(Received for publication, September 19, 1996, and in revised form, March 27, 1997)
From the Second Department of Hygienic Chemistry, Tohoku College of Pharmacy, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981, Japan
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,
Man1
3(Man
1
6)Man
1
2Man
1
2Man, in the presence of
GDP-mannose and Mn2+ ion at pH 6.0 gave a third type of
-1,2 linkage-containing mannohexaose, Man
1
2Man
1
3(Man
1
6)Man
1
2Man
1
2Man,
the structure of which was identified by means of a sequential
NMR assignment. The results of a substrate specificity study indicated
that the
-1,2-mannosyltransferase requires a mannobiosyl unit,
Man
1
3Man
1
, at the nonreducing terminal site. We
synthesized novel oligosaccharides using substrates possessing a
nonreducing terminal
-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,
Man
1
2Man
1
2Man
1
3(Man
1
6)Man
1
2Man
1
2Man, 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
-1,2-linked mannose units.
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 -1,2
linkage-containing side chains in its cell wall mannans. One is the
-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
-1,2-linked
mannose units attached to the nonreducing terminal of the
-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
-1,2 linkage-containing epitopes have been identified as
corresponding to antigenic factors 5 (14) and 6 (15), respectively. The
third type of
-1,2 linkage-containing side chains can be observed in
the mannan of Candida guilliermondii. This type of oligosaccharide contains
-1,2-linked mannose units attached to an
-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 -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
-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
-1,2-mannosyltransferases responsible for the formation of the
functional side chain.
In a previous study (22), we detected the -1,2-mannosyltransferase
II, which is responsible for the transfer of an additional
-1,2-linked mannose unit to the nonreducing terminal
-1,2-linked one attached to the
-1,2-linked mannotetraose. Although we tried to
detect the
-1,2-mannosyltransferase I responsible for the transfer
of the first
-1,2-linked mannose unit to the
-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
-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
-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
-1,2-linked mannose
unit from GDP-mannose to the nonreducing terminal
-1,3-linked
mannose unit of an
-linked mannopentaosyl side chain,
Man
1
3(Man
1
6)Man
1
2Man
1
2Man, synthesized from
Man
1
3Man
1
2Man
1
2Man corresponding to factor 34 (26),
takes place by participation of the
-1,2-mannosyltransferase IV, and
then, the elongation of a
-1,2-linked one to a resulting product,
Man
1
2Man
1
3(Man
1
6)Man
1
2Man
1
2Man,
takes place by participation of the
-1,2-mannosyltransferase V in
succession.
In the present study, we tried to detect and characterize the
-1,2-mannosyltransferase IV using several oligosaccharide substrates prepared from various yeast mannans.
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 -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, Man
1
2Man
1
2Man
1
2Man (AMan4), and
Man
1
3Man
1
2Man
1
2Man
1
2Man (AMan5)
were prepared from C. albicans mannan (24),
Man
1
3Man
1
2Man
1
2Man (SMan4) and
Man
1
3Man
1
3Man
1
2Man
1
2Man (SMan5)
were from S. cerevisiae serotype Ia mannan (27),
Man
1
3Man
1
2Man (KMan3) was from C. krusei mannan.1
Man
1
3(Man
1
6)Man
1
2Man (KaMan4),
Man
1
3(Man
1
6)Man
1
2Man
1
2Man (SaMan5),
Man
1
3(Man
1
6)Man
1
2Man
1
2Man
1
2Man
(AaMan6), and Man
1
3(Man
1
6)Man
1
3Man
1
2Man
1
2Man
(SaMan6) were enzymatically prepared from corresponding
linear oligosaccharides, KMan3, SMan4, AMan5, and SMan5, respectively, using the
-1,6-mannosyltransferase of C. albicans NIH B-792 cells
followed by the method described in a previous paper (23).
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 PreparationThe 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.
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 HPLC2An 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 AnalysisFor 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 SpectroscopyAll 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 DeterminationThe protein was determined using the bicinchoninic acid protein assay kit (Pierce) (30) with bovine serum albumin as the standard.
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·h1) 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.
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 -mannose unit (
= ~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
-1,3 linkage, the NOE cross-peak C3
was
found through the H-1-H-3-correlated cross-peak C3. Usually, an
-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
-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
-1,2-linked mannose unit,
Man-E. It has been shown that the H-1 signal of an
-linked mannose
unit substituted by a single
-1,2-linked unit causes a downfield
shift (
= 0.09-0.12 ppm) (10, 11, 17) and that the
-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
-mannose specific H-1-H-5-correlated cross-peak.
Moreover, SagMan6 resisted digestion with a jack bean
-mannosidase. These data also supported the fact that Man-D of SagMan6 is substituted by the
-1,2-linked mannose unit.
Therefore, we determined the structure of SagMan6 to be the
following.
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Since these findings indicate that the assay system is able to detect
the -1,2-mannosyltransferase IV, we further examined some properties
of this enzyme.
The effect of buffer pH on the transferase
activity was studied over the pH range of 3.5-8.5. The
-1,2-mannosyltransferase IV exhibits maximum activity at about pH
6.0 in 50 mM Tris/maleate (Fig. 3).
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.
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The enzyme activity is linear for at least
1.5 h at 30 °C under the standard conditions. The
Lineweaver-Burk plot of the -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.
Substrate Specificity
To assess the substrate specificity of
-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
-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
-1,2-mannosyltransferase IV and an
-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
-1,2 linkage (10,
11, 17). Since these
-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
-1,2-mannosyltransferase IV of C. guilliermondii. These results indicate that the
-1,2-mannosyltransferase IV requires the nonreducing terminal
-1,3-linked mannose unit as the substrate.
|
Furthermore, to investigate whether an elongation reaction by the
action of the -1,2-mannosyltransferase IV proceeds, we carried out
the enzyme reaction using the
-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
-linked mannose unit substituted by two
consecutive
-1,2-linked units causes an upfield shift (
= 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
-1,2-linked mannose units (10, 11,
17, 18). The elongation of the
-mannose unit was also confirmed by
-mannosidase treatment. Therefore, we determined the structure of
SaggMan7 to be the following.
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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 -1,3-linked mannose
unit, but do not contain the
-1,2-linked mannose unit in the side
chains. To confirm the addition of the
-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
-1,3-linked mannose units substituted by one and two
-1,2-linked mannose units, respectively (17, 18),
concomitant with the appearance of cross-peaks 8, 9, and 10, corresponding to the
-1,2-linked mannose units. However, the
reaction product did not give cross-peaks 6 and 7, corresponding to the
-1,2-linked mannose units substituted by one and two
-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
-1,2-mannosyltransferases, i.e. the
-1,3
linkage-containing side chains in the mannan were substituted by the
-1,2-linked mannose units.
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 -1,6-linked mannose unit to a
Man8GlcNAc2 core oligosaccharide (38, 42).
KRE2/MNT1 is an
-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
-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
-mannosyltransferases. In a preceding paper (19), we have
reported the
-1,2-mannosyltransferase II of C. albicans,
the enzyme which transfers one
-1,2-linked mannose unit to a
-1,2
linkage-containing side chain. Since there is no other study on the
-1,2-mannosyltransferase, this is the first report of the
-1,2-mannosyltransferase responsible for the introduction of
-1,2-linked mannose unit to an
-linked mannooligosaccharide.
The substrate specificity study indicates that the
-1,2-mannosyltransferase IV requires the nonreducing terminal
-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
-1,6-mannosyltransferase responsible for the synthesis of
-1,6-branched side chains (23). It is of interest to compare the
substrate recognition mechanisms of the two transferases or with
-1,3-mannosidase (48) or with the
-1,3-linked mannose-specific
lectin (28). Since C. guilliermondii cells contain the
-1,2- and
-1,6-mannosyltransferases judging from the structure of
its mannan (18), it is predictable that if we use an
-1,3
linkage-containing linear oligosaccharide as the substrate of the
enzyme reaction, we obtain at least two products that contain the
-1,2- or
-1,6-linked mannose unit. Therefore, we compared the
substrate specificity using pairs of oligosaccharides with or without
the
-1,6-linked branching mannose unit.
The substrate ability of linear oligosaccharides
(pyridylamino-KMan3, pyridylamino-SMan4,
pyridylamino-AMan5, and pyridylamino-SMan5) for
-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
-1,6-mannosyltransferase activity in fraction P from C. guilliermondii is significantly higher than the
-1,2-mannosyltransferase activity (Table II). The reason for the
absence of a linear
-1,2 linkage-containing side chains in the
mannan of C. guilliermondii seems to be in this relationship of the two enzymes. Since the
-1,2 linkage-containing unbranched oligosaccharides, Man
1
2Man
1
3Man
1
2Man
1
2Man
and Man
1
2Man
1
2Man
1
3Man
1
2Man
1
2Man, have
been isolated from the mannan of Candida saitoana (19),
which has antigenic factor 9, it is predictable that if we incubated an
-1,3 linkage-containing linear oligosaccharide with the enzyme
prepared from this strain, we obtain
-1,2 linkage-containing linear
oligosaccharides.
In this study, we could synthesize a pure -1,2 linkage-containing
oligosaccharide,
Man
1
2Man
1
3(Man
1
6)Man
1
2Man
1
2Man, using fraction P prepared from C. guilliermondii cells as
the enzyme and Man
1
3(Man
1
6)Man
1
2Man
1
2Man
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 -mannosyltransferases of S. cerevisiae (32, 33, 35).
However, this property of the enzyme is different from that previously
reported
-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
-1,2-mannosyltransferase I that is responsible for the introduction of the first
-1,2-linked mannose unit to an
-1,2-linked
mannotetraose side chain, of C. albicans. Although the
-1,2-mannosyltransferase IV recognizes only the nonreducing terminal
-1,3-linked mannose unit as identified in this study, the
-1,2-mannosyltransferase I does not seem to require only a
nonreducing terminal
-1,2-linked mannose unit. This is because the
-1,2-linked oligomannosyl moiety of the
-1,2 linkage-containing
side chains of C. albicans mannan is only tetraose and that
of the
-1,2 linkage-containing side chains of the mannans of
C. glabrata (13) and Candida
lusitaniae3 are predominantly triose.
Therefore, the
-1,2-mannosyltransferases of these species seem to
recognize the length of the
-1,2-linked oligomannosyl side chains
from the
-1,6-linked backbone mannose unit. Similar correlation
seems to be present for the recognition system of the
-1,3-mannosyltransferases of C. albicans and C. guilliermondii or S. cerevisiae.
We thank Dr. K. Hisamichi, the First Department of Medicinal Chemistry of the Tohoku College of Pharmacy, for recording the NMR spectra.