Characterization of beta -1,2-Mannosyltransferase in Candida guilliermondii and Its Utilization in the Synthesis of Novel Oligosaccharides*

(Received for publication, September 19, 1996, and in revised form, March 27, 1997)

Akifumi Suzuki Dagger , Nobuyuki Shibata , Mikiko Suzuki , Fumie Saitoh , Hiroko Oyamada , Hidemitsu Kobayashi , Shigeo Suzuki § and Yoshio Okawa

From the Second Department of Hygienic Chemistry, Tohoku College of Pharmacy, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

A particulate insoluble enzyme fraction containing mannosyltransferases from Candida guilliermondii IFO 10279 strain cells was obtained as the residue after extracting a 105,000 × g pellet of cell homogenate with 1% Triton X-100. Incubation of this fraction with a mannopentaose, Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow2Manalpha 1right-arrow2Man, in the presence of GDP-mannose and Mn2+ ion at pH 6.0 gave a third type of beta -1,2 linkage-containing mannohexaose, Manbeta 1right-arrow2Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow2Manalpha 1right-arrow2Man, the structure of which was identified by means of a sequential NMR assignment. The results of a substrate specificity study indicated that the beta -1,2-mannosyltransferase requires a mannobiosyl unit, Manalpha 1right-arrow 3Manalpha 1right-arrow, at the nonreducing terminal site. We synthesized novel oligosaccharides using substrates possessing a nonreducing terminal alpha -1,3-linked mannose unit prepared from various yeast mannans. Further incubation of the enzymatically synthesized oligosaccharide with the enzyme fraction gave the following structure, Manbeta 1right-arrow2Manbeta 1right-arrow2Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow 2Manalpha 1right-arrow2Man, which has been found to correspond to antigenic factor 9. Incubation of Candida albicans serotype B mannan with the enzyme fraction gave significantly transformed mannan, which contains the third type of beta -1,2-linked mannose units.


INTRODUCTION

We have studied the structures of the cell wall mannans of medically important Candida species for several years (1-3) and demonstrated that there are three types of beta -1,2 linkage-containing side chains in its cell wall mannans. One is the beta -1,2-linked mannooligomer, which is located in a phosphodiesterified oligosaccharide moiety, as the common epitope in the mannans of several Candida species (4-9). The second type is beta -1,2-linked mannose units attached to the nonreducing terminal of the alpha -1,2-linked oligomannosyl side chains in the mannans of Candida albicans serotype A (10, 11), Candida tropicalis (12), and Candida glabrata (13). These two beta -1,2 linkage-containing epitopes have been identified as corresponding to antigenic factors 5 (14) and 6 (15), respectively. The third type of beta -1,2 linkage-containing side chains can be observed in the mannan of Candida guilliermondii. This type of oligosaccharide contains beta -1,2-linked mannose units attached to an alpha -1,3-linked mannose unit, the presence of which has been demonstrated in the cell wall mannans of Saccharomyces kluyveri (16, 17), C. guilliermondii (18), and Candida saitoana (19) and have been identified to correspond to antigenic factor 9 (18).

The beta -1,2 linkage-containing oligomannosyl side chains are the specific epitopes of the mannans of the genus Candida and have not been found in mammalian cells. Therefore, the detection of such an antigen in the sera from patients using immunological procedures is useful for the diagnosis of invasive candidiasis. Furthermore, several workers reported that the beta -1,2 linkage-containing side chains participate in the adherence of fungal cells to host cells during the initial step of Candida infection (20, 21). Consequently, it is meaningful to characterize the beta -1,2-mannosyltransferases responsible for the formation of the functional side chain.

In a previous study (22), we detected the beta -1,2-mannosyltransferase II, which is responsible for the transfer of an additional beta -1,2-linked mannose unit to the nonreducing terminal beta -1,2-linked one attached to the alpha -1,2-linked mannotetraose. Although we tried to detect the beta -1,2-mannosyltransferase I responsible for the transfer of the first beta -1,2-linked mannose unit to the alpha -1,2-linked mannotetraose to synthesize antigenic factor 6, we could not detect the activity in the homogenate of C. albicans serotype A cells. Therefore, we tried to detect another beta -1,2-mannosyltransferase responsible for the formation of the antigenic factor 9, which have been found in the mannan of C. guilliermondii (18).

A postulated biosynthetic pathway of the oligosaccharide corresponding to factor 9 can be depicted as shown in Fig. 1 based on the results obtained by the structural analysis of C. guilliermondii mannan (18) and the substrate specificity of an alpha -1,6-mannosyltransferase of C. albicans (23) responsible for the formation of the branched oligosaccharides corresponding to factor 4 (24, 25). Namely, the introduction of a beta -1,2-linked mannose unit from GDP-mannose to the nonreducing terminal alpha -1,3-linked mannose unit of an alpha -linked mannopentaosyl side chain, Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow2Manalpha 1right-arrow2Man, synthesized from Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man corresponding to factor 34 (26), takes place by participation of the beta -1,2-mannosyltransferase IV, and then, the elongation of a beta -1,2-linked one to a resulting product, Manbeta 1right-arrow2Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow2Manalpha 1right-arrow2Man, takes place by participation of the beta -1,2-mannosyltransferase V in succession.


Fig. 1. Biosynthetic process of mannan side chains. The biosynthetic pathway of the side chains of C. guilliermondii mannan was deduced from the findings of the structural analysis obtained in our laboratory.
[View Larger Version of this Image (17K GIF file)]

In the present study, we tried to detect and characterize the beta -1,2-mannosyltransferase IV using several oligosaccharide substrates prepared from various yeast mannans.


EXPERIMENTAL PROCEDURES

Materials

The C. guilliermondii IFO 10279 strain was the same specimen used in a previous study (18). 2-Aminopyridine was obtained from Nakarai Tesque (Kyoto, Japan) and was recrystallized from n-hexane. Pyridylaminomannose was purchased from Ajinoki Co. (Aichi, Japan). GDP-mannose and jack bean alpha -mannosidase (EC 3.2.1.24) were obtained from Sigma. The TSK-Gel Amide-80 column (0.46 × 25 cm) and Toyopearl HW-40 (F) gel were obtained from Tosoh Co. (Tokyo, Japan). The cell wall mannan of C. albicans (serotype B) was prepared by the method described by a preceding paper (18). In this text, the oligosaccharides prepared from the mannans of C. albicans, S. cerevisiae, and Candida krusei were labeled with large letters, A, S, and K, respectively. Furthermore, the oligosaccharides synthesized by using enzymes of C. albicans and C. guilliermondii cells were labeled by the addition of small letters, a and g, respectively. Substrate oligosaccharides, Manalpha 1right-arrow2Manalpha 1right-arrow2Manalpha 1right-arrow2Man (AMan4), and Manalpha 1right-arrow 3Manalpha 1right-arrow2Manalpha 1right-arrow2Manalpha 1right-arrow2Man (AMan5) were prepared from C. albicans mannan (24), Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man (SMan4) and Manalpha 1right-arrow3Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man (SMan5) were from S. cerevisiae serotype Ia mannan (27), Manalpha 1right-arrow3Manalpha 1right-arrow2Man (KMan3) was from C. krusei mannan.1 Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow 2Man (KaMan4), Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow2Manalpha 1right-arrow2Man (SaMan5), Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow2Manalpha 1right-arrow2Manalpha 1right-arrow2Man (AaMan6), and Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man (SaMan6) were enzymatically prepared from corresponding linear oligosaccharides, KMan3, SMan4, AMan5, and SMan5, respectively, using the alpha -1,6-mannosyltransferase of C. albicans NIH B-792 cells followed by the method described in a previous paper (23).

Preparation of Pyridylamino-oligosaccharides

The pyridylamination of the oligosaccharide was performed using the method of Yamamoto et al. (29) as follows: to an oligosaccharide (1 mg), 600 µl of a 2-aminopyridine solution prepared by dissolving 1 g of 2-aminopyridine in 0.65 ml of concentrated hydrochloric acid was added. After being sealed, the tube was heated at 90 °C for 10 min. The tube was then opened, and 60 µl of the supernatant of a mixture of 100 mg of sodium cyanoborohydride and 60 µl of water, as the reducing reagent, was added. The tube was resealed and heated at 90 °C for 1 h. The reaction mixture was diluted with 2 ml of water and applied to a column (1 × 50 cm) of Toyopearl HW-40 and separated from free 2-aminopyridine by elution with 0.01 M ammonium acetate, pH 6.0.

Enzyme Preparation

The preparation of the mannosyltransferase fraction of C. guilliermondii strain cells was carried out by the method described in a previous paper (22) as follows. The cells were grown in the YPD medium (0.5% yeast extract, 1% peptone, and 2% glucose) at 28 °C until the mid-logarithmic growth phase (A600 = ~6). The cells were then harvested and washed with 5 mM Tris/HCl, pH 7.5, by centrifugation. The cells (about 40 g of wet cells) were resuspended in 15 ml of 5 mM Tris/HCl, pH 7.5, containing 3 mM MgCl2, 0.5% glycerol, 1.0% 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride and homogenized with a Bead Beater (Biospec Products) with 50 g glass beads. The homogenate was centrifuged for 20 min at 5,000 × g, and the supernatants were centrifuged for 20 min at 15,000 × g. Then the supernatants were recovered and were centrifuged for 1 h at 105,000 × g. The pellet was resuspended in 1 ml of 5 mM Tris/HCl, pH 7.5, containing 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride and extracted for 2 h at 4 °C. The mixture was centrifuged for 60 min at 105,000 × g. The pellet (fraction P) and supernatant (fraction S) were kept at -90 °C and were both assayed for protein contents and mannosyltransferase activity.

Mannosyltransferase Assay

The assay mixture containing fraction P (300 µg of protein), 5 mM pyridylamino-oligosaccharide, 20 mM GDP-mannose donor, 50 mM Tris/maleate, pH 6.0, 20 mM MnCl2, and 0.3% Triton X-100 in a total volume of 25 µl was incubated for 1 h at 30 °C (standard assay). The reaction was initiated by the addition of GDP-mannose and terminated by heating the mixture for 10 min at 100 °C. After removal of the denatured protein by centrifugation, each reaction mixture was analyzed by HPLC as will be described below. The amount of product was estimated by its fluorescence intensity using pyridylaminomannose as a standard.

Analysis of Enzyme Reaction Products by HPLC2

An Amide-80 column was used for the normal phase HPLC. The flow solvent was a 35:65 mixture of 3% acetic acid/triethylamine, pH 7.3, and acetonitrile, and the flow rate was 1.0 ml/min at 40 °C. Detection of the pyridylamino-oligosaccharides was fluorospectrometrically conducted with excitation and emission wavelengths of 320 and 400 nm, respectively.

Large Scale Enzyme Reaction for NMR Analysis

For the NMR analysis, the enzyme reaction was carried out in a total volume of 500 µl containing 5-10 mg of oligosaccharide or mannan, fraction P (about 12 mg of protein), 20 mM GDP-mannose, 50 mM Tris/maleate, pH 6.0, 20 mM MnCl2, and 0.3% Triton X-100. After incubation for 24-48 h at 30 °C, the reaction was stopped by boiling, and the denatured protein was removed by centrifugation. The enzyme-modified oligosaccharide was fractionated by HPLC and lyophilized. The enzyme-modified mannan was dialyzed against water and lyophilized.

NMR Spectroscopy

All NMR experiments were performed using a JEOL JNM-GSX 400 spectrometer at 400 MHz for 1H in D2O at a probe temperature of 45 °C. Acetone (2.217 ppm) was used as the internal standard for the 1H NMR.

Protein Determination

The protein was determined using the bicinchoninic acid protein assay kit (Pierce) (30) with bovine serum albumin as the standard.


RESULTS

Enzyme Preparation

The total amounts of protein in the membrane preparations, fractions P and S, prepared from the C. guilliermondii IFO 10279 strain cells were 48 and 38.5 mg, respectively. Transferase activities of the two fractions were assayed by incubation with GDP-mannose and pyridylamino-SaMan5. Although the two reaction systems gave the same single product corresponding to pyridylaminomannohexaose by HPLC, the total activity observed in fraction P (2597 nmol·h-1) was about three times higher than that in fraction S (923 nmol·h-1). Therefore, we used fraction P as the enzyme preparation for further studies. To determine the structure of the enzyme reaction product by NMR, a large scale reaction mixture with free SaMan5 was incubated for 36 h. The HPLC profile of the reaction products indicated that approximately 50% of the SaMan5 was transformed into a hexaose.

Sequential NMR Assignment of the Enzyme Reaction Product

The linkage sequence of the mannohexaose obtained by the enzyme reaction, abbreviated as SagMan6, was analyzed by a sequential NMR assignment method using rotating frame nuclear Overhauser effect spectroscopy (ROESY). This nonempirical assignment method has been demonstrated to give satisfactory results (8, 18, 24, 25, 31). The H-1 signal of the reducing terminal alpha -mannose unit (delta  = ~5.35 ppm) was able to be empirically assigned. Therefore, we started the sequential assignment of SagMan6 from the reducing terminal mannose unit. The boxed regions in Fig. 2 indicate intraresidue H-1-H-2 or H-1-H-3 connectivities, which were confirmed by relayed coherence transfer spectroscopy (relayed COSY) and two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA). On the other hand, cross-peaks labeled with primed letters indicate interresidue H-1-H-2' or H-1-H-3' connectivities between two adjacent mannose units. The numbers on the labels indicate the corresponding ring protons. Fig. 2, B and C, shows partial two-dimensional HOHAHA and ROESY spectra, respectively, of SagMan6. Since the H-1-H-2-correlated cross-peak A2 indicates the H-2 chemical shift of Man-A, the NOE cross-peak A2' between the H-2 of Man-A and the H-1 of Man-B was easily assigned. Similarly, the NOE cross-peak B2' between the H-2 of Man-B, which was assigned from cross-peak B2, and the H-1 of Man-C was assigned. Since Man-C is substituted by an alpha -1,3 linkage, the NOE cross-peak C3' was found through the H-1-H-3-correlated cross-peak C3. Usually, an alpha -1,3 linkage gives weak H-1-H-2' NOE cross-peak in addition to strong H-1-H-3' NOE cross-peak. Therefore, we can also assign through the H-1-H-2-correlated cross-peak C2 and the NOE cross-peak C2' between the H-2 of Man-D and the H-1 of Man-C. Additionally, we could find the NOE cross-peak D2' between the H-2 of Man-D and the H-1 of Man-E, of which the signal, 4.764 ppm, corresponds to the beta -1,2-linked mannose unit (10, 11, 17). Using this procedure, we could sequentially assign the H-1 signal from Man-A to Man-E as A2-A2'-B2-B2'-C3-C3'-(or -C2-C2'-)-D2-D2'-E2. Therefore, we assumed that Man-D of SagMan6 is substituted by a beta -1,2-linked mannose unit, Man-E. It has been shown that the H-1 signal of an alpha -linked mannose unit substituted by a single beta -1,2-linked unit causes a downfield shift (Delta delta  = 0.09-0.12 ppm) (10, 11, 17) and that the beta -1,2-linked mannose units gave H-1-H-5-correlated cross-peaks in a characteristic region (14, 17, 18). In the spectrum of SagMan6, the H-1 proton of Man-D at 5.239 ppm appeared at 0.1 ppm more downfield than that of SaMan5 at 5.129 ppm, and there is a beta -mannose specific H-1-H-5-correlated cross-peak. Moreover, SagMan6 resisted digestion with a jack bean alpha -mannosidase. These data also supported the fact that Man-D of SagMan6 is substituted by the beta -1,2-linked mannose unit. Therefore, we determined the structure of SagMan6 to be the following.
<AR><R><C><UP>Man</UP>&bgr;1<UP>→</UP>2<UP>Man</UP>&agr;1<UP>→</UP>3<UP>M</UP><UP>an</UP>&agr;1<UP>→</UP>2<UP>Man</UP>&agr;1<UP>→</UP>2<UP>Man</UP></C></R><R><C>                                             ↑ 6</C></R><R><C>                                 <UP> Man</UP>&agr;1</C></R></AR>
<UP><SC>Structure</SC> 1</UP>
The same hexaose has been found in O-linked oligosaccharides (16) and acetolysate (17) of the mannan of S. kluyveri.


Fig. 2. Partial two-dimensional HOHAHA and ROESY spectra of SagMan6 (B and C) and SaggMan7 (D and E) obtained from SaMan5 (A) by the action of the enzymes prepared from C. guilliermondii. The primed letters indicate interresidue H-1-H-2' or H-1-H-3' NOE cross-peaks and the unprimed letters indicate H-1-H-2 or H-1-H-3-correlated cross-peaks, which are boxed, due to J-coupling. Arrows indicate the direction of the sequential connectivity from the reducing terminal unit to the nonreducing terminal unit. The reaction was carried out at 30 °C for 36 h using SaMan5 as the substrate and fraction P prepared from C. guilliermondii IFO 10279 strain cells as the enzyme. Spectra were recorded using a JEOL JNM-GSX 400 spectrometer in D2O solution at 45 °C using acetone as the standard (2.217 ppm).
[View Larger Version of this Image (35K GIF file)]

Since these findings indicate that the assay system is able to detect the beta -1,2-mannosyltransferase IV, we further examined some properties of this enzyme.

Optimum pH

The effect of buffer pH on the transferase activity was studied over the pH range of 3.5-8.5. The beta -1,2-mannosyltransferase IV exhibits maximum activity at about pH 6.0 in 50 mM Tris/maleate (Fig. 3).


Fig. 3. Effect of pH on beta -1,2-mannosyltransferase activity. Assay conditions are described under "Experimental Procedures" except for the pH of the assay buffer. black-square; sodium acetate; bullet , Tris maleate.
[View Larger Version of this Image (15K GIF file)]

Metal Ion Requirement

The effect of several divalent cations, Mn2+, Mg2+, Ca2+, Ni2+, and Zn2+, on the enzyme activity was studied using their respective chlorides. As shown in Table I, the enzyme activity was enhanced by the addition of Mn2+, Mg2+, or Ca2+. The enzyme activity was not affected by the addition of EDTA. Furthermore, the enzyme activity was completely inhibited by the addition of 20 mM ZnCl2, and the lost activity could not be recovered by the addition of EDTA.

Table I. Effect of divalent metal ions and EDTA on the beta -1,2-mannosyltransferase activity in fraction P

The reaction conditions were the same as those given under "Experimental Procedures" except that the additional divalent metal ion to the reaction mixture was varied as indicated.

Metal salt (20 mM) Mannose incorporated

nmol · mg protein-1 · h-1
None 17
MnCl2 53
MgCl2 45
ZnCl2 0
CaCl2 40
NiCl2 19
EDTA 15
ZnCl2, EDTAa 0

a After 3-h incubation at 30°C with 20 mM ZnCl2, the same amount of 40 mM EDTA was added.

Enzyme Kinetics

The enzyme activity is linear for at least 1.5 h at 30 °C under the standard conditions. The Lineweaver-Burk plot of the beta -1,2-mannosyltransferase IV for pyridylamino-SaMan5 is shown in Fig. 4. The Km and the Vmax values for pyridylamino-SaMan5 calculated from this figure were about 18 mM and 200 nmol·mg protein-1·h-1, respectively.


Fig. 4. Effect of substrate concentration on beta -1,2-mannosyltransferase activity. Assay conditions are the same as those described under "Experimental Procedures" except for the concentration of GDP-mannose, 100 mM, and pyridylamino-SaMan5.
[View Larger Version of this Image (11K GIF file)]

Substrate Specificity

To assess the substrate specificity of beta -1,2-mannosyltransferase IV, we used the pyridylamino derivatives of the oligosaccharides prepared from the mannans of C. albicans, C. krusei, and S. cerevisiae and of enzymatically synthesized oligosaccharides as described under "Experimental Procedures." Table II shows that the oligosaccharides containing an alpha -1,3-linked mannose unit at the nonreducing terminal can serve as acceptors. Although KaMan4, AaMan6, and SaMan6 can also possibly act as the acceptors of both the beta -1,2-mannosyltransferase IV and an alpha -1,2-mannosyltransferase judging from the structure of C. guilliermondii mannan, their enzyme reaction products, designated as KagMan5, AagMan7, and SagMan7, exhibited a signal at about 4.76 ppm in the 1H NMR spectra (data not shown). Therefore, it is apparent that the transferred mannose unit is attached to each substrate with a beta -1,2 linkage (10, 11, 17). Since these beta -1,2 linkage-containing branched oligosaccharides have not been found in the acetolysate of yeast mannans, it is reasonable to say that we could prepare novel oligosaccharides using the beta -1,2-mannosyltransferase IV of C. guilliermondii. These results indicate that the beta -1,2-mannosyltransferase IV requires the nonreducing terminal alpha -1,3-linked mannose unit as the substrate.

Table II. Substrate specificity of the beta -1,2-mannosyltransferases

The reaction conditions were the same as those given under "Experimental Procedures" except that the substrate was varied as indicated.

Substrate (5 mM)
Mannose incorporateda
Abbreviation Structure  beta -1,2-Mannosyltransferase  alpha -1,6-Mannosyltransferase

nmol · mg protein-1 · h-1
Pyridylamino-KMan3 Manalpha 1right-arrow3Manalpha 1right-arrow2Man-pyridylamine 13 149
Pyridylamino-AMan4 Manalpha 1right-arrow2Manalpha 1right-arrow2Manalpha 1right-arrow2Man-pyridylamine 0 0
Pyridylamino-SMan4 Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man-pyridylamine 1 41
Pyridylamino-KaMan4 Manalpha 1right-arrow3Manalpha 1right-arrow2Man-pyridylamine 52
 up-arrow
Manalpha
Pyridylamino-AMan5 Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Manalpha 1right-arrow2Man-pyridylamine 1 49
Pyridylamino-SMan5 Manalpha 1right-arrow3Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man-pyridylamine 6 97
Pyridylamino-SaMan5 Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man-pyridylamine 52
 up-arrow  6 
Manalpha
Pyridylamino-AaMan6 Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Manalpha 1right-arrow2Man-pyridylamine 60
 up-arrow  6 
Manalpha
Pyridylamino-SaMan6 Manalpha 1right-arrow3Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man-pyridylamine 48
 up-arrow  6 
Manalpha

a The beta -1,2-mannosyltransferase and alpha -1,6-mannosyltransferase activities were calculated from the amount of the jack bean alpha -mannosidase-resistant and susceptible enzyme reaction products, respectively.

Furthermore, to investigate whether an elongation reaction by the action of the beta -1,2-mannosyltransferase IV proceeds, we carried out the enzyme reaction using the beta -1,2 linkage-containing oligosaccharide, SagMan6, as the substrate, and confirmed the structure of enzyme reaction product, SaggMan7. Fig. 2, D and E, indicate the assignment of SaggMan7. The H-1 and H-2 signals of SaggMan7 were sequentially assigned from Man-A to Man-F, A2-A2'-B2-B2'-C3-C3'-(or -C2-C2'-)-D2-D2'-E2-E2'-F2. It has been shown that the H-1 signal of an alpha -linked mannose unit substituted by two consecutive beta -1,2-linked units causes an upfield shift (Delta delta  = 0.02 ppm) (10, 11, 17, 18). The H-1 proton of the Man-D of SaggMan7 at 5.216 ppm appeared at about 0.02 ppm upfield from that of SagMan6 at 5.239 ppm. Moreover, SaggMan7 exhibits a signal at about 4.84 ppm, which corresponds to two consecutive beta -1,2-linked mannose units (10, 11, 17, 18). The elongation of the beta -mannose unit was also confirmed by alpha -mannosidase treatment. Therefore, we determined the structure of SaggMan7 to be the following.
<AR><R><C><UP>Man</UP>&bgr;1<UP>→</UP>2<UP>Man</UP>&bgr;1<UP>→</UP>2<UP>Man</UP>&agr;1<UP>→</UP>3<UP>M</UP><UP>an</UP>&agr;1<UP>→</UP>2<UP>Man</UP>&agr;1<UP>→</UP>2<UP>Man</UP></C></R><R><C>                                                                 ↑ 6</C></R><R><C>                                                      <UP> Man</UP>&agr;1</C></R></AR>
<UP><SC>Structure</SC> 2</UP>
The assignment results of SagMan6 and SaggMan7 are shown in Table III

Table III. Proton (H1 and H2) chemical shifts of mannooligosaccharides characterized in this study


 Abbreviation  Sugar residue
 Proton  Chemical shift
F E D C B A F E D C B A

ppm
SaMan5 Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man H1 5.129 5.022 5.232 5.346
 up-arrow  6                                                 
Manalpha 1 H1 4.913
H2 4.061 4.207 4.099 3.934
H2 3.996
SagMan6 Manbeta 1right-arrow2Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man H1 4.764 5.239 5.028 5.229 5.346
 up-arrow  6
Manalpha 1 H1 4.914
H2 4.039 4.274 4.213 4.100 3.932
H2 3.999
SaggMan7 Manbeta 1right-arrow2Manbeta 1right-arrow2Manalpha 1right-arrow3Manalpha 1right-arrow2Manalpha 1right-arrow2Man H1 4.845 4.845 5.216 5.026 5.233 5.347
 up-arrow  6
Manalpha 1 H1 4.915
H2 4.154 4.266 4.247 4.206 4.102 3.937
H2 3.997

Modification of Mannan by the beta -1,2-Mannosyltransferases

On the basis of these results, we performed a large scale enzyme reaction using the acid-treated mannan of C. albicans serotype B strain, which possess the nonreducing terminal alpha -1,3-linked mannose unit, but do not contain the beta -1,2-linked mannose unit in the side chains. To confirm the addition of the beta -1,2-linked mannose unit to the mannan, we compared the structures of the parent and enzyme modified mannans using two-dimensional HOHAHA (Fig. 5). As shown in Fig. 5B, the reaction product gave cross-peaks 4 and 5, which correspond to the alpha -1,3-linked mannose units substituted by one and two beta -1,2-linked mannose units, respectively (17, 18), concomitant with the appearance of cross-peaks 8, 9, and 10, corresponding to the beta -1,2-linked mannose units. However, the reaction product did not give cross-peaks 6 and 7, corresponding to the alpha -1,2-linked mannose units substituted by one and two beta -1,2-linked mannose units, respectively (17, 18). These results demonstrate that the mannan was changed to give complex structure by the action of the beta -1,2-mannosyltransferases, i.e. the alpha -1,3 linkage-containing side chains in the mannan were substituted by the beta -1,2-linked mannose units.


Fig. 5. Partial two-dimensional HOHAHA spectra of C. albicans NIH B-792 (serotype B) strain mannan (A) and the enzyme reaction product (B). Cross-peaks 1, 2, and 3 indicate the presence of alpha -1,6-linked branching mannose units (23-25), cross-peaks 4-10 indicate the presence of beta -1,2-linked mannose units (17-19).
[View Larger Version of this Image (24K GIF file)]


DISCUSSION

Most of the studies on mannosyltransferases concerning the synthesis of the cell wall mannoprotein of yeast have been done using Saccharomyces cerevisiae (32-47), and some of the structural genes coding for yeast mannosyltransferases have been isolated. OCH1 encodes the initiating mannosyltransferase that adds the first alpha -1,6-linked mannose unit to a Man8GlcNAc2 core oligosaccharide (38, 42). KRE2/MNT1 is an alpha -1,2-mannosyltransferase gene (39), and Kre2p/Mnt1p is responsible for the addition of mannose units to both N- and O-linked carbohydrate chains (40, 41). The MNN1 gene encodes an alpha -1,3-mannosyltransferase (43), and Mnn1p is also responsible for the synthesis of N- and O-linked carbohydrate chain (36, 43). Recently, YUR1, KTR1, and KTR2 genes were also found to corresponding to mannosyltransferases (45, 46). However, all of these enzymes were for alpha -mannosyltransferases. In a preceding paper (19), we have reported the beta -1,2-mannosyltransferase II of C. albicans, the enzyme which transfers one beta -1,2-linked mannose unit to a beta -1,2 linkage-containing side chain. Since there is no other study on the beta -1,2-mannosyltransferase, this is the first report of the beta -1,2-mannosyltransferase responsible for the introduction of beta -1,2-linked mannose unit to an alpha -linked mannooligosaccharide.

The substrate specificity study indicates that the beta -1,2-mannosyltransferase IV requires the nonreducing terminal alpha -1,3-linked mannose unit. However, the linkage of the penultimate mannose unit does not affect the substrate activity of the oligosaccharides. A similar substrate specificity has been found on the alpha -1,6-mannosyltransferase responsible for the synthesis of alpha -1,6-branched side chains (23). It is of interest to compare the substrate recognition mechanisms of the two transferases or with alpha -1,3-mannosidase (48) or with the alpha -1,3-linked mannose-specific lectin (28). Since C. guilliermondii cells contain the beta -1,2- and alpha -1,6-mannosyltransferases judging from the structure of its mannan (18), it is predictable that if we use an alpha -1,3 linkage-containing linear oligosaccharide as the substrate of the enzyme reaction, we obtain at least two products that contain the beta -1,2- or alpha -1,6-linked mannose unit. Therefore, we compared the substrate specificity using pairs of oligosaccharides with or without the alpha -1,6-linked branching mannose unit.

The substrate ability of linear oligosaccharides (pyridylamino-KMan3, pyridylamino-SMan4, pyridylamino-AMan5, and pyridylamino-SMan5) for beta -1,2-mannosyltransferase IV is very low in contrast to that of the corresponding branched oligosaccharides (pyridylamino-KaMan4, pyridylamino-SaMan5, pyridylamino-AaMan6, and pyridylamino-SaMan6). These results indicate that the alpha -1,6-mannosyltransferase activity in fraction P from C. guilliermondii is significantly higher than the beta -1,2-mannosyltransferase activity (Table II). The reason for the absence of a linear beta -1,2 linkage-containing side chains in the mannan of C. guilliermondii seems to be in this relationship of the two enzymes. Since the beta -1,2 linkage-containing unbranched oligosaccharides, Manbeta 1right-arrow2Manalpha 1right-arrow3Manalpha 1right-arrow 2Manalpha 1right-arrow2Man and Manbeta 1right-arrow2Manbeta 1right-arrow2Manalpha 1right-arrow3Manalpha 1right-arrow 2Manalpha 1right-arrow2Man, have been isolated from the mannan of Candida saitoana (19), which has antigenic factor 9, it is predictable that if we incubated an alpha -1,3 linkage-containing linear oligosaccharide with the enzyme prepared from this strain, we obtain beta -1,2 linkage-containing linear oligosaccharides.

In this study, we could synthesize a pure beta -1,2 linkage-containing oligosaccharide, Manbeta 1right-arrow2Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow2Manalpha 1right-arrow2Man, using fraction P prepared from C. guilliermondii cells as the enzyme and Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manalpha 1right-arrow2Manalpha 1right-arrow2Man as the substrate in the presence of MnCl2 and GDP-mannose. Since the synthesis of the oligosaccharides using specific glycosyltransferases does not require a complicated protecting procedure and does not produce by-products, it is an effective tool for the synthesis of oligosaccharides.

We will be able to synthesize many novel oligosaccharides by taking advantage of the substrate specificity of each enzyme obtained from several Candida species. The novel oligosaccharides seem to be useful for studying not only the substrate specificity of mannosyltransferases but also the specificity of lectins and mannosidases. Furthermore, this approach will become important for the synthesis of the sugar chains of medically important glycoproteins or glycolipids.

The requirement for Mn2+, Mg2+, or Ca2+ by the enzyme was similar to those previously reported for alpha -mannosyltransferases of S. cerevisiae (32, 33, 35). However, this property of the enzyme is different from that previously reported beta -1,2-mannosyltransferase II of C. albicans, which has no absolute requirement for metal ions (22).

In the preceding study (22), we could not detect the beta -1,2-mannosyltransferase I that is responsible for the introduction of the first beta -1,2-linked mannose unit to an alpha -1,2-linked mannotetraose side chain, of C. albicans. Although the beta -1,2-mannosyltransferase IV recognizes only the nonreducing terminal alpha -1,3-linked mannose unit as identified in this study, the beta -1,2-mannosyltransferase I does not seem to require only a nonreducing terminal alpha -1,2-linked mannose unit. This is because the alpha -1,2-linked oligomannosyl moiety of the beta -1,2 linkage-containing side chains of C. albicans mannan is only tetraose and that of the beta -1,2 linkage-containing side chains of the mannans of C. glabrata (13) and Candida lusitaniae3 are predominantly triose. Therefore, the beta -1,2-mannosyltransferases of these species seem to recognize the length of the alpha -1,2-linked oligomannosyl side chains from the alpha -1,6-linked backbone mannose unit. Similar correlation seems to be present for the recognition system of the alpha -1,3-mannosyltransferases of C. albicans and C. guilliermondii or S. cerevisiae.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed.
§   Present address: Sendai Research Institute for Mycology, 1-14-34 Toshogu, Aoba-ku, Sendai, Miyagi 981, Japan.
1   H. Oyamada, H. Kobayashi, M. Suzuki, N. Shibata, S. Suzuki, and Y. Okawa, manuscript in preparation.
2   The abbreviations used are: HPLC, high performance liquid chromatography; NOE, nuclear Overhauser effect; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; ROESY, rotating frame nuclear Overhauser effect spectroscopy; relayed COSY, relayed coherence transfer spectroscopy.
3   N. Shibata, N. Senbongi, H. Kobayashi, and Y. Okawa, manuscript in preparation.

ACKNOWLEDGEMENT

We thank Dr. K. Hisamichi, the First Department of Medicinal Chemistry of the Tohoku College of Pharmacy, for recording the NMR spectra.


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