Eur J Biochem 270, 2565–2575 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03622.x Existence of novel b-1,2 linkage-containing side chain in the mannan of Candida lusitaniae, antigenically related to Candida albicans serotype A Nobuyuki Shibata1, Hidemitsu Kobayashi2, Yoshio Okawa1 and Shigeo Suzuki3 Second Department of Hygienic Chemistry, Tohoku Pharmaceutical University, Sendai, Miyagi, Japan; 2Department of Nutrition, Faculty of Home Economics, Kyushu Women’s University, Kitakyushu, Fukuoka, Japan; 3Sendai Research Institute for Mycology, Sendai, Miyagi, Japan The antigenicity of Candida lusitaniae cells was found to be the same as that of Candida albicans serotype A cells, i.e both cell wall mannans react with factors 1, 4, 5, and sera of Candida Check However, the structure of the mannan of C lusitaniae was significantly different from that of C albicans serotype A, and we found novel b-1,2 linkages among the side-chain oligosaccharides, Manb1fi2Manb1fi 2Mana1fi2Mana1fi2Man (LM5), and Manb1fi2Manb1fi2Manb1fi2Mana1fi2Mana1fi2Man (LM6) The assignment of these oligosaccharides suggests that the mannoheptaose containing three b-1,2 linkages obtained from the mannan of C albicans in a preceding study consisted of isomers The molar ratio of the side chains of C lusitaniae mannan was determined from the complete assignment of its H-1 and H-2 signals and these signal dimensions More than 80% of the oligomannosyl side chains contained b-1,2-linked mannose units; no a-1,3 linkages or a-1,6-linked branching points were found in the side chains An enzyme-linked immunosorbent inhibition assay using oligosaccharides indicated that LM5 behaves as factor 6, which is the serotype A-specific epitope of C albicans Unexpectedly, however, LM6 did not act as factor The first description of the serological heterogeneity in Candida albicans was reported by Hasenclever and Mitchell [1] These researchers revealed that this species could be divided into two serotypes, A and B, by agglutination reaction between heat-killed whole cells of C albicans strains and rabbit anti-C albicans whole cell sera of both serotypes absorbed with whole cells of another serotype Later, Tsuchiya and collaborators [2] conducted an extensive serological study on a variety of yeasts based on the ability to agglutinate rabbit antiserum of other heat-killed fungal cells; from these data they developed a series of absorbed factor sera ÔCandida CheckÕ, a commercially available kit containing 10 factor sera is an effective tool for identifying the cells of Candida species in clinical specimens Candida lusitaniae can be pathogenic and cause nosocomial infection in immunocompromised patients, predominantly in granulocytopenic patients undergoing cytoreductive chemotherapy for acute leukemia and in bone marrow transplantation recipients [3–6] Although this species is less virulent than most other Candida species, it is important because of its propensity to develop resistance to antifungal agents, including amphotericin B [5–8] C albicans serotype A strains have been isolated predominantly from patients with invasive candidiasis However, C lusitaniae expresses the same antigenic pattern as that of C albicans serotype A, i.e both cell strains have antigenic factors 1, 4, and [9,10] Therefore, it is difficult to distinguish these strains from the antigenic pattern alone The cell wall mannan of C albicans serotype A is composed of an a-1,6-linked backbone moiety and an oligomannosyl side-chain moiety consisting of a-1,2-, a-1,3-, and b-1,2-linked mannose units Furthermore, a small number of a-1,6-linked branching mannose units may be present in the side chain In addition to this acid-stable structure, there is also a region composed of b-1,2-linked oligomannosyl residues linked through a phosphate group to the side chain Because the phosphodiester linkage is acidlabile, we can selectively release the b-1,2-linked mannooligosaccharides by treatment with 10 mM HCl We have shown that factor serum reacts with acid-stable oligomannosyl side chains containing b-1,2 and a-1,2 linkages – the serotype A-specific structure – and that factor serum reacts with the acid-labile b-1,2-linked oligomannosyl moieties [11] We have reported the presence of three kinds of b-1,2 linkage-containing side chains in the mannans from Candida species [11–17] Because the b-1,2-linked mannose unit is present among the pathogenic fungi only in the mannan of the genus Candida and the b-1,2 linkage-containing side chains behave as strong antigens, many workers [18–25] have produced monoclonal antibodies to b-1,2-linked mannose units to protect against candidiasis, for the serodiagnosis of candidiasis, or for the identification of mechanisms of Candida infection Furthermore, studies have indicated the b-1,2-linked mannose units participate in Correspondence to S Suzuki, Sendai Research Institute for Mycology, 1-14-34 Toshogu, Aoba-ku, Sendai, Miyagi 981–0908, Japan Fax: + 81 22 2754246, Tel.: + 81 22 2754680 (Received November 2002, revised 20 March 2003, accepted 16 April 2003) Keywords: Candida lusitaniae; mannan; NMR analysis; serotype A; b-1,2-linkage Ó FEBS 2003 2566 N Shibata et al (Eur J Biochem 270) the adherence of Candida cells to mammalian cells as the first step in infection [26–28] Poulain and his coworkers [29–32] reported that the b-1,2 linkage-containing oligosaccharides and phospholipomannans induce cytokine production and may act as a virulent factor in candidiasis Therefore, it is important to determine the detailed chemical structure of the cell wall mannan, especially the b-1,2 linkage-containing side chains In earlier structural studies by NMR, we identified almost all of the H-1–H-2-correlated cross-peaks of the C albicans serotype B [33], Candida stellatoidea [34], Candida guilliermondii [17], and Candida saitoana [35] mannans from assignment of the H-1 and H-2 signals of many mannooligosaccharides Although the H-1 proton signal provides the best information on the glycosidic linkage, several H-1 signals overlap The H-2 signal, a second structure reporter, can resolve the overlapped H-1 signals into H-1–H-2-correlated cross-peaks; therefore, we were able to determine the dimensions of the resolved H-1 signals using the two-dimensional (2D) HOHAHA spectrum Because the ratio of the mannose units is proportional to that of the corresponding signal dimensions, we can estimate the molar ratio of the side chains However, we have not completely assigned the H-1–H-2-correlated cross-peaks of the mannan of C albicans serotype A yet, because the mannan produces a spectrum containing a very complicated pattern of cross-peaks However, the 2D-HOHAHA spectrum of C lusitaniae mannan was simpler than that of the C albicans serotype A mannan, which prompted us to assign the cross-peaks of C lusitaniae mannan first These accumulated data will make it possible for us to predict the structures of other mannans from their 2D-HOHAHA spectra The object of this study was to determine the detailed chemical structure of the mannan of C lusitaniae, which is antigenically related to C albicans serotype A, using NMR techniques with the aim of elucidating the complete structure of the C albicans serotype A mannan Experimental procedures 2Mana1fi2Man and Manb1fi2Manb1fi2Mana1fi 2Mana1fi2Mana1fi2Man were obtained from the mannan of C albicans J-1012 (serotype A) [16] Preparation of mannan Yeast cells were grown at 28 °C with shaking in a liquid culture containing 0.5% yeast extract, 1% peptone, and 2% glucose Mannan was extracted from the cells with water at 135 °C for h and was separated by precipitation with Fehling’s solution [33] The mannan prepared from the cells of the C lusitaniae IFO 1019 strain was designated Fr L Acid treatment of mannan To determine the amount of acid-labile phosphodiesterified oligosaccharides in the mannan, 500 mg of Fr L were dissolved in 50 mL of 10 mM HCl, and held at 100 °C for h [13] After cooling, the reaction mixture was neutralized with 100 mM NaOH and was separated by column chromatography (2.5 · 100 cm) using Bio-Gel P-2 (extra fine) Elution was conducted with water, and aliquots of the eluates were assayed for carbohydrate content using the phenol–sulfuric acid method [36] Because this treatment cleaves the phosphodiester linkage, the acid-stable moiety of the mannan is eluted in the void volume and the mannooligosaccharides released from the mannan are retained by the column The acid-stable moiety of the mannan was designated Fr L-a Acetolysis of mannan Acetolysis under mild conditions [37] was performed as previously described [38] Briefly, acetylated mannan was dissolved in 100 : 100 : (v/v/v) acetic anhydride–acetic acid–sulfuric acid and held at 40 °C for 36 h After deacetylation using sodium methoxide, fractionation of the resultant mannooligosaccharide mixture was achieved by HPLC This treatment selectively cleaves the backbone a-1,6 linkages and yields an oligosaccharide mixture that originates from the oligomannosyl side-chain moieties Materials The C lusitaniae IFO 1019 strain was obtained from the Institute for Fermentation (Osaka, Japan) The C albicans J-1012 strain (serotype A) mannan was the same specimen used in preceding studies [11,16] Factors 1, 4, 5, 6, 9, and 13b sera of ÔCandida CheckÕ (lot number I675), a commercially available kit containing rabbit polyclonal antibodies to Candida cells, were purchased from Iatron (Tokyo, Japan) Except for factor serum, which is unabsorbed rabbit whole-cell serum against C albicans cells, factors 4, 5, 6, and 13b sera are anti-C albicans sera absorbed with cells of Candida parapsilosis, C guilliermondii, C stellatoidea, and Candida tropicalis, respectively Factor serum is anti-C guilliermondii serum absorbed with cells of C albicans [2] Jack bean a-mannosidase (EC 3.2.1.24) was obtained from Sigma Haptenic oligosaccharides, Manb1fi2Mana1fi3Mana1fi2Mana1fi2Man and Manb1fi2Manb1fi2Mana1fi3Mana1fi2Mana1fi2Man were prepared from the mannan of C guilliermondii [17] or C saitoana [35] and Manb1fi2Mana1fi2Mana1fi HPLC of oligosaccharides HPLC was carried out using a column (10 · 500 mm) of YMC-Pack PA-25 Elution was carried out with 52 : 48 (v/v) CH3CN–water, and the eluates monitored using a differential refractometer [17] 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 D2O at 45 °C with acetone (2.217 p.p.m.) [39] as the internal standard Enzyme-linked immunosorbent inhibition assay The enzyme-linked immunosorbent assay was conducted as described previously [11] Assays using factor sera were Ó FEBS 2003 conducted basically as described by Okawa et al [40] A haptenic oligosaccharide solution (50 lL) was mixed with a 100-fold dilution of factor serum (50 lL) and preincubated at 25 °C for h The reaction mixture was then added to the wells of a Fr L-a-coated microtiter plate and incubated at 25 °C for h After washing, a 1000-fold dilution of goat anti-(rabbit IgG) antibody–peroxidase conjugate (100 lL) was added to the wells and held at 25 °C for h Finally, a substrate solution of 0.01% o-phenylenediamine and 0.03% H2O2 in 150 mM citrate buffer (pH 5.0) (100 lL) was added, followed by the addition of M H2SO4 (50 lL), and the color measured at 492 nm Other methods For a-mannosidase treatment, the mannooligosaccharide mixture (200 mg) was dissolved in 50 mM sodium acetate buffer (pH 4.6; mL) containing 20 U of a-mannosidase After incubation at 37 °C for 48 h, the reaction mixture was boiled for to deactivate the enzyme Total carbohydrate content was determined by the phenol–sulfuric acid method of Dubois et al [36] with D-mannose as the standard Structure of C lusitaniae mannan (Eur J Biochem 270) 2567 serotype A [11] Therefore, we compared the structures of the two mannans using their 2D-HOHAHA spectra Figure shows the H-1 region (% 4.7–5.6 p.p.m.) of the one-dimensional (1D) NMR spectrum and the 2D-H-1– H-2-correlated cross-peaks of Fr L and the mannan of C albicans J-1012 The H-1 region of Fr L gave relatively simple signals in the a-anomeric region (% 4.9–5.6 p.p.m.) and signals at the b-anomeric region (% 4.7–4.9 p.p.m.) that were two times larger than the 1D-NMR signals of the mannan of C albicans J-1012 Several structural differences between the mannans are apparent The differences between cross-peaks 5, 6, and 8, which correspond to a-1,2-linked mannose units in the mannans, suggests differences in the lengths of the a-1,2-linked side-chain moieties The spectrum of Fr L does not show cross-peaks 3, 4, or 17, which correspond to a-1,3-linked mannose units or 3-O-substituted ones Furthermore, Fr L does not show cross-peaks 4, 8, 15, 18, 19 or 30, suggesting that the mannan does not have b-1,2-substituted a-1,3-linked mannose units or a-1,6linked branching mannose units [17,33–35,38,41] In contrast, both mannans contain cross-peaks 25, 27, and 28, which correspond to the b-1,2-linked mannose units [17,35,38] Acid treatment of Fr L Results Prediction of the structure of Fr L from its 2D-HOHAHA spectrum Fr L showed strong reactivity with factor 1, 4, and sera on ELISA (Fig 1), indicating that this mannan possesses the same antigenic determinants as those of C albicans To determine the structures and amounts of the phosphodiesterified oligosaccharides, we treated Fr L with 10 mM HCl at 100 °C for 60 As shown in Fig 3, the released oligosaccharide was predominantly triose The 1H NMR spectrum of the triose was identical to that obtained from the mannan of C albicans [14,42–44], indicating that it is the b-1,2-linked mannotriose The polysaccharide moiety eluted in the void volume fraction was designated Fr L-a Acetolysis of Fr L-a Figure 4A shows the HPLC elution pattern of the acetolysate of Fr L-a from a YMC PA-25 column Oligosaccharides up to hexaose were obtained from this fractionation These oligosaccharides were then digested with a-mannosidase, and the reaction products separated by HPLC (Fig 4B) The resistance of tetraose, pentaose, and hexaose to a-mannosidase degradation indicates that these oligosaccharides contain b-linkages The oligosaccharides from tetraose to hexaose obtained by a-mannosidase treatment were designated LM4 to LM6 H NMR analysis of oligosaccharides Fig Enzyme-linked immunosorbent assay of Fr L The assay was carried out using the factor sera of Candida Check s, factor serum; d, factor serum; n, factor serum; m, factor serum; h, factor serum; j, factor 13b serum These reactivities were exactly the same as those of the mannan of C albicans serotype A [11] Figure shows the H-1 region of the 1H NMR spectra of M3 and LM4 to LM6 As we expected, the signals corresponding to the b-1,2-linked mannose units from 4.776 to 4.918 p.p.m were present in LM4, LM5 and LM6 Although the signals corresponding to the b-1,2-linked mannose units of LM4 and LM5 (4.776–4.853 p.p.m) were the same as those of the one and two b-1,2 linkagecontaining oligosaccharides, respectively, obtained from Candida mannans, the : ratio of signal intensities at 4.918 and 4.845 p.p.m of LM6 was different from the corresponding three b-1,2 linkage-containing mannoheptaose and mannopentaose obtained from the mannans of 2568 N Shibata et al (Eur J Biochem 270) Ó FEBS 2003 Fig Two-dimensional HOHAHA spectra of Fr L and the mannan of C albicans J-1012 (serotype A) The boxed or circled regions in the spectra indicate the H-1–H-2-correlated cross-peaks, except for cross-peak 30, which indicates an H-1–H-4 correlated one The circled regions correspond to the b-mannose units in the phosphodiesterified acid-labile b-1,2-linked mannooligosaccharide Assignments of these cross-peaks are shown in Table Fig Determination of the phosphodiesterified acid-labile oligosaccharide Fr L was treated with 10 mM HCl at 100 °C for h, and the degradation products were separated using a column (2.5 · 100 cm) of Bio-Gel P-2 (A) In addition to a peak in the void volume (Fr L-a), one oligosaccharide peak (fractions 109–118) was recovered The 1H NMR spectrum (B) of the released oligosaccharide was identical to that of the b-1,2-linked mannotriose [43] Fig Elution patterns of oligosaccharides obtained from Fr L-a by acetolysis HPLC was performed with a YMC PA-25 column (10 · 500 mm) (A) before and (B) after a-mannosidase treatment Elution was carried out with 52 : 48 (v/v) CH3CN–water Acetolysis was performed with (CH3CO)2O–CH3COOH–H2SO4 (100 : 100 : v/v/v) at 40 °C for 36 h M1, M2, M3 and M4 indicate mannose, a-linked mannobiose, mannotriose, and mannotetraose, respectively LM4, LM5, and LM6 indicate the b-linkage-containing mannotetraose, mannopentaose, and mannohexaose, respectively Ó FEBS 2003 Structure of C lusitaniae mannan (Eur J Biochem 270) 2569 Fig The anomeric region of the 1H NMR spectra of oligosaccharides obtained from Fr L-a by acetolysis Spectra were recorded using a JEOL JNM-GSX 400 spectrometer in D2O at 45 °C using acetone as the internal standard (2.217 p.p.m.) LM4, LM5 and LM6 are designated as indicated in the legend of Fig C albicans serotype A [16] and Citeromyces matoritensis [45], respectively As the a-1,6-linked mannose unit also shows a signal at about 4.91 p.p.m [17,33,34], we cannot assign the signal from 1D-NMR Therefore, we analyzed these b-1,2 linkage-containing oligosaccharides using 2D-NMR Sequential NMR assignment Sequential assignment of the H-1 and H-2 signals of LM4, LM5, and LM6 was performed using the NOE cross-peaks of the NOESY spectrum by the method described by Hernandez et al [46] with slight modifications [17,43] (Fig 6) The NOE cross-peaks labeled with primed letters indicate Fig Partial NOESY spectra of LM4, LM5, and LM6 Sequential assignment of the H-1 and H-2 signals was performed using NOESY and COSY spectra Primed letters indicate inter-residue H-1–H-2¢-NOE cross-peaks and unprimed letters indicate intraresidue H-1– H-2-correlated cross-peaks, caused by J coupling, e.g A2 indicates the H-1–H-2-correlated cross-peak of the reducing terminal mannose unit, Man-A, and A2¢ indicates the inter-residue NOE cross-peak between the H-2 of ManA 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, A2–A2¢–B2–B2¢–C2–C2¢–D2 for LM4 through-space inter-residue H-1–H-2¢ connectivities between two adjacent mannose units The boxed cross-peaks labeled with unprimed letters indicate intraresidue H-1– H-2-correlated cross-peaks Inter-residue cross-peaks due to dipolar coupling are identified by their absence from a COSY spectrum (not shown), which shows only the intraresidue cross-peaks due to J coupling Using this procedure, the H-1 and H-2 signals of LM4 were sequentially assigned as shown in the leftmost panel of Fig The spectrum in this panel shows inter-residue H-1–H-2¢ connectivities between a reducing terminal mannose unit (Man-A) with an H-1 signal at 5.348 p.p.m and a mannose unit with an H-1 signal at 5.276 p.p.m (A2–A2¢–B2), and between the latter (Man-B) and a mannose unit with an Ó FEBS 2003 2570 N Shibata et al (Eur J Biochem 270) H-1 signal at 5.160 p.p.m (B2–B2¢–C2) Moreover, interaction between Man-C and the b-linked mannose unit with an H-1 signal at 4.776 p.p.m (C2–C2¢–D2) is seen, indicating that LM4 has the following structure: D C B A Manb1!2Mana1 !2Mana1!2Man (LM4) The H-1 and H-2 signals of LM5 and LM6 were also sequentially assigned from the H-1 of Man-A, A2–A2¢–B2– B2¢–C2–C2¢–D2–D2¢–E2 and A2–A2¢–B2–B2¢–C2-C2¢– D2–D2¢–E2–E2¢–F2, respectively Although the H-1 of Man-E and Man-F of LM6 gave almost the same chemical shifts, we could assign the H-2 signal (not shown) of the nonreducing terminal b-1,2-linked mannose unit as the signal with a chemical shift at about 4.14 p.p.m from the assignments of the phosphodiesterified b-1,2-linked mannooligosaccharides [42,43] Therefore, we could unambiguously assign the H-1–H-2-correlated cross-peaks E and F These results indicate that LM5 and LM6 have the following structures: E D C B A Manb1!2Manb1!2Mana1!2Mana1!2Man(LM5) F E D C B A Manb1!2Manb1!2Manb1!2Mana1!2Mana1!2Man (LM6) These assignment results indicate that cross-peaks 22, 23, and 24 on the 2D-HOHAHA spectrum in Fig correspond to Man-E, -F, and -D of LM6, respectively We also assigned other ring protons using the 2D-HOHAHA spectra; the results are shown in Table Although a small number of side chains with the same structure of LM4 has been found in the mannan of C glabrata [47], LM5 and LM6 are novel oligosaccharides Determination of the molar ratio of mannan side chains The molar ratio of the mannan side chains was calculated using the dimensions of the specific H-1 and H-2 signals corresponding to each side chain based on assignment results of the cross-peaks on the 2D-HOHAHA spectra (Fig 2) as previously described [17,34,35] The H-1 signal of the mannan from C lusitaniae at 5.152 p.p.m overlaps two signals at 5.160 p.p.m and 5.138 p.p.m corresponding to cross-peaks 20 and 21, respectively, as shown in Fig 2, and the amount of the two signals was 15.5% of the total H-1 signal dimension (Table 2) These signals were assigned as H-1 of the mannose unit substituted by b-1,2 linkages in LM4, LM5, and LM6 The H-1 signal at 4.778 p.p.m and H-2 signal at 4.401 p.p.m (not shown) simply correspond to cross-peaks 28 and 22, respectively, and both were 1.9% of the total dimension Cross-peaks 28 and 22 were identified as the one and three b-1,2 linkage-containing oligomannosyl side chains, LM4 and LM6, respectively Therefore, the amount of the two b-1,2 linkage-containing Table Chemical shifts of the oligosaccharides obtained by acetolysis ND, not determined Sugar residue Oligosaccharide LM4 F E Chemical shift D C B A F E D C B A 4.776 4.035 3.644 3.579 3.379 3.919 3.738 5.160 4.274 3.858 3.695 ND 3.858 3.759 5.276 4.118 3.956 3.673 ND 3.871 3.738 5.348 3.932 3.945 3.693 ND 3.860 3.499 4.841 4.148 3.610 3.567 3.348 3.914 3.728 4.853 4.261 3.655 3.598 3.389 3.916 3.756 5.140 4.251 3.887 3.605 ND 3.854 3.731 5.262 4.113 3.954 3.667 ND 3.866 3.733 5.340 3.933 3.944 3.692 ND 3.854 3.780 4.914 4.399 3.631 3.589 3.362 3.916 3.750 4.845 4.245 3.690 3.503 3.395 3.936 3.715 5.150 4.265 3.898 3.592 ND 3.854 3.760 5.267 4.119 3.959 3.675 ND 3.866 3.738 5.345 3.940 3.950 3.702 ND 3.865 3.769 Manb1fi2Mana1fi2Mana1fi2Mana H-1 H-2 H-3 H-4 H-5 H-6 H-6¢ LM5 Manb1fi2Manb1fi2Mana1fi2Mana1fi2Mana H-1 H-2 H-3 H-4 H-5 H-6 H-6¢ LM6 Manb1fi2Manb1fi2Manb1fi2Mana1fi2Mana1fi2Mana H-1 H-2 H-3 H-4 H-5 H-6 H-6¢ 4.918 4.147 3.613 3.575 3.379 3.929 3.737 Ó FEBS 2003 Structure of C lusitaniae mannan (Eur J Biochem 270) 2571 Table Assignments and dimensions of the NMR signals of Fr L Chemical shifts are for mannose residues shown in bold Signal dimensions were determined by integration of the peak area in the 1H NMR spectra or by calculation Cross peak Chemical shift (p.p.m.) H-1 5.554 5.542 5.373 5.302 H-2 4.210 4.189 4.090 4.100 5.286 5.261 5.152 5.233 4.099 4.087 4.126 4.096 10 5.130 5.121 4.065 4.017 11 5.104 4.029 12 5.093 3.997 13 5.074 4.008 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 5.047 4.920 4.909 5.033 5.246 5.218 5.160 5.138 4.915 4.918 4.844 4.857 4.849 4.844 4.778 4.768 H-1 4.920 H-1 4.906 4.850 4.064 4.009 3.991 4.204 4.276 4.250 4.275 4.256 4.401 4.147 4.247 4.263 4.269 4.152 4.038 4.044 H-4 3.668 H-2 4.290 4.173 30 a b Residue Signal dimension (%) Manb1fi2Mana1fiphosphate Manb1fi2Manb1fi2Mana1fiphosphate a1fi2Mana1fi3Mana1fi2 a1fi3Mana1fi2Mana1fi3Mana1fi2 ›6 ›6 Mana1 Mana1 Mana1fi2Mana1fi2 a1fi2Mana1fi2Mana1fi2 b1fi2Mana1fi2Mana1fi2 a1fi3Mana1fi2Mana1fi2Mana1fi2 ›6 Mana1 Mana1fi3 a1fi6Mana1fi6Mana1fi6Mana1fi6 ›2 Mana1 a1fi6Mana1fi6Mana1fi6Mana1fi6 > > ›2 ›2 ›2 > > = Mana1 a1fi6Mana1fi6Mana1fi6Mana1fi6 > ›2 > > > a1fi2Mana1 ; a1fi6Mana1fi6Mana1fi6Mana1fi6 ›2 ›2 ›2 a1fi2Mana1 Mana1fi2 Mana1fi6 a1fi6Mana1fi6 a1fi3Mana1fi2 Manb1fi2Mana1fi3 Manb1fi2Manb1fi2Mana1fi3 Manb1fi2Mana1fi2 Manb1fi2Manb1fi2Mana1fi2 Manb1fi2Manb1fi2Manb1fi2Mana1fi2 Manb1fi2Manb1fi2Manb1fi2Mana1fi2 Manb1fi2Manb1fi2Manb1fi2Mana1fi2 Manb1fi2Manb1fi2Mana1fi2 Manb1fi2Manb1fi2Mana1fi3 Manb1fi2Manb1fi2Mana1fi2(3) Manb1fi2Mana1fi2 Manb1fi2Mana1fi3 Mana1fi6 Manb1fi2Manb1fi2Mana1fiphosphate Manb1fi2Manb1fi2Mana1fiphosphate oligomannosyl side chain, LM5, can be estimated using the following formula 15:5 ðlM4 + lM5 + lM6ị 1:9 lM4 + lM6ị ẳ 11:7 ðlM5Þ Thus, the one, two, and three b-1,2 linkage-containing side chains, which correspond to LM4, LM5, and LM6, respectively, are in the ratio of 1.00 : 6.15 : 1.00 Similarly, we could estimate the molar ratio of the a-linked side chains 1.7 0 2.8 15.5 0 18.9 3.3 11.3 0 1.9 13.6 1.9 1.9 1.9 11.7 11.7 1.9 0 1.7 1.7 From these results, we propose the chemical structure and the molar ratio of the side chains of the cell wall mannan of C lusitaniae as shown in Fig Surprisingly, more than 80% of the oligomannosyl side chains were substituted by b-1,2-linked mannose units We could also identify that the circled cross-peaks a and b in Fig correspond to the middle and the nonreducing terminal b-1,2-linked mannose units, respectively, of phosphodiesterified b-1,2-linked mannotriose 2572 N Shibata et al (Eur J Biochem 270) Ó FEBS 2003 Fig Possible structure of C lusitaniae IFO 1019 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 shown in Fig Haptenic activity of the b-1,2 linkage-containing oligosaccharides The reactivity of factor serum to several b-1,2 linkagecontaining oligosaccharides was tested using an enzymelinked immunosorbent inhibition assay between Fr L-a and factor serum As we expected, the b-1,2-substituted a-1,3-linked mannose-containing oligosaccharides, Manb1fi2Mana1fi3Mana1fi2Mana1fi2Man and Manb1fi 2Manb1fi2Mana1fi3Mana1fi2Mana1fi2Man, obtained from the mannan of C guilliermondii [17] or C saitoana [35] did not behave as factor (Fig 8) In contrast, LM4 showed the same inhibition activity as that of the C albicans-derived pentaose, Manb1fi2Mana1fi 2Mana1fi2Mana1fi2Man The inhibition activity of LM5 was twice that of LM4, but was the same as that of the C albicans-derived hexaose, Manb1fi2Manb1fi 2Mana1fi2Mana1fi2Mana1fi2Man Unexpectedly, LM6 showed no inhibition activity at all, suggesting that the three-b-1,2 linkage-containing oligomannosyl side chain does not behave as factor Discussion In previous papers, we reported two different lengths of three b-1,2 linkage-containing oligosaccharides, Manb1fi2Manb1fi2Manb1fi2Mana1fi2Mana1fi2Mana1fi 2Man (AM7), and Manb1fi2Manb1fi2Manb1fi 2Mana1fi2Man (CM5), from the mannans of C albicans serotype A [15,16] and Cit matritensis [45], respectively In these reports, the ratio of the signals at 4.91 and 4.84 p.p.m were about : However, the sequential assignment study of LM6, which also contains three b-1,2-linked mannose units, indicated that both Man-E and Man-F gave a signal at 4.918 p.p.m., Man-D showed a signal at 4.845 p.p.m., and the ratio of the dimensions of the two signals was : These results suggest that both AM7 and CM5 were mixtures of a two-b-1,2 linkage-containing and a three-b-1,2 linkage-containing oligosaccharide and that the assignment results for the b-mannose units were incorrect Fig Enzyme-linked immunosorbent inhibition assay of b-1,2 linkagecontaining oligosaccharides An inhibition assay of the reaction between Fr L-a and factor serum was performed Haptenic oligosaccharides; h, LM4; , LM5; j, LM6; s, Manb1fi2Mana1fi 2Mana1fi2Mana1fi2Man; d, Manb1fi2Manb1fi2Mana1fi2Man a1fi2Mana1fi2Man; n, Manb1fi2Mana1fi3Mana1fi2Mana1fi 2Man; m, Manb1fi2Manb1fi2Mana1fi3Mana1fi2Mana1fi 2Man Because we could assign cross-peaks 22, 23, and 24 of Fr L, we could also calculate the molar ratios of the threeb-1,2 linkage-containing oligosaccharide side chains of the mannan of C albicans serotype A However, this mannan also includes both b-1,2-substituted a-1,3-linked mannosecontaining oligomannosyl side chains and a-1,6-branched oligomannosyl side chains, which is apparent from crosspeaks 8, 15, 18, 19, and 30 Therefore, we need to Ó FEBS 2003 separate the side-chain oligosaccharides for precise structure determination and for the calculation of the molar ratio of the side chains We will clarify the structure of the mannan of C albicans serotype A based on these results in a later study Enzyme-linked immunosorbent inhibition assay of the reaction system between Fr L-a and factor serum using several pure b-1,2 linkage-containing oligosaccharides was performed to identify the antigenic activity Although reaction was not inhibited by the b-1,2 and a-1,3 linkage-containing oligosaccharides, Manb1fi2Mana1fi 3Mana1fi2Mana1fi2Man and Manb1fi2Manb1fi 2Mana1fi3Mana1fi2Mana1fi2Man, strong inhibition was observed with LM4 and LM5 In a preceding paper [48], we tested the haptenic activity of the b-1,2 linkagecontaining oligosaccharides using a cell agglutination inhibition assay, but had not used the b-1,2-substituted a-1,3-linked mannose-containing oligosaccharides Therefore, in this quantitative inhibition assay, we confirmed that the antibody to factor recognizes the a-1,2 linkage of the mannose unit substituted by the b-1,2-linked mannose unit Furthermore, the lack of reactivity of the three-b-1,2 linkage-containing oligosaccharide, LM6, indicates that factor serum does not react with the intermediary linkages fi2Manb1fi2Mana1fi2, but contains at least two kinds of antibodies which recognize LM4 and LM5 from the nonreducing terminal The weak agglutination inhibition activity of the b-1,2 linkage-containing heptaose obtained from C albicans serotype A mannan in a preceding paper [48] also suggests that the heptaose was comprised of a mixture of two and three b-1,2 linkage-containing oligosaccharides The above findings indicate that the recognition size limit of the antibody to these oligosaccharides is four mannose units, Manb1fi2Manb1fi2Mana1fi2Mana1fi, and that more than two b-1,2 linkage-containing oligosaccharides behave as factor [11] instead of as factor Nevertheless, these side chains correspond to a serotype A-specific structure because b-1,2-substituted a-1,2-linked mannose-containing oligomannosyl side chains are not present in the serotype-B mannan A series of studies on the epitope structure of factor has been carried out by Han and Cutler and his coworkers where they isolated monoclonal antibodies B6.1 (IgM) [22] and C3.1 (IgG) [25], both of which react with phosphodiesterified b-1,2-linked oligomannosyl moieties (factor 5) These monoclonal antibodies enhance the resistance of mice to disseminated candidiasis and protect against Candida vaginal infection [22–25] They determined the minimal epitope of these monoclonal antibodies to be b-1,2-linked mannotriose by agglutination inhibition assays [23,25] Furthermore, they showed, using synthetic oligomers, that these epitopes adopt a compact helical conformation [49] In a preceding study [50], we detected and characterized b-1,2-mannosyltransferase II in the cell homogenate of C albicans, but could not detect b-1,2-mannosyltransferase I, which is responsible for the transfer of the first b-1,2linked mannose unit to an a-1,2-linked mannose unit and is the key enzyme for the synthesis of factor Because the a-1,2-linked oligomannosyl moieties of b-1,2 linkagecontaining side chains of the mannans of C albicans, C lusitaniae, and Cit matritensis are strictly fixed at Structure of C lusitaniae mannan (Eur J Biochem 270) 2573 tetraose, triose and biose, respectively, there is a possibility that the b-1,2-mannosyltransferase I responsible for the synthesis of these side chains recognizes the length of the side chain from the a-1,6-linked backbone mannose units If we use the backbone a-1,6-linked mannose-containing side-chain oligosaccharide as the substrate, we may be able to detect b-1,2-mannnosyltransferase I References Hasenclaver, H.F & Mitchell, W.O (1961) Antigenic studies of Candida I Observation of two antigenic groups in Candida albicans J Bacteriol 82, 570–573 Tsuchiya, T., Fukazawa, Y., Taguchi, M., Nakase, T & Shinoda, T (1974) Serologic aspects of yeast classification Mycopathol Mycol Appl 53, 77–91 Pappagianis, D., Collins, M.S., Hector, R & Remington, J (1979) Development of resistance to amphotericin B in Candida lusitaniae infecting a human Antimicrob Agents Chemother 16, 123–126 Merz, W.G (1984) Candida lusitaniae: frequency of recovery, colonization, infection, and amphotericin B resistance J Clin Microbiol 20, 1194–1195 Hadfield, T.L., Smith, M.B., 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Kobayashi, H., Shibata, N & Suzuki, S (1992) Evidence for oligomannosyl residues containing both b-1,2 and a-1,2 linkages as a serotype A-specific epitope(s) in mannan of Candida albicans Infect Immun 60, 2106–2109 Structure of C lusitaniae mannan (Eur J Biochem 270) 2575 49 Nitz, M., Ling, C.-C., Otter, A., Cutler, J.E & Bundle, D.R (2002) The unique solution structure and immunochemistry of the Candida albicans b-1,2-mannopyranan cell wall antigens J Biol Chem 277, 3440–3446 50 Suzuki, A., Takata, Y., Oshie, A., Tezuka, A., Shibata, N., Kobayashi, H., Okawa, Y & Suzuki, S (1995) Detection of b-1,2mannosyltransferase in Candida albicans cells FEBS Lett 373, 275–279  ... b1fi2Mana1fi2Mana1fi2 a1 fi3Mana1fi2Mana1fi2Mana1fi2 ›6 Mana1 Mana1fi3 a1 fi6Mana1fi6Mana1fi6Mana1fi6 ›2 Mana1 a1 fi6Mana1fi6Mana1fi6Mana1fi6 > > ›2 ›2 ›2 > > = Mana1 a1 fi6Mana1fi6Mana1fi6Mana1fi6 > ›2 > > > a1 fi2Mana1 ; a1 fi6Mana1fi6Mana1fi6Mana1fi6... mannan of C glabrata [47], LM5 and LM6 are novel oligosaccharides Determination of the molar ratio of mannan side chains The molar ratio of the mannan side chains was calculated using the dimensions... a1 fi6Mana1fi6Mana1fi6Mana1fi6 ›2 ›2 ›2 a1 fi2Mana1 Mana1fi2 Mana1fi6 a1 fi6Mana1fi6 a1 fi3Mana1fi2 Manb1fi2Mana1fi3 Manb1fi2Manb1fi2Mana1fi3 Manb1fi2Mana1fi2 Manb1fi2Manb1fi2Mana1fi2 Manb1fi2Manb1fi2Manb1fi2Mana1fi2 Manb1fi2Manb1fi2Manb1fi2Mana1fi2