The J ournal of Dermatologv Vol. 3: 25-29, 1976
PURIFICATION AND ISOLATION OF A BIOLOGICALLY ACTIVE PEPTIDO-RHAMNOGALACTAN FROM SPOROTHRIX SCHENCKII YOICHI NAKAMURA ABSTRACT
A biologically active peptido-rhamnogalactan was isolated from disrupted Sporothrix schenckii yeast cells by sodium deoxycholate extraction and was further purified by DEAE-Sephadex A-50 chromatography. This fraction, 60.6% sugar and 3.0% nitrogen, was active in delayed hypersensitivity skin tests in sporotrichosis patients and also cross-reacted serologically. with a peptido-rhamnomannan isolated from Sporothrix schenckii culture filtrates. These results suggest strong that L-rhamnose, (X-I, 2 linked, is more important in determining the serological specificity of Sporothrix schenckii than the (X-I, 3 linked L-rhamnose or the mannan backbone of the peptidorhamnomannans so far studied by others.
Previous immunochemical studies of Sporothrix schenckii have shown that immunologically active substances are polysaccharide-peptide complexes, containing rhamnose and mannose as the main sugar components. In the present research, peptido-rhamnogalactans were isolated from disrupted Sporothrix schenckii yeast cells by sodium deoxycholate extraction, and their chemical and immunological properties were investigated. MATERIALS AND METHODS
Organism. Sporothrix schenckii (ATCC 10268) was grown on brain heart infusion broth for four days at 36°C with constant shaking. Thimerosal, 0.01 % final concentration, was then added and the culture maintained at room temperature for 24 hours. The yeast phase organisms were then collected by filtration of filter paper, washed 3 times with acetone, dried, and stored. Preparation of antigen (Fig. I). Acetone-dried cells were defatted by continuous extraction with absolute ethanoljdiethyl ether (I: I) in a Soxhlet Received July 27, 1975; accepted for publication October 20, 1975. From the Department of Dermatology (Director: Prof. R. Fukushiro), Kanazawa University School of Medicine, Takara-machi, Kanazawa 920, Japan.
Acetone dried cells I defatted with EtOH-Ether (I: I) for 2 days Defa tted cells disrupted in a Ribi-Sorvall refrigerated cell-fractionator and centrifuged Residue extracted with a solution containing KCI and centrifuged; the above procedure repeated several times i Residue 5 volumes of 0.2% DOC added, stirred overnight, centrifuged Extract 10 volumes of acetone (-20°C) added and kept overnight; centrifuged Precipitate (Fr 0) f
I
I
Fig. I.
Preparation of antigen
extracter for 2 days. The defatted cells were suspended in 0.1 M Tris-HCI buffer, pH 7.5 (80 gj 500 ml) and disrupted in a Ribi-Sorvall refrigerated cell-fractionator at 20,000 to 30,000 psi. All operations were carried out at DoC to 5°C. The disrupted cell suspension was centrifuged at 17,000 to 18,000 g for 30 minutes. The residue was extracted for 5 to 10 minutes, while stirring with a solution containing potassium chloride of 74.56 g, iodoacetic acid of 186 mg, and anhydrous sodium citrate of 10 g per liter, adjusted to pH 4.7 with concentrated hydrochloric acid. The suspension was centrifuged at 17,000 to 18,000 g for 30 minutes, and the extracts were discarded. The extraction procedure was
26
NAKAMURA
repeated until the extracts yielded no precipitate upon treatment with 5% trichloracetic acid. To the residue were added five volumes of 0.2% sodium deoxycholate (DOC) solution. The mixture was stirred overnight and then centrifuged at 17,000 to 18,000 g for 30 minutes. The overnight extraction was repeated and the extracts combined. Ten volumes of acetone (-20°C) were added slowly with stirring to the DOC extract and the mixture was maintained overnight at -20°C. The white precipitate (Fr 0), collected by centrifugation at 12,000 g for 20 minutes, was dissolved in 7 ml of 0.5 M Tris-HCI buffer, pH 7.5. Chromatography. Preparations were chromatographed by anion-exchange on a 3.5 X 45 em DEAE-Sephadex A-50 column eluted with 0.5 M Tris-HCI buffer, pH 7.5, collecting 3.5 ml fractions. Skin tests were performed on five sporotrichosis patients and five normal volunteers. The antigen, 0.1 ml of 100 fLg/ml dissolved in physiological saline, was injected intradermally and the reaction was read after 48 hours. Double immunodiffusion was carried out using rabbit anti Fr I serum. Chemical analysis. Nitrogen was measured by a modified Kjeldahl-Nesslerization procedure (I). Sugars were determined by the anthrone reaction using galactose as standard, and by gas liquid chromatography as described previously (2). Amino acids were measured on a commercial amino acid analyzer (HITACHI-KLA-3B) after hydrolysis of 10 mg samples in I ml 6 N HCI in sealed tubes at 105°C for 22 hours, followed by removal of acid by evaporation. RESULTS
Results of the chromatographic separation of the DOC extract fraction on a DEAESephadex A-50 column are shown in Fig. 2. Two major and one minor peaks were observed. Composition of fractions. The first major peak, Fr 1 (99.4 mg), contained mostly carbohydrate, whereas the second major peak, Fr 2 (216 mg), appeared to contain predominantly protein. The sugar composition of the two fractions is shown in Table 1. Both fractions contained rhamnose, galactose, and only
- - anthrone (620nm) - - - 2BOnm Fe 2
~
1.5
,
Fr 1
I'
t-----
PURIFICATION AND ISOLATION OF A BIOLOGICALLY ACTIVE PEPTIDO-RHAMNOGALACTAN FROM SPOROTHRIX SCHENCKII YOICHI NAKAMURA ABSTRACT
A biologically active peptido-rhamnogalactan was isolated from disrupted Sporothrix schenckii yeast cells by sodium deoxycholate extraction and was further purified by DEAE-Sephadex A-50 chromatography. This fraction, 60.6% sugar and 3.0% nitrogen, was active in delayed hypersensitivity skin tests in sporotrichosis patients and also cross-reacted serologically. with a peptido-rhamnomannan isolated from Sporothrix schenckii culture filtrates. These results suggest strong that L-rhamnose, (X-I, 2 linked, is more important in determining the serological specificity of Sporothrix schenckii than the (X-I, 3 linked L-rhamnose or the mannan backbone of the peptidorhamnomannans so far studied by others.
Previous immunochemical studies of Sporothrix schenckii have shown that immunologically active substances are polysaccharide-peptide complexes, containing rhamnose and mannose as the main sugar components. In the present research, peptido-rhamnogalactans were isolated from disrupted Sporothrix schenckii yeast cells by sodium deoxycholate extraction, and their chemical and immunological properties were investigated. MATERIALS AND METHODS
Organism. Sporothrix schenckii (ATCC 10268) was grown on brain heart infusion broth for four days at 36°C with constant shaking. Thimerosal, 0.01 % final concentration, was then added and the culture maintained at room temperature for 24 hours. The yeast phase organisms were then collected by filtration of filter paper, washed 3 times with acetone, dried, and stored. Preparation of antigen (Fig. I). Acetone-dried cells were defatted by continuous extraction with absolute ethanoljdiethyl ether (I: I) in a Soxhlet Received July 27, 1975; accepted for publication October 20, 1975. From the Department of Dermatology (Director: Prof. R. Fukushiro), Kanazawa University School of Medicine, Takara-machi, Kanazawa 920, Japan.
Acetone dried cells I defatted with EtOH-Ether (I: I) for 2 days Defa tted cells disrupted in a Ribi-Sorvall refrigerated cell-fractionator and centrifuged Residue extracted with a solution containing KCI and centrifuged; the above procedure repeated several times i Residue 5 volumes of 0.2% DOC added, stirred overnight, centrifuged Extract 10 volumes of acetone (-20°C) added and kept overnight; centrifuged Precipitate (Fr 0) f
I
I
Fig. I.
Preparation of antigen
extracter for 2 days. The defatted cells were suspended in 0.1 M Tris-HCI buffer, pH 7.5 (80 gj 500 ml) and disrupted in a Ribi-Sorvall refrigerated cell-fractionator at 20,000 to 30,000 psi. All operations were carried out at DoC to 5°C. The disrupted cell suspension was centrifuged at 17,000 to 18,000 g for 30 minutes. The residue was extracted for 5 to 10 minutes, while stirring with a solution containing potassium chloride of 74.56 g, iodoacetic acid of 186 mg, and anhydrous sodium citrate of 10 g per liter, adjusted to pH 4.7 with concentrated hydrochloric acid. The suspension was centrifuged at 17,000 to 18,000 g for 30 minutes, and the extracts were discarded. The extraction procedure was
26
NAKAMURA
repeated until the extracts yielded no precipitate upon treatment with 5% trichloracetic acid. To the residue were added five volumes of 0.2% sodium deoxycholate (DOC) solution. The mixture was stirred overnight and then centrifuged at 17,000 to 18,000 g for 30 minutes. The overnight extraction was repeated and the extracts combined. Ten volumes of acetone (-20°C) were added slowly with stirring to the DOC extract and the mixture was maintained overnight at -20°C. The white precipitate (Fr 0), collected by centrifugation at 12,000 g for 20 minutes, was dissolved in 7 ml of 0.5 M Tris-HCI buffer, pH 7.5. Chromatography. Preparations were chromatographed by anion-exchange on a 3.5 X 45 em DEAE-Sephadex A-50 column eluted with 0.5 M Tris-HCI buffer, pH 7.5, collecting 3.5 ml fractions. Skin tests were performed on five sporotrichosis patients and five normal volunteers. The antigen, 0.1 ml of 100 fLg/ml dissolved in physiological saline, was injected intradermally and the reaction was read after 48 hours. Double immunodiffusion was carried out using rabbit anti Fr I serum. Chemical analysis. Nitrogen was measured by a modified Kjeldahl-Nesslerization procedure (I). Sugars were determined by the anthrone reaction using galactose as standard, and by gas liquid chromatography as described previously (2). Amino acids were measured on a commercial amino acid analyzer (HITACHI-KLA-3B) after hydrolysis of 10 mg samples in I ml 6 N HCI in sealed tubes at 105°C for 22 hours, followed by removal of acid by evaporation. RESULTS
Results of the chromatographic separation of the DOC extract fraction on a DEAESephadex A-50 column are shown in Fig. 2. Two major and one minor peaks were observed. Composition of fractions. The first major peak, Fr 1 (99.4 mg), contained mostly carbohydrate, whereas the second major peak, Fr 2 (216 mg), appeared to contain predominantly protein. The sugar composition of the two fractions is shown in Table 1. Both fractions contained rhamnose, galactose, and only
- - anthrone (620nm) - - - 2BOnm Fe 2
~
1.5
,
Fr 1
I'
t-----
