©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Existence of Branched Side Chains in the Cell Wall Mannan of Pathogenic Yeast, Candida albicans
STRUCTURE-ANTIGENICITY RELATIONSHIP BETWEEN THE CELL WALL MANNANS OF CANDIDA ALBICANS AND CANDIDA PARAPSILOSIS(*)

(Received for publication, July 11, 1994; and in revised form, November 9, 1994)

Nobuyuki Shibata (1) Kyoko Ikuta (1) Tomonori Imai (1) Yohko Satoh (1) Richi Satoh (1) Atsuko Suzuki (1) Chizuko Kojima (1) Hidemitsu Kobayashi (1) Kanehiko Hisamichi (2) Shigeo Suzuki (1)(§)

From the  (1)Second Department of Hygienic Chemistry and (2)First Department of Medicinal Chemistry, Tohoku College of Pharmacy, Sendai 981, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Isolation of side chain oligosaccharides from mannans of Candida albicans NIH B-792 (serotype B) and Candida parapsilosis IFO 1396 strains has been conducted by acetolysis under mild conditions. Structural study of these oligosaccharides by ^1H and C NMR and methylation analyses indicated the presence of novel branched side chains with the following structures in C. albicans mannan.


INTRODUCTION

and

It was observed that the H-1 proton chemical shifts of the second and the third mannose units from the reducing terminus in each oligosaccharide are shifted upfield by substitution with an alpha-linked mannose unit at position 6 of the 3-O-substituted mannose unit. An agglutination inhibition assay between factor 4 serum and cells of Candida stellatoideaIFO 1397 lacking the beta-1,2-linked mannose unit, with oligosaccharides obtained from these mannans, indicated that only the branched oligosaccharides were active. This finding suggests that the branched oligosaccharides correspond to the epitope of antigenic factor 4. The presence of the branched structure in other mannans was detected by the characteristic H-1-H-2-correlated cross-peak of the alpha-1,2-linked mannose unit connected with the 3,6-di-O-substituted one by two-dimensional homonuclear Hartmann-Hahn spectroscopy.

Yeasts of the genus Candida, especially of Candida albicans species are known to be pathogenic in man. Candidiasis is an opportunistic infectious disease in early childhood and in adults with predisposing conditions such as diabetes, cancer, AIDS, and treatment with immunosuppressive agents after organ transplantation(1, 2) . The antigenicity of Candida cell walls resides in the mannan. Moreover, mannan is highly soluble and can be detected in the sera of some patients with candidiasis by various techniques, including immunologic procedures(3, 4, 5, 6, 7, 8, 9) . Thus, the detection of circulating mannan is important for diagnosis of invasive candidiasis. However, the cell wall components in sera from patients with other infections, such as Mycobacterium tuberculosis(10) , Serratia marcescens(11) , and Salmonella thompson(12) may cross-react with anti-Candida mannan antibody if it is not specific to Candida species. On the other hand, there are many reports(13, 14, 15, 16, 17, 18, 19, 20) about immunomodulatory effects by the mannan or oligosaccharide of C. albicans, including the induction of suppressive effects against both B- and T-cell-mediated immune responses. Although the mechanism of the immunosuppressive effects of these components is still unknown, several reports (21, 22, 23) suggest that side chain oligomannosyl moieties participate in adherence of the C. albicans cells to mammalian cells in the initial step of Candida infection. Therefore, the fine chemical structure of cell wall mannans must be known to develop an accurate serodiagnostic procedure for candidiasis and to understand these diverse host-parasite interactions.

In 1961, Hasenclever and Mitchell (24) reported two serotypes in C. albicans strains designated serotypes A and B. Later, Tsuchiya et al.(25) proposed the relationship between antigenic structures of many yeasts, including seven medically important species of Candida, based on 10 cell surface antigenic factors. In recent years, structural analysis of cell wall mannans of C. albicans has been developed extensively(26, 27) , and we have demonstrated the presence of phosphodiesterified beta-1,2-linked oligomannosyl residues as a group of common epitopes throughout the two serotype strains(28, 29) . Furthermore, a beta-1,2-linked mannose unit connected to an alpha-1,2-linked unit was found to correspond to a specific epitope for serotype A strains(30, 31) . Although these two groups of beta-1,2 linkage-containing epitopes were identified as corresponding to antigenic factors 5 and 6(32, 33) , the structure of factor 4 has not been determined. From the results of agglutination assays of monoclonal anti-factor 4 antibodies with cells of many Candida strains, it was speculated by Kagaya et al.(34) that the antigenic factor 4 corresponds to treebranch-like structures proposed by Suzuki et al.(35) . However, the results of our structural studies for C. albicans mannans provided evidence that the mannans have a comb-like structure with an alpha-1,6-linked backbone(26, 27) .

The C. albicans serotype B and Candida parapsilosis cells used in this study have antigenic factors 1, 4, and 5 and factors 1, 13, and 13b, respectively(25) . Therefore, the difference in structures of the mannans of both species correlates with the presence or the absence of antigenic factors, 4 or 13 and 13b, although the previous study by Funayama et al.(36) could not reveal any structural difference. This result could be attributable to the acetolysis conditions, since these workers prepared the side chain oligosaccharides by a conventional acetolysis procedure that cleaves all alpha-1,6 and beta-1,2 linkages. Our recent study (37) of Saccharomyces kluyveri mannan indicated that acetolysis under mild conditions released oligosaccharides retaining both beta-1,2 linkage and part of the alpha-1,6 linkages of the branching mannose unit. Therefore, we applied the mild acetolysis method to analyze structural difference(s) between oligomannosyl side chains of the two Candida mannans. Although several reports suggest the presence of branched side chains in the mannans of C. albicans, no isolation of any branched oligosaccharide has been achieved(35, 38, 39, 40) . In the present study, we demonstrate the existence of novel branched side chains that dominate the antigenic factor 4 specificity in the mannan of a C. albicans serotype B strain.


EXPERIMENTAL PROCEDURES

Materials

Candida guilliermondii IFO 10279, C. parapsilosis IFO 1396, C. stellatoidea IFO 1397 (type I), and S. kluyveri IFO 1685 strains were obtained from the Institute for Fermentation, Osaka (IFO), Japan. C. albicans NIH B-792 (serotype B) and C. albicans J-1012 (serotype A) strains are stock cultures in our laboratory. Factor 1, 4, 5, 6, and 13b sera of ``Candida Check'' (lot L261), a commercially available kit of rabbit polyclonal antibodies against Candida cells, were purchased from Iatron (Tokyo, Japan). Except for the factor 1 serum, which is an unabsorbed rabbit whole-cell serum against C. albicans cells, factor 4, 5, 6, and 13b sera are the sera to C. albicans absorbed with cells of C. parapsilosis, C. guilliermondii, C. stellatoidea, and Candida tropicalis, respectively(25) .

Preparation of Mannans

Yeast cells were cultivated in a 500-ml flask containing 0.5% yeast extract-supplemented Sabouraud liquid medium on a reciprocal shaker at 28 °C for 48 h. From the cells, which were washed and dehydrated with acetone, the crude mannan was extracted with water at 135 °C for 3 h. After dialysis, mannan was separated using Fehling solution to form a water-insoluble precipitate of the Cu-mannan complex. The mannan was recovered from the copper complex by treatment with Amberlite IR-120 (H) resin, neutralization, dialysis, and lyophilization. The mannans prepared from the cells of C. parapsilosis IFO 1396 and C. albicans NIH B-792 strains were designated as fractions P and A, respectively.

Acetolysis of Mannan

Acetolysis under mild conditions (31) was carried out in the same manner for the analysis of S. kluyveri mannan(37) . Namely, prior to acetolysis, the mannan was converted into its O-acetyl derivative. Mannan (100 mg) was dissolved in anhydrous formamide (5 ml). To the solution was added a 1/1 (v/v) mixture of (CH(3)CO)(2)O and anhydrous pyridine (50 ml), and the mixture was kept at 40 °C for 12 h. After evaporation under high vacuum using an oil diffusion pump, the residual O-acetyl mannan was dissolved in a 100/100/1 (v/v) mixture of (CH(3)CO)(2)O, CH(3)COOH, and H(2)SO(4) (50 ml), and the resultant solution was kept at 40 °C for 36 h. The O-acetylated mannooligosaccharide mixture was extracted from the reaction mixture with CHCl(3) and de-O-acetylated with CH(3)ONa. Fractionation of the resultant mannooligosaccharide mixture was achieved using a column (2.5 times 100 cm) of Bio-Gel P-2 (-400 mesh). Elution was carried out with water, and aliquots of eluates were assayed for carbohydrate content by the phenol-sulfuric acid method(41) . Eluates corresponding to each peak were combined and rechromatographed on the same column.

Acetolysis under conventional conditions was carried out as described by Kocourek and Ballou (42) using a 10/10/1 (v/v) mixture of (CH(3)CO)(2)O, CH(3)COOH, and H(2)SO(4), and the resultant solution was kept at 40 °C for 12 h.

Nuclear Magnetic Resonance Spectroscopy

All NMR experiments were performed with a JEOL JNM-GSX 400 spectrometer at 400 MHz for ^1H and 100 MHz for C. The spectra were recorded using a 1% (w/v) solution of each mannan or oligosaccharide in 0.7 ml of D(2)O at 45 °C. Acetone (2.217 ppm) (43) and CD(3)OD (49.00 ppm) were used as the internal standards for ^1H and C NMR, respectively.

Slide Agglutination Inhibition Test

The assay was conducted as described by Miyakawa et al.(44) as follows. Factor 4 serum (50 µl) was preincubated for 2 h at 30 °C in the presence of serially 2-fold diluted oligosaccharide inhibitor solution (50 µl). To this solution, heat-killed C. stellatoidea IFO 1397 strain cells (10^8/100 µl) were added and incubated at 30 °C for 1 h.

Methylation Analysis

Methylation of oligosaccharides was performed according to Ciucanu and Kerek(45) . Gas chromatography of O-methyl-O-acetyl-D-mannitols was performed using a glass column (3 mm times 200 cm) containing 3% OV-210 on Supelcoport (100-200 mesh) at 185 °C using N(2) as the carrier gas at a flow rate of 20 ml/min.

Other Methods

Total carbohydrate was determined by the phenol-sulfuric acid method of Dubois et al.(41) with D-mannose as the standard. Total phosphate was determined by the method of Ames and Dubin(46) , using KH(2)PO(4) as the standard.


RESULTS

Acid Treatment of Mannans

Acid treatment of C. albicans mannans with 10 mM HCl at 100 °C for 1 h selectively cleaves phosphodiester linkage to release beta-1,2-linked mannooligosaccharides(28) . The C. albicans serotype B mannans contain beta-1,2-linked mannose units in only acid-eliminable oligosaccharide moieties, and therefore the resultant acid-modified mannans consist of alpha-linked mannose units. Since the beta-1,2-linked oligosaccharides correspond to one of the major epitopes(29) , antigenic factor 5(32) , for C. albicans mannans, those of C. albicans NIH B-792 strain, fraction A, and C. parapsilosis IFO 1396 strain, fraction P, were subjected to treatment with 10 mM HCl to compare the ratios of these oligosaccharides(28, 47) . Although acid hydrolysis of fraction A gave mannose and mannooligosaccharides from biose to heptaose as reported (28) , release of neither mannose nor oligosaccharides from fraction P occurred under the same conditions (data not shown). The absence of acid-labile oligosaccharides in fraction P is consistent with the lack of an H-1 ^1H NMR signal at about 5.55 ppm corresponding to the 1-O-alpha-phosphorylated mannose unit(28, 32) , unreactivity to factor 5 serum(32) , and a negative result for phosphate analysis.

Acetolysis of Mannans

The polysaccharides recovered from the partial hydrolysates of fractions P and A were subjected to acetolysis under conventional and mild conditions. Fig. 1shows elution patterns of the acetolysates of the acid-modified mannans from a Bio-Gel P-2 column. It is apparent that the amounts of higher oligosaccharides (heptaose and either hexaose or pentaose) in the acetolysates obtained under the mild conditions were increased as compared with those obtained under the conventional conditions. The oligosaccharides from tetraose to heptaose obtained from the C. parapsilosis and C. albicans mannans by mild acetolysis were designated as PM(4), PM(5), PM(6), and PM(7) and AM(4), AM(5), AM(6), and AM(7), respectively.


Figure 1: Elution patterns of oligosaccharides obtained from C. parapsilosis (A and B) and C. albicans (C and D) mannans by acetolysis. A and C, acetolysis was performed with (CH(3)CO)(2)O, CH(3)COOH, H(2)SO(4) (10:10:1, v/v) at 40 °C for 12 h (conventional conditions). B and D, acetolysis was performed with (CH(3)CO)(2)O, CH(3)COOH, H(2)SO(4) (100:100:1, v/v) at 40 °C for 36 h (mild conditions). M, M(2), M(3), M(4), M(5), M(6), and M(7) indicate mannose, mannobiose, mannotriose, mannotetraose, mannopentaose, mannohexaose, and mannoheptaose, respectively. Vo, void volume.



^1H NMR Analysis of Oligosaccharides

^1H NMR spectra of the oligosaccharides obtained from the two acid-modified mannans by conventional acetolysis were essentially the same as those reported by Funayama et al.(36, 48) . The oligosaccharides consisted of alpha-1,2- and alpha-1,3-linked mannose units (data not shown). On the other hand, as shown in Fig. 2, the ^1H NMR spectra of the oligosaccharides higher than tetraose, except for PM(6), obtained by mild acetolysis gave other signals at about 4.91 ppm corresponding to alpha-1,6-linked mannose units(49) . Furthermore, signals at 5.22 and 5.24 ppm were present only in AM(6) and AM(7). These chemical shifts are only slightly different from that of an alpha-1,2-linked mannose unit connected with a 3,6-di-O-substituted mannose unit in the branched oligosaccharides obtained from the mannan of S. kluyveri, 5.23 ppm(37) . The spectra of AM(5), AM(6), and AM(7) indicated that these oligosaccharides were mixtures of isomers with or without an alpha-1,6-linked mannose unit, judging from the ratio of dimensions of signals corresponding to the reducing terminal and alpha-1,6-linked mannose units. Thus, we tried to separate the isomers by high pressure liquid chromatography on a normal or a reverse phase column after modification of the oligosaccharides to 2-aminopyridyl derivatives by the method of Hase et al.(50, 51) . Unfortunately, however, the isomers could not be separated from each other by these procedures. Therefore, we attempted structural analysis of these oligosaccharides without further separation. Assignment of the ^1H NMR signals for the isomeric oligosaccharides that did not contain the alpha-1,6 linkage, which had been obtained by the conventional acetolysis in the present and the preceding (29) studies, was readily achieved. Therefore, it seemed possible to assign the signals of other alpha-1,6-linkage-containing oligosaccharides in the mixture.


Figure 2: The anomeric region of the ^1H NMR spectra of oligosaccharides obtained from C. parapsilosis (A) and C. albicans (B) mannans by acetolysis under the mild conditions. Spectra were recorded using a JEOL JNM-GSX 400 spectrometer in D(2)O solution at 45 °C using acetone as the standard (2.217 ppm). M(4)-M(7) are designated as in the legend to Fig. 1.



C NMR Analysis of Oligosaccharides

To confirm the existence of the branched structure, DEPT (^1)135 C NMR spectra of the oligosaccharides were recorded (Fig. 3). Negative signals in these spectra imply that the carbon atom must have two protons and therefore correspond to the C-6 of a mannose unit(52) . The downfield shifted negative signals at 66.14 and 66.39 ppm in the spectra of AM(5), AM(6), and AM(7) indicate that the mannose unit is substituted at the 6-O-position. Reduction of AM(7) with NaBH(4) caused a downfield shift of the two signals from 61.96 ppm to 64.09 ppm and from 66.39 ppm to 69.79 ppm, whereas the signal at 66.14 ppm did not shift (Fig. 3E). This result indicates that the mannose unit with a C-6 signal at 66.14 ppm is not located on the reducing terminus and that AM(6) and AM(7) each contain one branching alpha-1,6-linked mannose unit with a C-1 signal at 100.30 ppm. It was further demonstrated from the presence of cross-peaks in the HMBC spectrum between an H-1 proton at 4.907 ppm and the downfield shifted negative signals from 61.96 ppm in DEPT 135, based on a three-bond coupling across an O-glycosidic linkage(53) , that the shifted signals should correspond to the C-6 carbons of the 6-O-substituted mannose unit (Fig. 3F). On the other hand, PM(5) and PM(7) gave only one downfield shifted negative signal at 66.39 ppm (data not shown). These results indicate that the alpha-1,6-linked mannose unit in PM(5) and PM(7) must be connected to a reducing terminus. Therefore, it is apparent that the C-1 signals at 100.50 ppm of PM(5) and PM(7) correspond to alpha-1,6-linked mannose units that originated from the backbone of this mannan.


Figure 3: C NMR spectra of oligosaccharides obtained from fraction A. DEPT 135 C NMR spectra of AM(4) (A), AM(5) (B), AM(6) (C), AM(7) (D), and AM(7)-H (E), and HMBC of AM(7)-H (F) are shown. Spectra were recorded using a JEOL JNM-GSX 400 spectrometer in D(2)O solution at 45 °C using CD(3)OD as the standard (49.00 ppm). Negative signals on DEPT 135 spectra correspond to C-6 carbon. The dashed lines among those spectra indicate downfield shift of C-6 signals by substitution with another mannose unit or by reduction on the reducing terminal mannose unit.



Two-dimensional Homonuclear Hartmann-Hahn Spectroscopy of Oligosaccharides

To determine the location of a mannose unit substituted by an alpha-1,6-linked mannose unit, two-dimensional HOHAHA (54) analysis of PM(6), PM(7), AM(6), and AM(7) was carried out. It has been shown in the previous papers that the H-1 proton chemical shift of a mannose unit does not change by phosphorylation or glycosylation at the 6-O-position but affects the chemical shift of some proton signals allocated around the substituted position(37, 52, 55, 56) . In the present study, it was revealed that cross-peaks of PM(7) that correlated with H-1 protons at 5.348 and 5.264 ppm, which have been assigned to those of Man-A and Man-B, respectively (Table 1), were significantly shifted as compared with those of PM(6) (Fig. 4). Although two-dimensional HOHAHA spectra of PM(6)-H and PM(7)-H, the reduction products of PM(6) and PM(7) with NaBH(4), respectively, show an upfield shift of the H-1 signals corresponding to Man-B, the difference between the cross-peaks has disappeared. This finding also indicates that the alpha-1,6-linked mannose unit is attached to the reducing terminal unit, Man-A, and that the difference in the cross-peaks correlated with the H-1 protons of Man-B of PM(6) and PM(7) results from a steric effect. Despite some difference between cross-peaks correlated with the H-1 proton at about 5.03 ppm (corresponding to the 3-O-substituted mannose unit, Man-D, of PM(6) and AM(6)), it is not significant enough to determine the substituting position.




Figure 4: Partial two-dimensional HOHAHA analysis of mannooligosaccharides PM(6), PM(7), AM(6), and AM(7), obtained by mild acetolysis and the reduction products PM(6)-H, PM(7)-H, AM(6)-H, and AM(7)-H. The cross-peaks for all ring protons, H-2 to H-6, are indicated by a vertical line correlated with the H-1 signal of each mannose unit. The dashed line between the cross-peaks of parent and reduced oligosaccharides indicates a shift of the H-1 signal caused by the reduction by NaBH(4).



To identify the H-1 proton of the second mannose unit from the reducing terminal unit, Man-B, AM(6) and AM(7) were reduced with NaBH(4). The H-1 and H-2 signals of Man-B were easily assigned based on their upfield shift, Delta = 0.06 and 0.1 ppm, respectively(37) . Namely, the signals at 5.24 and 5.27 ppm of AM(6) and AM(7) were assigned to the H-1 proton of Man-B. These results indicate that the upfield shift of the H-1 signal of Man-C from 5.28 to 5.22 ppm, Delta = 0.06 ppm, by the attachment of an alpha-1,6-linked mannose unit to Man-D is larger than that of Man-B from 5.27 to 5.24 ppm, Delta = 0.03 ppm. The shift value of the H-1 signal of Man-C is similar to that observed on the branched oligosaccharide from S. kluyveri mannan, Delta = 0.05 ppm (37) . Therefore, we propose that the chemical structures of the branched oligosaccharides in the isomer mixtures, AM(6) and AM(7), are as follows (Structures 1 and 2, respectively).

Sequential NMR Assignment

To confirm the structure of the branched oligosaccharides in AM(6) and AM(7), a sequential assignment study of the H-1 and H-2 signals of their reduction products, AM(6)-H and AM(7)-H, was performed by the method described by Hernandez et al.(55) with slight modification(47) . The right side of the diagonal of each panel in Fig. 5shows COSY, whereas the left side shows rotating frame NOE spectroscopy. In this figure, cross-peaks labeled with primed letters indicate through-space interresidue H-1-H-2` connectivities between two adjacent mannose units except D`, which indicates interresidue H-1-H-3` connectivities. On the other hand, cross-peaks labeled with unprimed uppercase and lowercase letters indicate intraresidue H-1-H-2- and H-2-H-3-correlated cross-peaks, respectively, caused by J-coupling. By this procedure, H-1 and H-2 signals were sequentially assigned from the H-1 of the Man-B, B-B`-C-C`-D-d-D`-E for AM(6)-H (Fig. 5A) and B-B`-C-C`-D-d-D`-E-E`-F for AM(7)-H (Fig. 5B). The results summarized in Table 1clearly demonstrate that attachment of an alpha-1,6-linked mannose unit to Man-D causes an upfield shift not only of the H-1 proton of Man-C but also that of Man-B by a steric effect, such as that observed on an alpha-1,3-linked mannose unit in S. cerevisiae mannan(57) .


Figure 5: Sequential connectivities of mannose units of AM(6)-H (A) and AM(7)-H (B). The right side of the diagonal shows COSY, and the left side of the diagonal shows rotating frame NOE spectroscopy. Primed letters indicate interresidue H-1-H-2` or H-1-H-3` NOE cross-peaks, and unprimed uppercase and lowercase letters indicate the H-1-H-2 and the H-2-H-3-correlated cross-peaks, respectively, caused by J-coupling; e.g.B indicates the H-1-H-2-correlated cross-peak of the second mannose unit, Man-B, and B` indicates the interresidue NOE cross-peak between H-2 of Man-B and H-1 of an adjacent mannose unit, Man-C. By this procedure, H-1 and H-2 signals were sequentially assigned from the H-1 of the Man-B, B-B`-C-C`-D-d-D`-E for AM(6)-H and B-B`-C-C`-D-d-D`-E-E`-F for AM(7)-H.



Methylation Analysis

The presence of branching points in these oligosaccharides was further confirmed by methylation analysis. The results of PM(6)-H, PM(7)-H, AM(6)-H, and AM(7)-H indicate that almost all of the alpha-1,6-linked mannose units in the latter two oligosaccharides were connected to a 3-O-substituted mannose unit, i.e. only 1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl mannitol was detected as a di-O-methyl mannitol derivative by gas-liquid chromatography (Table 2).



Comparison of Two-dimensional Homonuclear Hartmann-Hahn Spectroscopy of Several Mannans

Fig. 6shows the two-dimensional HOHAHA spectra of fractions P and A and four other mannans obtained from related yeasts, C. stellatoidea IFO 1397, C. albicans J-1012 (serotype A), S. kluyveri IFO 1685, and C. guilliermondii IFO 10279 strains. Cross-peak 1 in the two-dimensional HOHAHA of fractions P and A, those connected with a dotted line to a cross-peak in H-C COSY with a C-1 signal at 100.49 ppm, corresponds to the consecutive alpha-1,6-linked mannose units of the mannan backbone. On the other hand, cross-peak 2, connected to a C-1 signal at 100.29 ppm through a cross-peak in H-C COSY, was not present in the spectrum of fraction P but existed in that of fraction A. Although cross-peak 1 is observed in the two-dimensional HOHAHA spectra of all six tested mannans, cross-peak 2 was absent in that of C. parapsilosis. Furthermore, cross-peak 3, corresponding to an alpha-1,2-linked mannose unit substituted by a 3,6-di-O-substituted unit with an alpha-1,2 linkage, was observed distinctly in the two-dimensional HOHAHA spectra of mannans of C. albicans serotype B, C. stellatoidea, C. guilliermondii, and S. kluyveri, and faintly in that of C. albicans serotype A, but it was absent from the spectrum of fraction P.


Figure 6: Partial two-dimensional HOHAHA spectra of fractions P and A and other mannans of related yeasts. H-C COSY of fractions P and A are also shown. Cross-peaks 1 and 2 indicate alpha-1,6-linked backbone and alpha-1,6-linked branching mannose units, respectively. Cross-peak 3 indicates an alpha-1,2-linked mannose unit substituted by a 3,6-di-O-substituted mannose unit.



Haptenic Activity of Branched Oligosaccharides

The presence of cross-peaks 2 and 3 in the two-dimensional HOHAHA spectra of mannans correlates well with the reactivities of the cells of these strains with factor 4 serum of Candida Check. Therefore, we examined the inhibitory effect of oligosaccharides obtained by acetolysis of the two acid-modified mannans on the agglutination reaction between factor 4 serum and cells of the C. stellatoidea IFO 1397 strain expressing only factors 1 and 4. As shown in Table 3, only AM(6) and AM(7) showed inhibitory effects at an amount of 0.1 µmol or greater. These results indicate that the branched oligosaccharide side chains present in fraction A behave as the antigenic factor 4.




DISCUSSION

The present comparative study between the structures of the mannans of C. parapsilosis and C. albicans serotype B strains clearly demonstrates the existence of branched side chains in the latter mannan. Now we propose the entire chemical structures of the mannans of C. parapsilosis IFO 1396 and C. albicans NIH B-792 strains based on the results of the present and recent studies (29, 52) as shown in Fig. 7. There was a report (35) on the presence of a treebranch-like structure in the mannan of C. albicans serotype A strain. Based on our structural studies of C. albicans mannan, we find no evidence for such a structure. The branched structure is predominant in the mannan of the C. albicans serotype B strain, but small amounts occur in that of the C. albicans serotype A strain. Therefore, the structures recognized by monoclonal antibodies specific for antigenic factor 4 proposed by Kagaya et al.(34) were also speculative. Funayama et al.(36) reported that antigenic factors 13 and 13b correspond to a mannohexaose moiety with a linear structure, Manalpha12Manalpha13Manalpha12Manalpha12Manalpha1 2Man. Therefore, it is apparent that the antigenic factor 4 corresponding to the branched mannoheptaose moiety would be degraded to structures corresponding to antigenic factor 13b by conventional acetolysis(29, 30, 36, 58) . The above results suggest that the C. parapsilosis IFO 1396 strain cannot synthesize branched side chains and express antigenic factors 13 and 13b but not factor 4. However, in the cells of the C. albicans serotype B strain, these side chains are branched by the addition of alpha-1,6-linked mannose units to make the epitope corresponding to antigenic factor 4 instead of 13b. Therefore, we can say that the relationship between the structures of antigenic factors 13b and 4 is the same as that seen in blood groups H and A or B.


Figure 7: Possible structure of C. parapsilosis IFO 1396 and C. albicans NIH B-792 strain mannans. M denotes a D-mannopyranose unit. The side chain sequence is not specified. alpha-1,6-Linked mannose units in brackets indicate partial absence of this unit.



Factor 4 serum is prepared by absorption of anti-C. albicans serotype A strain whole-cell serum with C. parapsilosis cells(25) . Because C. albicans serotype A and C. parapsilosis cells have epitopes corresponding to antigenic factors 1, 4, 5, and 6 and factors 1, 13, and 13b, respectively, the factor 4 serum contains antibodies against antigenic factors 5 and 6 as well as factor 4(32) . Therefore, some Candida species react strongly with factor 4 serum, despite a low density of the real antigenic factor 4 in the mannans. For example, the C. albicans serotype A strain mannan seems to contain only a small amount of branched side chain as judged from the intensity of cross-peak 3 in the two-dimensional HOHAHA spectrum. However, because C. albicans serotype A strain mannans contain beta-1,2-linked mannose units connected to an alpha-1,2-linked mannose to give antigenic factor 6, strong cross-reactions with factor 4 serum can be observed. As additional evidence, Kobayashi et al.(59) and Okawa et al.(60) observed a significant decrease in the reactivity of C. albicans serotype A strain cells to factor 4 serum in addition to the disappearance of reactivities to factor 5 and 6 sera when the cells were cultivated at low pH or at high temperature. This result suggests that the presence of two types of beta-1,2-linkage-containing side chains, corresponding to antigenic factors 5 and 6, are responsible for the strong reactivity of the C. albicans serotype A strain cells to factor 4 serum.

The upfield shift of the H-1 proton of an alpha-1,2-linked mannose unit by a steric effect, found in branched mannooligosaccharides obtained from the mannan of S. kluyveri(37) , was also observed in the branched oligosaccharides of C. albicans mannan. In the case of the latter oligosaccharides, the alpha-1,6-linked branching mannose unit affects the H-1 proton chemical shifts of both the third mannose unit and the second one from the reducing terminal. Therefore, the three-dimensional structure of these branched oligosaccharides in aqueous solution would be of interest.

A strong cross-peak 3 in two-dimensional HOHAHA spectra of the mannans of C. guilliermondii and C. stellatoidea suggests that these mannans contain significant amounts of branched side chains. The structural analysis of the mannans of these strains is in progress.


FOOTNOTES

*
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§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: DEPT, distortionless enhancement by polarization transfer; two-dimensional HOHAHA, two-dimensional homonuclear Hartmann-Hahn spectroscopy; HMBC, two-dimensional heteronuclear multiple bond connectivity; COSY, two-dimensional ^1H-^1H- correlated spectroscopy; NOE, nuclear Overhauser effect.


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