Sap7 calculated by SWISS-MODEL.

<p>The structure of Sap7 was calculated using SWISS-MODEL based on the crystal structure of Sap2 (1eag), and visualized using Pymol. Red: D244 and D464, green: amino acids within 3 Å of D244, blue: amino acids within 3 Å of D464, pale yellow: fragment 1, orange: fragment 2, and pink: N422–N451.</p