©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Existence of Novel Branched Side Chains Containing -1,2 and -1,6 Linkages Corresponding to Antigenic Factor 9 in the Mannan of Candida guilliermondii(*)

(Received for publication, November 3, 1995; and in revised form, January 30, 1996)

Nobuyuki Shibata (1)(§) Rieko Akagi (1) Tomoko Hosoya (1) Kumi Kawahara (1) Akifumi Suzuki (1) Kyoko Ikuta (1) Hidemitsu Kobayashi (1) Kanehiko Hisamichi (1) Yoshio Okawa (1) Shigeo Suzuki (1)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Isolation of beta-linkage-containing side chain oligosaccharides from the mannan of Candida gilliermondii IFO 10279 strain has been conducted by acetolysis under mild conditions. A structural study of these oligosaccharides by one- and two-dimensional NMR and methylation analyses indicated the presence of extended oligosaccharide side chains with two consecutive beta-1,2-linked mannose units at the nonreducing terminal of alpha-linked oligosaccharides. The linkage sequence present in this mannan, Manbeta12Manalpha13Manalpha, has also been found in the mannan of Saccharomyces kluyveri but not in the mannan of Candida species. Furthermore, these oligosaccharides are branched at position 6 of the 3-O-substituted mannose units as follows.

and

The H-1 signals of the mannose units substituted by a 3,6-di-O-substituted unit showed a significant upfield shift (Delta = 0.04-0.08 ppm) due to a steric effect. The inhibition of an enzyme-linked immunosorbent assay between the mannan of C. guilliermondii and factor 9 serum with oligosaccharides obtained from several mannans indicated that only the oligosaccharides with the above structure were active, suggesting that these correspond to the epitope of antigenic factor 9.


INTRODUCTION

We have reported the presence of two types of beta-1,2-linked mannose units in the cell wall mannans of the genus Candida. One is located in a phosphodiesterified oligosaccharide moiety as one of the major epitopes for Candida albicans serotypes A and B(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) and Candida tropicalis(12) strains. The beta-1,2-linked oligosaccharides can be released selectively from these mannans by treatment with weak acid (10 mM HCl)(1) . The resultant acid-modified mannans of C. albicans serotype A and C. tropicalis strains still contain beta-1,2-linked mannose units attaching to alpha-1,2-linked mannotetraose side chains(3, 4, 12) . These are the second type of beta-1,2-linked mannose units corresponding to a serotype A-specific epitope for C. albicans. The first and second types of beta-1,2 linkage-containing side chains have been identified as corresponding to antigenic factors 5 (13) and 6(14) , respectively.

In an earlier paper(15) , we demonstrated the presence of a third type of a beta-1,2-linked mannose unit attaching to an alpha-1,3-linked one in the cell wall mannan of Saccharomyces kluyveri and speculated on the presence of the same type of beta-1,2-linked unit in those of C. albicans serotype A and Candida guilliermondii based on the presence of characteristic H-1-H-2-correlated cross-peaks in their two-dimensional HOHAHA (^1)spectra.

There are several reports on the responsibility of alpha-linked side chains(16) , beta-linked ones(9) , or complex side chains with alpha- and beta-linkages (17) of cell wall mannan for the adherence of C. albicans cells to host cells in the initial step of Candida infection. Furthermore, mannans or mannooligosaccharides of C. albicans cells are known to stimulate cytokine production(18, 19, 20, 21, 22, 23) . Therefore, the identification of the third type of beta-1,2 linkage containing mannan side chains is important for understanding the pathogenecity of C. guilliermondii and its accurate serodiagnosis.

C. guilliermondii, which is one of the causes of human candidiasis in immunocompromised hosts, has antigenic factors 1, 4, and 9(24, 25) . Although the structure corresponding to antigenic factor 4 was recently identified to be the following(26) ,

and

there is no report of the chemical structure corresponding to antigenic factor 9 except a study by Ataoglu et al.(27) . They showed that the factor 9 serum reacts with Saccharomyces cerevisiae X2180-1A-5 (mnn2) mutant strain cells, which have a linear alpha-1,6-linked mannan corresponding to the backbone in their cell wall. Therefore, we tried to detect a novel side chain corresponding to antigenic factor 9 in the cell wall mannan of a pathogenic yeast C. guilliermondii. For fragmentation of the mannan, we applied a mild acetolysis, which selectively cleaves backbone alpha-1,6 linkages to retain beta-1,2 and branched alpha-1,6 linkages as well as alpha-1,2 and alpha-1,3 linkages(4, 15, 26) . Structures of the resultant oligosaccharides were determined by one- and two-dimensional NMR techniques. Consequently, we demonstrated the presence of the third type of beta-1,2 linkage containing side chains in Candida species mannan corresponding to antigenic factor 9.


EXPERIMENTAL PROCEDURES

Materials

The C. guilliermondii IFO 10279 strain was obtained from the Institute for Fermentation, Osaka (IFO), Japan. Linear alpha-1,6-linked mannan prepared from the cells of S. cerevisiae X2180-1A-5 (mnn2) strain, which was developed by Ballou and co-workers(28, 29) , was the same specimen used in an earlier paper(30) . Factors 5 and 9 sera of ``Candida Check'' (lot number I751), a commercially available kit of rabbit polyclonal antibodies against Candida cells, were purchased from Iatron (Tokyo, Japan). Jack bean alpha-mannosidase (EC 3.2.1.24) was obtained from Sigma.

Preparation and Acetolysis of Mannan

Yeast cells were grown at 28 °C in shaking liquid culture containing 0.5% yeast extract, 1% peptone, and 2% glucose. Mannan was extracted from the cells with water at 135 °C for 3 h and was separated by precipitation with Fehling's solution(26) . The mannan prepared from the cells of C. guilliermondii IFO 10279 strain was designated as fraction G. Acetolysis under conventional (31) and mild (4) conditions was performed as described in a preceding paper(26) . Fractionation of the resultant mannooligosaccharide mixture was achieved using a column (2.5 times 100 cm) of Bio-Gel P-2 (extra fine). Elution was carried out with water, and aliquots of eluates were assayed for carbohydrate content by the phenol-sulfuric acid method(32) . Separation of higher oligosaccharides by HPLC was carried out with a column (10 times 500 mm) of YMC-Pack PA-25. Elution was done with a 52:48 (v/v) mixture of CH(3)CN and water, and the eluates were monitored with a differential refractometer. Eluates corresponding to each peak were rechromatographed on the same column.

Nuclear Magnetic Resonance Spectroscopy

All ^1H NMR experiments were performed with a JEOL JNM-GSX 400 spectrometer at 400 MHz. 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) (33) was used as the internal standard for ^1H NMR.

Inhibition Test of Enzyme-linked Immunosorbent Assay

Enzyme-linked immunosorbent assay was conducted as described in a preceding paper(13) . Enzyme-linked immunosorbent assay-inhibition test using a factor serum was basically conducted as described by Okawa et al.(34) . A haptenic oligosaccharide solution (50 ml) was mixed with 5-fold-diluted factor 9 serum (50 ml) and preincubated for 2 h at 25 °C. The reaction mixture was then added to the wells of a fraction G-coated microtiter plate and incubated for 2 h at 25 °C. After washing, 1000-fold-diluted goat anti-rabbit IgG antibody peroxidase-conjugate was added to the wells and kept for 2 h at 25 °C. Finally, a substrate solution of 0.01% o-phenylenediamine and 0.006% H(2)O(2) in 150 mM citrate buffer (pH 5.0) (100 ml) was added, and the color was measured at 492 nm after the addition of 2 M H(2)SO(4) (50 ml).

Methylation Analysis

Methylation of oligosaccharides was performed according to Ciucanu and Kerek(35) . 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

For alpha-mannosidase treatment, the mannooligosaccharide mixture (200 mg) was dissolved in 50 mM sodium acetate buffer (pH 4.6) (2 ml) containing 20 units of alpha-mannosidase. After incubation for 48 h at 37 °C, the reaction mixture was boiled for 5 min to inactivate the enzyme. Total carbohydrate was determined by the phenol-sulfuric acid method of Dubois et al.(32) with D-mannose as the standard. Total phosphate was determined by the method of Ames and Dubin(36) , using KH(2)PO(4) as the standard.


RESULTS

Preparation of Mannan

The cell wall mannan prepared from the crude extract of C. guilliermondii IFO 10279 strain cells by the Fehling's solution method was designated as fraction G. Clearly, this mannan contains no phosphodiesterified oligosaccharides, which have been found in the mannan of C. albicans(1, 26) , judging from the lack of an H-1 signal at about 5.55 ppm on ^1H NMR corresponding to the 1-O-alpha-phosphorylated mannose unit(1, 13, 37) , unreactivity to factor 5 serum(25, 38) , and a negative result for phosphate analysis.

Acetolysis of Mannan

Fig. 1A shows the elution pattern of the acetolysate of fraction G from the Bio-Gel P-2 column obtained under the conventional conditions. Each oligosaccharide from biose to heptaose was rechromatographed by the same column and designated as AM(2) to AM(7). To obtain side chain oligosaccharides with beta-1,2-linked mannose units and alpha-1,6-linked branching ones, we also carried out the mild acetolysis of fraction G. By this fragmentation, oligosaccharides up to decaose were obtained (Fig. 1B). These oligosaccharides were then digested with alpha-mannosidase, and the enzyme-resistant oligosaccharides were applied on a column of Bio-Gel P-2. However, it was difficult to separate the higher oligosaccharides from each other (Fig. 1B). Therefore, these oligosaccharides were separated by HPLC and rechromatographed by the same column. The oligosaccharides from hexaose to decaose were designated as BM(6) to BM.


Figure 1: Elution patterns of oligosaccharides obtained from fraction G by acetolysis. A, B, elution was performed with a column (2.5 times 100 cm) of Bio-Gel P-2 before (bullet) and after (circle) alpha-mannosidase treatment. A, acetolysis was performed with (CH(3)CO)(2)O/CH(3)COOH/H(2)SO(4) (10:10:1, v/v/v) at 40°C for 12 h (conventional conditions). B, acetolysis was performed with (CH(3)CO)(2)O/CH(3)COOH/H(2)SO(4) (100:100:1, v/v/v) at 40°C for 36 h (mild conditions). C, elution pattern of alpha-mannosidase-treated acetolysate B by HPLC with a column of YMC-Pack PA-25. AM(2)-AM(7) in panel A indicate mannobiose to mannoheptaose obtained by the conventional acetolysis. BM(6)-BM in panel C indicate mannohexaose to mannodecaose obtained by the mild acetolysis followed by alpha-mannosidase treatment.



^1H NMR Analysis of Oligosaccharides

The ^1H NMR spectra of the oligosaccharides from AM(2) to AM(4) obtained by the conventional acetolysis were the same as those obtained from the mannans of S. cerevisiae(33, 39) or S. kluyveri(15, 40) . Namely, AM(2) and AM(3) consist only of alpha-1,2 linkages, and AM(4) contains a nonreducing terminal alpha-1,3-linked mannose unit. Because AM(5) shows signals at 5.379 and 5.042 ppm, the alpha-1,3-linked mannose unit of this oligosaccharide is substituted by an alpha-1,2-linked mannose unit (40, 41) (Fig. 2). AM(6) shows an additional signal at 5.139 ppm, and the signal dimension at 5.027 ppm was doubled compared with that of AM(5). This result suggests that AM(6) contains two alpha-1,3-linked mannose units at the first and the third positions from the nonreducing terminal. AM(7) shows signals corresponding to a beta-1,2-linked mannose unit, 4.758 ppm, and an alpha-1,3-linked mannose unit substituted by a beta-1,2-linked one, 5.245 ppm, both of which were found in the mannan of S. kluyveri(15) . The conventional acetolysis conditions cleave beta-1,2 and alpha-1,6 linkages; therefore, part of these oligosaccharides seems to correspond to a degradation product of the parent side chains.


Figure 2: The anomeric region of the ^1H NMR spectra of oligosaccharides obtained from fraction G by acetolysis under the conventional (A) and the mild (B) 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). AM(3)-AM(7) and BM(6)-BM are designated as in the legend to Fig. 1.



On the other hand, the ^1H NMR spectra of oligosaccharides higher than pentaose obtained by the mild acetolysis commonly show signals at about 4.84 ppm corresponding to two consecutive beta-1,2-linked mannose units(3, 4, 15) . As shown in the preceding papers(4, 15) , the H-1 proton of an alpha-1,2-linked mannose unit substituted by a consecutive beta-1,2-linked one, 5.138 ppm, appears at about 0.02 ppm upfield from that substituted by a single beta-1,2-linked one, 5.160 ppm. Therefore, the signal at 5.236-5.245 ppm of the H-1 proton of an alpha-1,3-linked mannose unit substituted by a single beta-1,2-linked mannose unit (15) seems to shift to about 5.22 ppm with the addition of consecutive beta-1,2-linked units. Because BM(6) shows a signal corresponding to an alpha-1,2-linked mannose unit substituted by an alpha-1,3-linked mannose unit, 5.033 ppm, it is reasonable to assign the signal at 5.218 ppm to the alpha-1,3-linked mannose unit substituted by consecutive beta-1,2-linked mannose units. Therefore, we can propose that the chemical structure of BM(6) is as follows.

BM(7) shows a new signal corresponding to an alpha-1,6-linked mannose unit, 4.914 ppm, in addition to the signals of BM(6). Furthermore, as observed on the branched oligosaccharides obtained from the mannans of C. albicans(26) and S. kluyveri(15) , the signal at 5.276 ppm corresponding to an alpha-1,2-linked mannose unit, Man-B, of BM(6) was also shifted upfield to 5.232 ppm on BM(7). This result suggests that the alpha-1,6-linked mannose unit is attached to the 3-O-substituted one, Man-C, of BM(7).

BM(8) shows new signals at 5.223, 4.846, and 4.838 ppm in addition to those of AM(6). This indicates that the structure of BM(8) was that of AM(6) with two beta-1,2-linked mannose units at the nonreducing terminal as follows.

It is obvious that BM(9) and BM contain one and two alpha-1,6-linked mannose units, respectively, judging from the dimension of the signals at about 4.91 ppm. In the spectrum of BM(9), about two-thirds of the signal at 5.368 ppm corresponding to Man-D seems to be shifted upfield to 5.311 ppm by the addition of an alpha-1,6-linked mannose unit to Man-E of BM(8). Furthermore, about one-third of the signal at 5.269 ppm corresponding to Man-B is also shifted upfield to 5.231 ppm as the result of the attachment of an alpha-1,6-linked mannose unit to Man-C. Namely, BM(9) seems to be a mixture of two isomers with a difference in the branching point. Finally, the two alpha-1,6-linked mannose units of BM seem to attach on Man-C and Man-E judging from the presence of two upfield-shifted signals at 5.291 and 5.226 ppm.

Determination of Branching Point

From the structural study of S. kluyveri(15) and C. albicans(26) mannans, we can speculate that the alpha-1,6 branching point of the side chain of C. guilliermondii mannan is the 3-O-substituted mannose units. Although the H-1 signal of a mannose unit does not shift by phosphorylation or glycosylation at the O-6 position, the attachment affects the chemical shift of some ring proton signals allocated around the substituted position(15, 26, 42, 43, 44) . Therefore, to detect the branching point of oligosaccharides, we recorded one- and two-dimensional HOHAHA spectra, and we found that the shifted ring protons correlated only with the 3-O-substituted mannose units of BM(7), BM(9), and BM compared with those of BM(6) and BM(8) (data not shown). These findings demonstrate that the branching point of these oligosaccharides is the 3-O-substituted mannose unit. This result was confirmed by methylation analysis of the oligosaccharides as shown in Table 1. Namely, BM(7), BM(9), and BM gave 2,4-di-O-methyl-1,3,5,6-tetra-O-acetyl mannitol, which corresponds to the 3,6-di-O-substituted mannose unit. On the other hand, 2,4,6-tri-O-methyl-1,3,5-tri-O-acetyl mannitol, which corresponds to the 3-O-substituted mannose unit, was obtained from BM(6), BM(8), and BM(9) but was not obtained from BM(7) and BM.



Sequential NMR Assignment

A sequential assignment study of the H-1 and H-2 signals of these oligosaccharides was performed to confirm the structure by the method described by Hernandez et al.(42) with slight modification(8, 26) . The right side of the diagonal of each panel in Fig. 3shows the relayed COSY, whereas the left side shows the rotating frame NOE spectroscopy. In this figure, cross-peaks labeled with primed letters indicate through-space interresidue H-1-H-2` or H-1-H-3` connectivities between two adjacent mannose units. On the other hand, cross-peaks labeled with unprimed letters indicate intraresidue H-1-H-2- or H-1-H-3-correlated cross-peaks caused by J-coupling. By this procedure, the H-1 and H-2 signals of BM(6) were sequentially assigned from the H-1 of the Man-A, A-A`-B-B`-C-C`-D-D`-E-E`-F (Fig. 3). Similarly, the H-1 and H-2 signals of BM(7), BM(8), BM(9), and BM were assigned sequentially (Fig. 3). Because an alpha-1,3 linkage gives a weak H-1-H-2` NOE cross-peak in addition to a strong H-1-H-3` NOE cross-peak, we can use the H-1-H-2` NOE cross-peak c` instead of the H-1-H-3` NOE cross-peak C` for the assignment or to confirm the connectivity. Especially for BM, two H-1-H-2-correlated cross-peaks c and e of 3-O-substituted mannose units, Man-C and Man-E, were better separated than the corresponding two H-1-H-3-correlated ones, C and E. Therefore, it was effective to use the cross-peaks c and e and H-1-H-2` NOE cross-peaks c` and e`.


Figure 3: Sequential connectivities of the mannose units of BM(6), BM(7), BM(8), and BM. The right side of the diagonal shows the relayed COSY, and the left side of the diagonal shows the rotating frame NOE spectroscopy. Primed letters indicate interresidue H-1-H-2` or H-1-H-3` NOE cross-peaks, and unprimed letters indicate the H-1-H-2- and the H-1-H-3-correlated cross-peaks, caused by J-coupling; e.g. A indicates the H-1-H-2-correlated cross-peak of the reducing terminal mannose unit, Man-A, and A` indicates the interresidue NOE cross-peak between the H-2 of Man-A and the H-1 of an adjacent mannose unit, Man-B. By this procedure, the H-1 and H-2 signals were sequentially assigned from the H-1 of Man-A, A-A`-B-B`-C-C`-(or c-c`-)D-D`-E-E`-F for BM(6).



The results summarized in Table 2clearly demonstrate that the attachment of an alpha-1,6-linked mannose unit to Man-C and Man-E causes an upfield shift of the H-1 signals of Man-B and Man-D, respectively, due to a steric effect(15, 26) .



Two-dimensional Homonuclear Hartmann-Hahn Spectroscopy of Mannan

The molar ratio of the mannan side chains was calculated from the dimensions of the H-1 and H-2 signals of fraction G (Fig. 4A) based on the assignment result of the cross-peaks on the two-dimensional HOHAHA spectrum (Fig. 4B) followed by the method described previously(15) . As shown in Table 3, the cross-peaks 1 and 3 in the two-dimensional HOHAHA spectrum of fraction G were assigned to correspond to 2-O-substituted alpha-1,3- and alpha-1,2-linked mannose units, respectively. The cross-peaks 2 and 5 were found to correspond to the upfield-shifted ones of cross-peaks 1 and 3, respectively, by the effect of the presence of an alpha-1,6-linked branching mannose unit as shown in Table 2. Cross-peaks 4 and 6 correspond to alpha-1,3-linked mannose units substituted with a beta-1,2 linkage by mannose and beta-1,2-linked mannobiose unit(s), respectively. On the other hand, cross-peaks 7 and 8 correspond to alpha-1,2-linked mannose units substituted by beta-1,2-linked one(s)(15) . The absence of these cross-peaks indicates that this mannan has no side chains corresponding to the C. albicans serotype A-specific epitope, antigenic factor 6. Cross-peak 12 indicates the presence of the 3-O-substituted alpha-1,2-linked mannose unit. Although the H-1 signal at 5.037 ppm overlaps those of cross-peaks 11 and 12, the H-2 signal at 4.213 ppm corresponds only to the cross-peak 12. Therefore, we can determine the molar ratio of the 3-O-substituted alpha-1,2-linked mannose units or that of the alpha-1,3-linked ones in mannan as shown in Table 3. To distinguish the NMR spectra of the two kinds of alpha-1,6-linked mannose units, branch and backbone forming ones, the signals of the ring protons of both mannose units were compared. Fig. 4C indicates the one-dimensional HOHAHA spectrum of BM(7) recorded by the irradiation of the signal at 4.914 ppm corresponding to the branching alpha-1,6-linked mannose unit. On the other hand, Fig. 4D shows a normal ^1H NMR spectrum of a linear alpha-1,6-linked backbone mannan prepared from S. cerevisiae X2180-1A-5 (mnn2) mutant strain cells. As shown in Fig. 4C, the H-4 signal of the former mannose unit appeared at a characteristic region, 3.66-3.68 ppm, and gave cross-peaks 18 and 19 corresponding to the H-1-H-4- and H-2-H-4-correlated ones, respectively. This finding suggests that the cross-peaks 13, 18, and 19 correspond to the branching alpha-1,6-linked mannose unit, and cross-peak 14 corresponds to the backbone alpha-1,6-linked mannose unit.


Figure 4: Assignment of H-1 and H-2 signals of fraction G. (A) Normal ^1H NMR spectrum of fraction G, (B) two-dimensional HOHAHA spectrum of fraction G, (C) one-dimensional HOHAHA spectrum of BM(7) recorded by the irradiation of the signal at 4.914 ppm corresponding to the branching alpha-1,6-linked mannose unit, (D) normal ^1H NMR spectrum of linear alpha-1,6-linked mannan obtained from the cells of the S. cerevisiae X2180-1A-5 (mnn2) strain.





Although it is difficult to determine the dimension of the H-1 signal of the branched mannose unit because of the overlapping of cross-peaks 13 and 14, we can estimate it from the dimensions of the H-1 signals of cross-peaks 2 and 5. Because the H-1 signal dimensions of cross-peaks 4 and 6 are the same as those of cross-peaks 17 and 16 (half of the signal at 4.849 ppm), respectively, the H-1 signal dimension of cross-peak 5 can be determined by subtraction of the H-1 signal dimension of cross-peaks 4 and 6 from that of the signal at 5.218 ppm as shown in Table 3. These results indicate that the amount of the alpha-1,6-linked branching mannose units is slightly smaller than that of the total alpha-1,3-linked ones but is sufficient to attach to all of the 3-O-substituted ones in the beta-1,2 linkage-containing side chains. Namely, BM(6), BM(8), and BM(9) correspond to the degradation products of BM(7) or BM on mild acetolysis. From these results, we can propose the chemical structure of the cell wall mannan of C. guilliermondii IFO 10279 strain as shown in Fig. 5.


Figure 5: Possible structure of C. guilliermondii IFO 10279 strain mannan. M denotes a D-mannopyranose unit. The side-chain sequence is not specified. The molar ratio of the side chains in the mannan is expressed as a percentage of the total side chains. The values are calculated from the dimensions of the ^1H NMR signals in Fig. 4.



Haptenic Activity of Side Chain Oligosaccharides

C. guilliermondii cells have antigenic factors 1, 4, and 9(24, 25) . Therefore, we examined the inhibitory effect of BM(6) to BM on the reactivity of factor 9 serum to fraction G on enzyme-linked immunosorbent assay. As shown in Fig. 6, these oligosaccharides showed the same strong inhibitory effect despite the presence or the absence of the alpha-1,6-linked branching mannose unit. On the other hand, Manbeta12Manbeta12Manbeta12Man (antigenic factor 5), Manbeta12Manalpha12Manalpha12Manalpha1 2Man, and Manbeta12Manbeta12Manalpha12Manalpha12Manalpha1 2Man (antigenic factor 6) showed no inhibitory effect. These results indicate that the third type of beta-1,2 linkage containing oligosaccharide moieties, Manbeta12Manbeta12Manalpha1 3Manalpha1, in fraction G behaves as the antigenic factor 9.


Figure 6: Inhibition of enzyme-linked immunosorbent assay by mannooligosaccharides. To the fraction G-coated microtiter plate, factor 9 serum pretreated with or without haptenic mannooligosaccharides, BM(6) (circle), BM(7) (bullet), BM(8) (box), BM (), betaM4 (Manbeta12Manbeta1 2Manbeta12Man) (times), alphabetaM5 (Manbeta12Manalpha12Manalpha12Manalpha1 2Man) (Delta), and alphabetaM6 (Manbeta12Manbeta12Manalpha12Manalpha1 2Manalpha12Man) (), for 2 h, at 25 °C, was added. After the mixture was allowed to stand for 2 h, 1000-fold-diluted goat anti-rabbit IgG antibody was added, and binding was detected as described under ``Experimental Procedures.''




DISCUSSION

In 1988, Kogan et al.(45) reported the presence of a 2,3-di-O-substituted mannose unit in the side chain of C. albicans and C. guilliermondii mannans based on the results of methylation analysis of polysaccharides. Later, Kagaya et al.(38) suggested the presence of a branching structure (46) in the C. guilliermondii mannan from the cross-reactivity of a monoclonal antibody against factor 4. Recently, we found that the antigenic factor 4 corresponds to an alpha-1,6-branched side chains of the mannan with a comb-like structure(26) .

Ataoglu et al.(27) reported that the antigenic factor 9 corresponds to a consecutive alpha-1,6-linked mannose unit from the reactivity of factor 9 serum to the cells of the S. cerevisiae X2180-1A-5 (mnn2) mutant strain, which have linear alpha-1,6-linked mannan. It is true that fraction G exposes about 50% of the alpha-1,6-linked backbone mannose units (Fig. 5). Because factor 9 serum was prepared simply by absorption of the anti-C. guilliermondii whole cell serum with C. albicans serotype A cells(24) , it is reasonable to expect that it contains antibodies against several epitopes, including the backbone mannose units. In this study, however, we could demonstrate the existence of novel side chains containing a third type of beta-1,2-linked mannose unit, BM(7) and BM, as the specific structure for C. guilliermondii mannan corresponding to antigenic factor 9.

In 1981, Zhang and Ballou (40) reported the presence of O-linked branching mannooligosaccharides up to octaose in S. kluyveri mannoprotein. In the study, they analyzed the structure of oligosaccharides by the methylation technique. However, because it is impossible to determine the linkage sequence from the methylation analysis data, they proposed the structure of mannopentaose to mannooctaose based on that of the shorter ones. On the other hand, the sequential assignment method of oligosaccharides through HMBC (15, 47) or NOE (8, 26, 42) cross-peaks between the glycosylated two mannose units was demonstrated to be suitable for assigning and determining the linkage sequence by this and the preceding studies.

The upfield shift effect of the H-1 signal of an alpha-1,2-linked mannose unit substituted by a 3,6-di-O-substituted one, the effect of which was first found on the branched side chains of the mannan of S. kluyveri (Delta = 0.047 ppm) (15) and later on that of C. albicans (Delta = 0.055 ppm)(26) , was observed on BM(7), BM(9), and BM (Delta of Man-B = 0.044-0.049 ppm). In this study, we also found the same effect of the H-1 signal of an alpha-1,3-linked mannose unit, Man-D, substituted by a 3,6-di-O-substituted one (Delta of Man-D = 0.062 ppm for BM(9) and 0.082 ppm for BM). The large upfield shift effect found in Man-D of BM seems to be due to the attachment of two alpha-1,6-linked mannose units to the neighboring 3-O-substituted ones of Man-D at the reducing and the nonreducing sides. The upfield shift of the H-1 signals seems to be the result of a steric effect; therefore, it is of interest to identify the conformation of these oligosaccharides.

From the results of this and the preceding (12, 26) structural studies of the mannans containing alpha-1,6-branched side chains, we can speculate that the alpha-1,6-mannosyltransferase responsible for the biosynthesis of branched side chains requires oligosaccharides containing an alpha-1,3 linkage as an acceptor. This hypothesis is supported by the results of Pang et al.(48) . They reported that the mannan of an S. kluyveri (mnn1) mutant strain that lacks alpha-1,3-mannosyltransferase activity also lacks the branching alpha-1,6-linked mannose unit. To understand the timing of the transfer of an alpha-1,6-linked mannose unit to the side chain, however, we need to determine the substrate specificity of the alpha-1,6-mannosyltransferase. Recently, we detected a beta-1,2-mannosyltransferase responsible for the synthesis of the second type of beta-1,2 linkage (antigenic factor 6)(49) . Now we are interested in an alpha-1,6-mannosyltransferase and a beta-1,2-mannosyltransferase responsible for the synthesis of the branch and the third type of beta-1,2 linkage (antigenic factor 9), respectively.


FOOTNOTES

*
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§
To whom correspondence should be addressed: Second Dept. of Hygienic Chemistry, Tohoku College of Pharmacy, 4-4-1 Komatsushima, Sendai, Aoba-ku, Miyagi 981, Japan. Tel.: 81-22-234-4181; Fax: 81-22-275-2013.

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The abbreviations used are: HOHAHA, homonuclear Hartmann-Hahn spectroscopy; relayed COSY, relayed coherence transfer spectroscopy; NOE, nuclear Overhauser effect; HPLC, high performance liquid chromatography.


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