Candida tropicalis NBRC 1400 strain was obtained from the Biological Resource Center, National Institute of Technology and Evaluation, Japan. This strain was maintained on Sabouraud agar slants. Jack bean α-mannosidase (EC 3.2.1.24) was obtained from Sigma Chemical Co. (St. Louis, Mo.). Column packing for gel filtration chromatography (Bio-Gel P-2; 400 mesh), with a fractionation range of 100 to 1800 Da, was obtained from Bio-Rad (Richmond, Calif.).

Cultivation of C. tropicalis NBRC 1400 and preparation of mannan were performed as described for C. albicans J-1012 [25]. This strain was cultivated in Sabouraud liquid medium at 27˚C for 72 h on a reciprocal shaker. Preparation of mannan was conducted by a combination of hot-water extraction and Fehling solution method [13]. The purified mannan obtained from the cells of the C. tropicalis NBRC 1400 was designated Fr 1400.

Treatment of Fr 1400 was done as described by Shibata et al. [26]. Briefly, mannan was dissolved in 10 mM HCl, and the solution was heated in a boiling water bath for 1 h. The solution was neutralized with 100 mM NaOH concentrated in vacuo. The hydrolysate was applied to a column of Bio-Gel P-2 (2.5 by 100 cm) and eluted with water (0.25 ml/min). The acid-modified Fr 1400 was designated Fr 1400-a.

Conventional acetolysis of Fr 1400-a was done by a modification [27] of the method of Kocourek and Ballou [28]. A 10:10:1 (vol/vol/vol) mixture of (CH₃CO)₂O, CH₃COOH, and H₂SO₄ was used for the acetolysis. After de-O-acetylation, the resultant oligosaccharides were fractionated on a column (2.5 by 100 cm) of Bio-Gel P-2.

Mild acetolysis of Fr 1400-a was done with a 100:100:1 (vol/vol/vol) mixture of (CH₃CO)₂O, CH₃COOH, and H₂SO₄ as described previously [27]. Separation of the region containing longer-chain oligosaccharides than hexaose was unsatisfactory in the case of mild acetolysis of Fr 1400-a. This was due to the presence of several isomers as judged by observation of the peak shape in the elution profile. This region was further treated with jack bean α-mannosidase to degrade the isomer(s) consisting of α linkages as described below.

This treatment was conducted by the method of Shibata et al. [29]. Briefly, each longer-chain oligosaccharide fraction was dissolved in 50 mM sodium acetate buffer (pH 4.6), to a concentration of 5 mg/ml, and 10 U of α-mannosidase per ml was added to the solution. After incubation at 37˚C for 48 h, each reaction mixture was applied to a column (2.5 by 100 cm) of Bio-Gel P-2 and eluted with water.

Total carbohydrate was determined by the phenol-sulfuric acid method [30] with D-mannose as the standard. Total protein was determined by the Folin method of Lowry et al. [31] with bovine serum albumin (Sigma) as the standard. Total phosphate was determined by the method of Ames and Dubin [32] with KH₂PO₄ as the standard. Four-hundred-megahertz ¹H-NMR spectrum analyses were conducted exactly as described previously [13] with acetone as the standard (2.217 ppm). Specific rotations were determined by means of a JAS DIP-360 digital polarimeter. The sample was dissolved in water, and measurement was done after 3 h of dissolution of each sample in water.

As shown in Table 1, Fr 1400 was mostly composed of carbohydrates (applox. 90%) and contained a small amount of protein and phosphate groups. The low specific rotation value of mannan, +36.1 degree, indicates existence of β-linked mannose residues in Fr 1400.

Fr 1400 was treated with 10 mM HCl at 100˚C for 1 h to isolate oligosaccharides linked through phosphate. Each hydrolysate was fractionated on a column of Bio-Gel P-2. As shown in Figure 1, acid treatment of Fr 1400 resulted in two oligosaccharides, triose (M3) and tetraose (M4), in amounts of 1.1%, on the basis of parent mannan. The ¹H-NMR spectra of two oligosaccharides were apparently identical to those of oligosaccharides isolated from mannans of Candida species, which was described previously [13] [14] [19] [32] [33] (H1 signals of oligosaccharides were not shown). Therefore, triose and tetraose were identified as Manβ1-2Manβ1-2Man and Manβ1-2Manβ1-2Manβ1-2Man, respectively, on the basis of the assignment of H1 signals [34] [35] (Table 2). The acid-stable fraction, Fr 1400-a, was obtained as the void-volume (Vo) regions in the gel filtration patterns (Figure 1).

^aDetermined by the phenol-H₂SO₄ method [30]. ^bDetermined by the Folin method of Lowry et al. [31]. ^cDetermined by the Ames-Dubin method [32] as-KH₂PO₄. ^d1% (wt/vol) solution in water. ^eWeight basis of the acetone-dried whole cells.

^aI, acid-labile oligosaccharide; II, acetolysis-labile oligosaccharide. ^bM denotes a D-mannose unit. ^cChemical shift was indicated on the basis of a value of acetone (2.217 ppm) as an internal standard [13].

Figure 2(a) shows the ¹H-NMR spectra (H1 region) of Fr 1400, demonstrating that these signals closely resemble those of the mannan of C. albicans serotype Astrain [36]. The weak signals, 5.542 and 5.563 ppm, and a strong signal, 4.839 ppm, indicate the presence of phosphate-bound side chains corresponding to Candida antigenic factor 5 and the β-1,2-linkage-containing side chain corresponding to Candida antigenic factor 6, respectively (Figure 2(a)). On the

¹H-NMR spectra of Fr 1400-a, loss of the weak signals mentioned above is evidence that the phosphate-bound side chains corresponding to Candida antigenic factor 5 were eliminated from each parent mannan by acid treatment (Figure 2(b)).

To obtain the α-linked oligosaccharides corresponding to side chains from the acid-stable domain of Fr 1400-a was at first subjected to conventional acetolysis, and the acetolysate was fractionated on a column of Bio-Gel P-2. The products isolated from this acetolysate were mannose (M), oligosaccharides, biose (M2) to hexaose (M6), and a phosphorylated oligosaccharide(s) eluted in the Vo region (Figure 3(a)). On the other hand, to isolate the β-1,2-linkage-containing oligosaccharide, Fr 1400-a was acetolysed under mild conditions. The elution pattern of the degradation products by this procedure indicates that a large amount of phosphorylated oligosaccharide(s) was eluted in the Vo region, and this oligosaccharide was followed by the fraction consisting of oligosaccharide isomers with longer chains than hexaose, the oligosaccharides with shorter chains than pentaose (M2 to M5), and mannose (M) (Figure 3(b)). The fraction consisting of longer-chain isomers than hexaose was then digested with the α-mannosidase, and the products were fractionated by gel filtration chromatography to remove the α-linked oligosaccharides from this fraction. Consequently, the α-mannosidase-resistant oligosaccharides, hexaose (M6) and heptaose (M7) remained (Figure 3(c)).

All oligosaccharides were analyzed by ¹H-NMR (Figure 4 and Table 2). The lower oligosaccharides, tetraose, triose, and biose were identified as Manα1-2Manα1-2Manα1-2Man, Manα1-2Manα1-2Man, and Manα1-2Man, respectively (signals are not shown), by correlation with data in the literature [13] [37] [38]. However, higher oligosaccharides, hexaose and pentaose, contain nonreducing terminal α-1,3-linked mannose units, because of the deposition of strong signals at 5.149 ppm; therefore, these oligosaccharides identified as Manα1-3Manα1-2Manα1-2Manα1-2Manα1-2Man, Manα1-3Manα1-2Manα1-2Manα1-2Man, respectively (Figure 4(a) and Table 2). The H1 signals of M6 and M7 obtained from Fr 1400-a by mild acetolysis followed by α-mannosidase revealed that these oligosaccharides were identified as Manβ1-2Manβ1-2Manα1-2Manα1-2Manα1-2Man and

Manβ1-2Manβ1-2Manβ1-2Manα1-2Manα1-2Manα1-2Man, respectively, on the basis of assignment of the same oligosaccharides isolated from C. albicans serotype A and C. stellatoidea type II strains [13] [19] [36] (Figure 4(b) and Table 2). Additionally, the signals of the phosphorylated oligosaccharide(s) fraction

(Vo) obtained by acetolysis under two conditions indicated that the sugar moiety of this fraction was composed of both α-1,2- and β-1,2-linked mannose units (Figure 4(a) and Figure 4(b)).