Most of the antigenic activity of pathogenic Candida yeasts is carried out by N-linked polysaccharides composed of mannose that cover the outermost layer of their cell walls . Therefore, structural studies on cell wall mannan of pathogenic Candida yeast have been actively conducted for the purpose of diagnosing candidiasis and searching for target antigens for yeast species identification    .
Nuclear magnetic resonance (NMR) analysis plays a major role in the study of the chemical structure of Candida yeast cell wall mannan. In recent years, it has become possible to determine the approximate overall structure of a mannan simply by performing a two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) analysis of intact mannan molecule  . This is because the nuclear Overhauser effect (NOE) cross-peak of various intact mannans and/or derived manno-oligosaccharides were sequentially assigned in two-dimensional maps such as nuclear Overhauser enhancement and exchange spectroscopy (NOESY) by Shibata et al.    and Kobayashi et al.  
Candida tropicalis is one of the species that is often clinically isolated as a deep-seated mycosis-causing yeast. The cell wall mannan of this yeast is basically a comb-like structure in which several side chains are linked to linear backbone composed of α-1,6-linked mannose residues . There are two types of these side chains, one consisting of α-1,2- and β-1,2-linked mannose residues , and the other containing α-1,3-linked mannose residues in addition to these mannose residues .
In this short report, we note that information on the two types of mannan structures from Candida tropicalis can be fully analyzed by two-dimensional 1H-13C correlation (H-C COSY) spectroscopy, which is not inferior to two-dimensional HOHAHA.
2. Materials and Methods
Candida tropicalis NBRC 0199 and 1400 strains were obtained from the National Institute of Technology and Evaluation, Chiba, Japan. These strains were maintained on Sabouraud agar slants. Cultivation of two C. tropicalis strains and preparation of mannan were performed as described . These strains were 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 . The purified mannans obtained from the cells of the C. tropicalis NBRC 0199 and 1400 were designated Fr. 0199 and Fr. 1400, respectively.
¹H-NMR spectrum (internal acetone, 2.217 ppm) was measured with a Jeol JNM-GSX 400 spectrometer on solutions (3 - 10 mg sample/0.7mL) in D2O at 70˚C . 13C-NMR spectrum (internal CD3OD, 49.00 ppm) was measured with the same spectrometer on solutions (15 - 25 mg sample/0.7mL) in D2O at 55˚C . Two-dimensional H-C COSY was also recorded under the same conditions as for the 1H- and 13C-NMR spectra in accordance with previous description .
3. Results and Discussion
Eight anomeric H1-C1 cross-peaks on a H-C COSY two-dimensional map of Fr. 0199 (Figure 1(a) and Table 1) were assigned based on the previous report . The presence of cross-peak 1, 2, and 7 indicates that mannan contain β-1,2-linked oligomannosyl side chains. Cross-peaks 5 or 3 correspond to the 2-O-substituted or unsubstituted forms of the backbone in which α-1,2-linked mannose residues are polymerized, respectively. The existence of internal α-1,2-linked mannose residues was confirmed by the appearance of cross-peaks 8, and 9. The appearance of cross-peak 4 indicates the presence of a non-reducing terminal α-1,2- linked mannose residue of long side chain. On the other hand, in the two-dimensional map of Fr. 1400 (Figure 1(b) and Table 1), the appearance of an additional cross-peak 6 indicates the presence of non-reducing terminal α-1,3-linked mannose residue. Summarizing these analysis results, the overall structure of C. tropicalis NBRC 0199 and 1400 strain mannans can be proposed as shown in Figure 2.
Table 1. Identification of chemical shifts of 13C-1H COSY spectrum of Fr. 0199 and Fr. 1400.
aMeasured using acetone (2.217 ppm) as a standard; bM denotes a D-mannose residue.
Figure 1. H-C COSY (anomeric region) spectra of Fr. 0199 (a) and Fr. 1400 (b).
Figure 2. Approximate overall structures of Fr. 0199 and Fr. 1400. M denotes a D-mannose. Polymerization of mannan unit r was not determined. The estimated polymerization degrees of the side chains were m= 0 - 4, n≥ 2, x≥ 1 and y≥ 0, respectively.
In the previous study , it was shown to be suitable to analyze the comb-like yeast mannan composed of α-1,2-, β-1,2- and α-1,6-linked mannose residues by two-dimensional H-C COSY. In this report, we have shown that this procedure is also useful in the structural analysis of similar mannan composed of α-1,2-, α-1,3-, β-1,2-, and α-1,6-linked mannose residues. However, in the single NMR analysis of undegraded mannan, the degrees of polymerization of the α-1,2- and β-1,2-linked mannose residues constituting the side chains (x and y values in Figure 2) cannot be identified. The only way to obtain such accurate information is to perform a hydrolysis method such as acetolysis on mannan and analyze the resultant oligosaccharides corresponding to the mannan side chains.
In the NMR analysis of yeast cell wall mannan, the H1-C1 cross-peaks of H-C COSY two-dimensional map give almost the same value of information as the H1-H2 cross-peaks of two-dimensional HOHAHA map. However, if there is a need to measure the degree of polymerization of the side chains in the mannan molecules, since not enough two-dimensional NMR analysis, will occur need to perform a partial acid-hydrolysis such as acetolysis on the mannan.
NMR: Nuclear magnetic resonance
H-C COSY: 1H-13C correlation spectroscopy
HOHAHA: Nuclear Hartmann-Hahn
NOE: Nuclear Overhauser effect
NOESY: Nuclear Overhauser enhancement and exchange spectroscopy
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