(Received for publication, December 23, 1996, and in revised form, May 13, 1997)
From the National Institute of Bioscience and Human
Technology, Tsukuba, Ibaraki 305, Japan and the § Asahi
Chemical Industry Co., Ltd., Fuji, Shizuoka 416, Japan
In yeast Saccharomyces cerevisiae the
N-linked sugar chain is modified at different positions by
the addition of mannosylphosphate. The mnn6 mutant is
deficient in the mannosylphosphate transferase activity toward
mannotetraose (Karson, E. M., and Ballou, C. E. (1978) J. Biol. Chem. 253, 6484-6492). We have cloned the MNN6 gene by complementation. It has encoded a 446-amino acid
polypeptide with the characteristics of type II membrane protein. The
deduced Mnn6p showed a significant similarity to Kre2p/Mnt1p, a Golgi -1,2-mannosyltransferase involved in O-glycosylation.
The null mutant of MNN6 showed a normal cell growth, less
binding to Alcian blue, hypersensitivity to Calcoflour White and
hygromycin B, and diminished mannosylphosphate transferase activity
toward the endoplasmic reticulum core oligosaccharide acceptors
(Man8GlcNAc2-PA and Man5GlcNAc2-PA) in vitro, suggesting
the involvement of the MNN6 gene in the endoplasmic reticulum core oligosaccharide phosphorylation. However, no differences were observed in N-linked mannoprotein oligosaccharides
between
och1
mnn1 cells and
och1
mnn1
mnn6 cells, indicating the existence of
redundant genes required for the core oligosaccharide phosphorylation. Based on a dramatic decrease in polymannose outer chain phosphorylation by MNN6 gene disruption and a determination of the
mannosylphosphorylation site in the acceptor, it is postulated that the
MNN6 gene may be a structural gene encoding a
mannosylphosphate transferase, which recognizes any oligosaccharides
with at least one
-1,2-linked mannobiose unit.
In yeast Saccharomyces cerevisiae the biosynthesis of
N-linked oligosaccharides has been studied in detail. The
core oligosaccharide (Man8GlcNAc2) synthesized in the endoplasmic
reticulum (ER)1 is identical in yeast and
mammals. The outer chain attached to the core-like oligosaccharide
contains an -1,6-linked polymannose backbone with branches of
-1,2-linked mannobiose capped with terminal
-1,3-linked mannose
residues (1-5). In yeast, N-linked sugar chains are also
modified at different positions by the addition of mannosylphosphate
(6-9). Although this oligosaccharide modification significantly
contributes to a major negative charge of the cell wall (8-10), less
is known about its biosynthesis and function in yeast.
Ballou and co-workers (11) have isolated mnn mutants that
are blocked at various stages of outer chain elongation. The
mnn1 mutant lacks -1,3-mannosyltransferase activity and
is defective in adding terminal
-1,3-linked mannose to both
N-linked and O-linked oligosaccharides (11, 12).
The
-1,3-mannosyltransferase has a property that competes with
mannosylphosphate transferase (10, 14, 15). The mnn4 and
mnn6 mutants are known to produce phosphate-deficient mannan
relative to wild type cells, presenting a phenotype of less binding to
the phthalocyanin dye, Alcian blue (10, 15). The MNN4 gene
has been cloned and predicted to encode a large protein containing
1,178 amino acids functioning as a positive regulator for
mannosylphosphate transferase (16). The mnn6 is a recessive
mutation, indicating a lack of mannosylphosphate in the branches of the
mannose outer chain in vivo, and is deficient in the
mannosylphosphate transferase activity toward mannotetraose in
vitro (15). Although mannosylphosphate transferase activity was
decreased in the mnn6 mutant, it was still uncertain whether the mnn6 mutation affected the N-linked core
oligosaccharide phosphorylation.
In this work, we have cloned the MNN6 gene by functional
complementation and analyzed the effects of the mnn6 null
mutation on enzymatic activities toward the core oligosaccharide
acceptors. The mnn6 mutation does not affect the apparent
profiles of N-linked core oligosaccharide phosphorylation
in vivo, suggesting the presence of redundant functional
genes. Characterization of enzymatic reaction product suggested that
the MNN6 gene may encode a mannosylphosphate transferase,
which recognizes any oligosaccharides with at least one
-1,2-linked
mannobiose unit.
Strain TO3-6D, used for the cloning of MNN6, was a meiotic segregant from a cross of LB1425-1B, kindly provided by C. E. Ballou (University of California, Berkeley) and a strain of LB1-10B, purchased from the American Type Culture Collection (ATCC). KK4 and its isogenic mnn6 disruptant strain XW44 were used for the Calcoflour White (CFW; Sigma) and hygromycin B (Sigma) sensitivity test. Strains YS125-15B, XW27, YS131-30A, and YS131-30D were used for microsomal membrane preparation. YS126-47D and its isogenic mnn6 disruptant strain XW43 were used for oligosaccharide analysis. Yeast strains used in this study are summarized in Table I. Yeast strains with multigene disruptions were constructed by standard genetic methods (17, 18). Yeast strains were grown either in YPD (2% Bacto-peptone, 1% yeast extract, and 2% glucose) or in a complete minimal medium containing 4% glucose, 0.67% Bacto-yeast nitrogen base without amino acids (Difco), 0.3 M sorbitol and supplemented with the appropriate auxotrophic requirements (18). CFW and hygromycin B were separately added to autoclaved YPD/agar to a final concentration of 50 µg/ml just prior to pouring plates.
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Yeast genomic DNA library in YCp50, "CEN BANK" A and B, were purchased from ATCC. Transformation of yeast cells was carried out by the lithium acetate procedure (19). Transformants were selected and maintained on an SD-ura plate. The colonies from master plates were transferred to nitrocellulose filter and incubated for one more day at 30 °C. Those colonies on the filter were fixed by autoclaving at 120 °C for 1 h and stained by immersion into 0.1% Alcian blue solution until the blue color was developed on the wild type (MNN6) colonies at room temperature (20-30 min). Positive clones were screened as those providing a blue stain (wild type phenotype). The putative clones were further reassayed and confirmed individually by conventional Alcian blue assay (11).
DNA SequencingBacterial strain JM109 was used for the preparation of plasmids. Restriction fragments containing a portion of the MNN6 gene were subcloned into pRS316 vector (20). Sequencing was performed by the dideoxy chain termination method with dye primers (21) and done with the SequiThermTM Long-Read Cycle Sequencing Kit-LC by the LI-COR model 4000L Automated Sequencer. Sequence comparisons against the GenBank or GenPept sequence data bases were performed using the FASTA (22) and BLAST (23) programs. The hydrophobicity plot was generated by the method of Kyte and Doolittle (24).
Gene DisruptionDisruption of MNN6 gene was made
by the single-step gene replacement procedure (25). The 4.3-kilobase
pair HpaI-SacI fragment containing
MNN6 from pSA9-7 was digested with BglII and
BclI restriction endonucleases. The BglII site is
located 333 base pairs upstream from the ATG, and the BclI
site is found 558 base pairs upstream from the stop codon (see Fig.
4A). This digestion removed a 1116-base pair fragment
encompassing 261 amino acids of the MNN6 sequence and
further replaced it with a BglII fragment containing the
complete ADE2 gene from pASZ11 (26). Haploid yeast strains
were used to transform with the linearized
mnn6::ADE2 DNA fragments (Fig. 4A). The
disruption of MNN6 was confirmed by Southern hybridization (data not shown). Selection of mnn6 disruptants
(mnn6) was done as follows: for strains carrying the
ade2 mutation,
mnn6 was selected on an SD-ade
plate; for strains carrying no ade2 mutation (like KK4),
mnn6 was selected by QAE-Sepharose adsorption according to the method described by Ballou (11).
Construction of High Copy Plasmid Carrying MNN6
A complementing fragment (HpaI/NruI fragment) containing the entire MNN6 gene was excised by digestion with KpnI/SacI, whose sites are located in multicloning sites of pRS316-based plasmid pRSMNN6 and then inserted into the multicopy vector of pET351 and named pETMNN6. The pET351 high copy vector was constructed based on YEp351 (27), in which a BamHI/HpaI fragment containing the LEU2 gene was replaced with a BamHI/PvuII fragment carrying TRP1 gene from pJJ246 vector (28).
Mannosylphosphate Transferase AssayThe microsomal membrane
proteins containing mannosylphosphate transferase activity were
prepared according to the previous method (29), except that the cell
pellets were frozen at 20 °C for 1 h before the cells were
destroyed by glass beads using a B. Braun homogenizer. The enzyme assay
was carried out by using 400 µg of protein of the high speed pellet
(centrifugation at 100,000 × g for 60 min) in 50 µl
of 50 mM Tris-HCl (pH 6.0), 10 mM
MnCl2, 25 or 50 pmol of acceptor (depending on the acceptor used), 0.6% Triton X-100, 1 mM GDP-mannose, and 0.5 mM 1-deoxy-mannojirimycin as an inhibitor of
-mannosidase in yeast (30) at 30 °C for 60 min. The enzyme
reaction was terminated by boiling for 5 min, and the reaction mixture
was ultrafiltrated with ultrafree C3LGC (Millipore). The filtrated
solution was lyophilized and used for high performance liquid
chromatography (HPLC) analysis. Man8GlcNAc2-PA, purchased from Takara
Shuzo Co. (Kyoto, Japan), was used as the acceptor (50 pmol) in the
enzyme assay. Man5GlcNAc2-PA acceptor (25 pmol) prepared from strain
YS133-1D mannoproteins was used for the enzyme assay.
Preparation of oligosaccharides was the same as described previously (31). In brief, yeast cells were grown in YPAD medium at 25 °C and harvested at stationary phase. Mannoproteins were hot citrate buffer-extracted, ethanol-precipitated, and further purified by concanavalin A-Sepharose. The N-linked oligosaccharides were liberated from the bulk yeast mannoproteins using glycopeptidase A (Seikagaku Kogyo Co., Tokyo, Japan), an enzyme specific to release N-linked oligosaccharides from glycoprotein or glycopeptide. Pyridylamination of the oligosaccharides was performed using a commercial reagent kit (Takara Shuzo Co., Kyoto, Japan). The PA-oligosaccharides were obtained by gel filtration of pyridylaminated products on a Toyopearl HW-40F column (1.0 × 40 cm) and used for detection by fluorescence (excitation = 310 nm; emission = 380 nm).
HPLC AnalysisThe separation of PA-oligosaccharides was carried out by HPLC using a Tosoh CCPM-II pump, a Tosoh PX-8020 controller, and a Shimadzu spectrofluorometric detector, RF-550. Phosphorylated oligosaccharides were fractionated by their size and polarity with Asahipak NH2P-50 (0.46 × 25 cm) (Asahi Chemical Co., Tokyo, Japan) at a flow rate of 1 ml/min. The retention time of oligosaccharide largely depends on the number of sugar residues in the amine-modified column chromatography. Samples were resuspended in buffer A and injected in up to 20-µl aliquots. For analysis of mannosylphosphorylated Man8GlcNAc2-PA, the ratio of 200 mM acetic acid adjusted with triethylamine (pH 7.3) to acetonitrile was 30:70 (v/v) for buffer A and 70:30 (v/v) for buffer B. Initial solvent composition was 80% buffer A with 20% buffer B, and a linear gradient was run over 40 min, in which the percentage of buffer B increased from 20 to 100% with a flow rate of 1 ml/min. For analysis of mannosylphosphorylated Man5GlcNAc2-PA, buffer A contained a 10:90 (v/v) ratio of 200 mM acetic acid adjusted with triethylamine (pH 7.3) to acetonitrile, and buffer B was 100% 200 mM acetic acid adjusted with triethylamine (pH 7.3). The initial solvent of 100% buffer A (0% buffer B) was run for 5 min, and then the percentage of buffer B was linearly increased from 0 to 50% within 30 min; finally, the percentage of buffer B was linearly increased from 50 to 100% for another 15 min with a flow rate of 1 ml/min.
Samples (enzymatic reaction
product or in vivo acidic oligosaccharide product prepared
from och1
mnn1 cells) were dissolved in 8 µl of 0.1 M sodium acetate buffer (pH 5.0). One microunit/2 µl of
-1,2-mannosidase solution (from Aspergillus saitoi;
Oxford Glycosystems, Inc.) was added and then incubated for 15 h
at 37 °C.
Matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) mass spectrometry was performed
in the negative ion mode by using -cyano-4-hydroxy cinnamic acid as
a matrix. The mass spectrometer used in this work was a Finnigan
Lasermat (Finnigan MAT Ltd., Hempstead, United Kingdom). Samples
(100-1000 pmol) were desalted by HPLC using a Tosoh TSK-GEL Carbon-500
column (0.46 × 10 cm). Two solvents, A and B, were used. Solvent
A was water containing 0.1% trifluoroacetic acid. The column was
equilibrated with solvent A. After the sample injection, the proportion
of solvent B was increased linearly up to 100% over 60 min.
PA-oligosaccharides were detected by fluorescence (excitation = 320 nm; emission = 400 nm).
1H NMR spectra of
PA-oligosaccharide were measured on a JNM-A500 (JEOL Co.) at 50 °C.
Samples (~10 nmol) were dissolved in 99.96% D2O and
lyophilized. After three repetitions of the above procedure, the
samples were finally dissolved in 700 µl of 99.996% D2O.
The chemical shifts () are expressed in ppm downfield from internal
sodium 4,4-dimethyl-4-silapentane-1-sulfonate, but they were actually
measured by reference to internal acetone (
= 2.217 ppm).
The original mnn6 mutant exhibited a reduced amount of phosphomannan on the cell wall and showed less binding to Alcian blue, a dye that binds to a phosphate moiety of cell surface mannoproteins (11). The wild type cells exhibited a blue color after staining with Alcian blue, while the mnn6 mutant showed as white. This characteristic was used to clone the wild type MNN6 gene by complementation. Strain TO3-6D (mnn1 mnn6) was transformed with a yeast genomic DNA library constructed in the centromeric vector YCp50, which carries the URA3 as a selective marker. Transformants were selected and maintained on an SD-ura plate. About 1.2 × 104 transformants were screened, and two positive clones (blue) were selected from among a majority of colorless white transformants.
Restriction maps of the insert DNA on the plasmids from two positive
clones were identical. A ~10-kb DNA fragment that can complement the
mnn6 mutation was isolated. To identify the smallest complementing region, further subcloning into pRS316 vector was carried
out. Plasmid pA9 (Fig. 1) contained a ~10-kb
Sau3AI fragment insert at the BamHI site on YCp50
vector. pSA series plasmids ranging from pSA9-1 to pSA9-7 and
pRSMNN6 (Fig. 1) were the subclones derived from plasmid pA9
and inserted into single-copy yeast vector pRS316. These plasmids were
introduced into strain TO3-6D, and the complementation analysis was
carried out by Alcian blue staining. The complementing region was
assigned into a 2.7-kb HpaI-NruI fragment
(pRSMNN6) (Fig. 1). To exclude the possibility of the suppressor gene cloning of mnn6 mutation, strain XW13
(mnn6::ADE2) (Table I and Fig.
4A) was crossed with strain TO3-6D (mnn6), and
after sporulation, tetrads were analyzed by Alcian blue staining. All
of the segregants derived from 20 tetrads showed the mutant phenotype,
which was not able to bind to Alcian blue, demonstrating the gene
disruption at the original mnn6 locus and confirming the
cloning of the MNN6 gene.
MNN6 Is a Member of the KRE2/MNT1 Mannosyltransferase Gene Family
Sequence analysis of a 2.7-kb fragment revealed one open
reading frame with 1338 base pairs, which was translated to a protein of 446 amino acids (MNN6 accession number U43922). From the GenBankTM data base, the MNN6 gene was identical
with the KTR6 gene, which was reported by the genome
sequencing as a family of killer toxin related genes (accession number
U39205). Two potential N-glycosylation sites were found in
the Mnn6p sequence (Fig. 2A). Kyte-Doolittle hydrophobicity analysis showed a potential membrane-spanning region near the N-terminus suggesting a type II membrane protein
(Fig. 2B).
In addition, a homology search of the MNN6 sequence revealed
that Mnn6p shares 38% identity and 79% similarity with Kre2p/Mnt1p, an -1,2-mannosyltransferase responsible for O-linked
glycosylation in yeast (32, 33). Sequence alignment of the Mnn6p and
Kre2 protein families (Ktr1p, Ktr2p, Ktr3p, and Yur1p) (34-36) is
shown in Fig. 3. Six cysteine residue positions in the
latter half of Mnn6p were identical to those of the other proteins,
suggesting a similarity of three-dimensional structures. Interestingly,
Mnn6p has an additional cysteine at the 120th residue, which shares an
identical position with Kre2p, but lacks one cysteine at the 229th
residue, which is commonly located in the other five proteins.
Disruption of the MNN6 Gene Results in Calcoflour White and Hygromycin B Sensitivities
To study the function of Mnn6p, the
MNN6 gene was disrupted by inserting the ADE2
gene (Fig. 4). MNN6 gene disruption did not
affect the cell morphology and the rate of cell growth, indicating a
nonessential gene for normal cell growth. The mnn6 null
(mnn6) mutant displayed the same phenotype as the
original mnn6 mutant, which provides a prominent loss of
Alcian blue binding ability, while the isogenic wild type cells
strongly bound the dye. A single copy plasmid containing the
MNN6 gene restored the Alcian blue binding, which was lost
in the
mnn6 mutant (data not shown), suggesting the
involvement of MNN6 in oligosaccharide phosphorylation. A
sensitivity to a negatively charged fluorescent dye CFW and an
aminoglycoside antibiotic hygromycin B was examined. As shown in Fig.
4B, the
mnn6 mutant was sensitive to CFW and
hygromycin B, while the isogenic wild type was not affected. A single
copy of the MNN6 gene recovered the growth defect of the
mnn6 mutant by CFW and hygromycin B, respectively. Since
CFW binds to nascent chains of chitin and prevents both microfibril
formation and cell wall assembly (37), the result may suggest a lesser
charge repulsion between CFW and the cell surface in
mnn6
mutant.
The original
mnn6 mutant was deficient in the mannosylphosphate addition
to the mannose outer chain (15). This is supported by the Alcian blue
staining of the isogenic pairs with or without the MNN6 gene
(mnn1 cells and
mnn1
mnn6 cells in Table
II, G-1). To further examine whether the MNN6
gene may affect phosphorylation of the oligosaccharide lacking a
mannose outer chain, other isogenic pairs of double and triple mutant
cells (
och1
mnn1 and
och1
mnn1
mnn6) were constructed. The
och1
mnn1 cells showed a
significant dye binding, while the
och1
mnn1
mnn6
cells failed to bind the dye (Table II, G-2). The introduction of a
multicopy plasmid containing the MNN6 gene
(pETMNN6) into
och1
mnn1
mnn6 cells
recovered the dye binding ability, but the effect of multicopy gene
dosage was not observed on Alcian blue staining (Table II, G-2). These
results suggest that the MNN6 gene may be involved in the
phosphorylation in vivo not only at the outer chain portion
but also the N-linked core and/or O-linked
oligosaccharides. In contrast, the Alcian blue staining was not changed
by the introduction of the
mnn6 mutation into
och1
mnn1
kre2 cells, which produces the
N-linked core oligosaccharide (Man8GlcNAc2) (31) and
truncated O-linked chains (Man2) (32), suggesting the
possibility of no apparent effect of the MNN6 gene on
N-linked core oligosaccharide phosphorylation in
vivo.
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The assay
conditions for the mannosylphosphate transferase were established by
using 1 mM GDP-mannose as a donor and 50 pmol of
pyridylaminated core oligosaccharide Man8GlcNAc2-PA (M8-PA; see
structure shown in Fig. 8, A-1) as an acceptor in 50 µl of reaction mixture (see "Experimental Procedures"). Under these assay
conditions, microsomal membranes from MNN6 wild type cells (och1
mnn1, strain YS125-15B) showed two reaction
products (peaks 1 and 2) (Fig. 5, A-1), which
were already identified as a monomannosylphosphorylated Man8GlcNAc2-PA
(ManP-M8-PA) (16). In contrast, microsomal membranes from isogenic
mnn6 cells (
och1
mnn1
mnn6, strain
XW27) diminished corresponding peaks (Fig. 5, A-2), and the
enzyme activity was restored after the introduction of the
MNN6 gene into
mnn6 cells (Fig. 5,
A-3), indicating more directly the involvement of the MNN6 gene in the mannosylphosphate transferase activity
toward Man8GlcNAc2-PA in vitro. However, introduction of
MNN6 in a multicopy plasmid did not produce any higher
enzymatic activities in wild type cells (Fig. 5, A-4),
suggesting the presence of some limiting factors for this enzyme
reaction.
Furthermore, the enzyme activity toward the N-linked
core-like oligosaccharide derivative Man5GlcNAc2-PA (prepared from
och1
mnn1
alg3 cells, 25 pmol) (see structure shown
in Fig. 8, A-2), was also compared by using microsomal
membranes prepared from isogenic pairs of
mnn1 cells
(strain YS131-30A) and
mnn1
mnn6 cells (strain YS131-30D). The MNN6 cell exhibited the enzyme activity
toward the Man5GlcNAc2-PA (M5-PA) acceptor and gave the reaction
product of peak 3 (Fig. 5, B-1). This reaction product was
identified as a monomannosylphosphorylated Man5GlcNAc2PA
(ManP-M5-PA) by alkaline phosphatase digestion and subsequent mild acid
treatment (data not shown). Disruption of the MNN6 gene
diminished mannosylphosphate transferase activity toward M5-PA
(Fig. 5, B-2), indicating the involvement of MNN6
in mannosylphosphorylation of core-like oligosaccharide Man5GlcNAc2.
Taken together, these data strongly suggest that the MNN6 gene may encode a mannosylphosphate transferase, which is involved in the oligosaccharide phosphorylation not only of the mannose outer chain but also of the N-linked core portion.
Characterization of Enzymatic Reaction ProductsTo determine
the structure of reaction products, peaks 1 and 2 in Fig. 5 were
analyzed by MALDI-TOF mass spectrometry. The molecular ion peaks were
observed at m/z 2048.9 for peak 1 and 2042.5 for
peak 2, respectively. These mass values were nearly identical to the
molecular mass of ManP-M8-PA (calculated Mr
2041.8). The 1H NMR spectra of peak 2 show the
mannosylphosphate signal at 5.44 (Fig.
6B). The intensity of this signal indicates
the presence of one mannosylphosphate group in peak 2. Measurement of
1H NMR spectra of peak 1 was not successful due to the loss
of material during the purification process.
Since the core-like oligosaccharide has two mannosylphosphorylation
sites (38), two structures of ManP-M8-PA are possible. One is the
structure in which mannosylphosphate attaches to the side of the
-1,6-branch of core Man8GlcNAc2 (structure I); the other is the
structure in which mannosylphosphate attaches to the side of
-1,3-branch of the same Man8GlcNAc2 (structure II). By
-1,2-mannosidase digestion, structure I releases three mannoses and
yields ManP-M5-PA, but structure II releases two mannoses and yields
ManP-M6-PA. The
-1,2-mannosidase digestion products of peaks 1 and 2 were analyzed by MALDI-TOF mass spectrometry. The molecular ion peak of
each product was detected at m/z 1724.7 (ManP-M6-PA, calculated Mr 1717.5) for peak 1 and 1558.9 (ManP-M5-PA, calculated Mr 1555.4)
for peak 2, respectively. These results revealed the site of
monomannosylphosphorylation at Man8GlcNAc2. Peak 1 product was
mannosylphosphorylated at the
-1,3-branch of core Man8GlcNAc2
(structure II) and peak 2 product was mannosylphosphorylated at the the
-1,6-branch of core Man8GlcNAc2 (structure I).
Further to examine the effect of
MNN6 gene disruption on the mannosylphosphate addition in
the N-linked core portion, we analyzed the oligosaccharides
of mannoproteins prepared from och1
mnn1 (strain
YS126-47D) and
och1
mnn1
mnn6 (strain XW43) cells. In HPLC analysis, a peak of neutral M8-PA, two peaks of ManP-M8-PA (peaks
1 and 2), and unknown peak x were observed in
och1
mnn1 cells (Fig. 7). Unexpectedly,
the same oligosaccharide pattern was observed in
och1
mnn1
mnn6 cells (data not shown). Especially, the ratio of peak area corresponding to ManP-M8-PA to neutral M8-PA was
not changed in both cells. These results suggested the presence of
functionally redundant gene(s), which may interfere the appearance of
mutant phenotype due to MNN6 gene disruption in
vivo.
We also analyzed the structure of peak x, which was eluted
at 39.5 min in Fig. 7. The molecular ion peaks were observed at m/z 2294.7, which was nearly identical to the
molecular mass of dimannosylphosphorylated Man8GlcNAc-PA (calculated
Mr 2283.9). 1H NMR spectra of peak
x showed a mannosylphosphate signal at 5.44, and the
intensity of this signal indicated the presence of two
mannosylphosphate groups (Fig. 6C). To determine the
mannosylphosphorylation sites, the
-1,2-mannosidase digestion
product of peak x was first subjected to time-of-flight mass
spectrometry analysis. However, the molecular ion peak was not
observed, presumably due to the increase of negative charge in the
molecule. Then the above digestion product was subjected to mild acid
treatment followed by alkaline phosphatase to convert to neutral
PA-oligosaccharide. This product showed a retention time of 9.5 min in
amino column HPLC (Asahipack NH2P-50), which was identical
to the authentic Man6GlcNAc2-PA (Takara PA-sugar chain 018) (data not
shown). Therefore, the sites of two mannosylphosphates in peak
x were confirmed as shown by the star in Fig.
8, A-1.
We have reported the cloning and analysis of the MNN6 gene. For the cloning, the original Alcian blue dye binding assay in a test tube was not appropriate for the colony screening due to the laborious work. To solve this problem, a modified procedure for Alcian blue staining was developed on plates. The method established in this work should be applicable to clone other yeast genes, especially genes related to the biosynthesis of cell wall components.
Sequence analysis of the MNN6 gene predicted a type II
membrane protein with 446 amino acids, which is highly homologous to -1,2 mannosyltransferase, Kre2p/Mnt1p, involved in
O-glycosylation in S. cerevisiae (32, 33). A
significant homology between Mnn6p and KRE2/MNT1 gene family
proteins suggests the presence of similar domain involved in the
recognition of a common structure of the acceptor (
-1,2-linked
mannobiose) recognized by all of these enzymes. The functional
relations between Mnn6p and Kre2p/Mnt1p will be investigated in future
work.
Disruption of MNN6 caused a hypersensitivity to CFW and hygromycin B (Fig. 4). The former phenotype is caused by the loss of charge repulsion between the cell surface and drug, leading to the penetration of drug through the outermost mannoprotein portion in the cell wall. It is noteworthy that hygromycin B-sensitive mutants involve not only the defects in sugar chain length, as reported (39, 40), but also in oligosaccharide phosphorylation, although its mechanism is still unclear.
We have shown that MNN6 is involved in core oligosaccharide
phosphorylation by demonstrating the loss of mannosylphosphate transferase activity in vitro toward Man8GlcNAc2 and
Man5GlcNAc2 in mnn6 cells. Two reaction products (peaks 1 and 2) corresponding to ManP-M8-PA were identified when M8-PA was used
as an acceptor. The mannosylphosphorylation site was determined by
time-of-flight mass spectrometry after the
-1,2-mannosidase
treatment. These sites were identical to the phosphorylation sites
observed in dimannosylphosphorylated oligosaccharide in vivo
(peak x compound in Fig. 7) described in this paper and to
those reported for the N-linked core-like Man10GlcNAc2
oligosaccharide from carboxypeptidase Y and mnn1 mnn9 strain
mannoproteins (38, 41). When M5-PA was used for acceptor substrate,
only one peak corresponding to monomannosylphosphorylated product was
observed (Fig. 5, peak 3, B-1). Although the
phosphorylation site could not be determined by
-1,2-mannosidase
treatment due to the limited amount of purified material, based on
combined results on both the phosphorylation sites determined for
ManP-M8-PA in vitro and the structure of dimannosylphosphorylated oligosaccharide determined in vivo,
the most reasonable phosphorylation site in ManP-M5-PA is shown in Fig.
8 (A-2).
Apparently, -1,2-linked mannotriose
(Man
1,2Man
1,2Man) (mannose residue for the
phosphorylation is shown in boldface type) is a common structure for
the phosphorylation of Man8GlcNAc2, Man5GlcNAc2, and mannose outer
chain branch. Consistent with the previous result (15), we found that
mnn6 mutant diminished the enzyme activity toward the
-1,2-linked mannotriose (Man
1,2Man
1,2Man), which
mimics both the N-linked outer chain branch and the
O-linked oligosaccharides (data not shown). As described,
mnn6 also showed a defect in the core oligosaccharide
phosphorylation in vitro at the site of the
-1,6-branch
(Man
1,2Man
1,6Man) (Fig. 8, A-1). Based on
these data, we have proposed that MNN6 is involved in the
phosphorylation of any oligosaccharides containing at least one
-1,2-linked mannobiose (Fig. 8B). In mammalian cells,
GlcNAc-1-phosphate transferase showed a requirement for the presence of
at least one Man
1,2Man sequence on the glycoprotein acceptor (42).
Since the acceptor sites for the core oligosaccharide are identical in
yeast and mammals (38), the mannosylphosphate transferase in yeast may
share some similar acceptor requirements with the mammalian
GlcNAc-1-phosphate transferase.
Oligosaccharide profiles of total mannoproteins were compared between
och1
mnn1 and
och1
mnn1
mnn6 cells.
Disruption of MNN6 did not exhibit any significant defect in
the oligosaccharide phosphorylation in vivo (data not
shown), consistent with the Alcian blue staining of the cells (Table
II, G-3), suggesting the presence of redundant enzymes required for
N-linked core oligosaccharide phosphorylation. Since the
MNN6 is a member of the KRE2/MNT1 gene family, it
is most likely that some genes in this family may function as
homologues. Disruption of MNN6 in the
mnn1
cells caused a dramatic loss of Alcian blue staining (Table II),
indicating the involvement of MNN6 in the phosphorylation of
N-linked outer chain in vivo. Since
O-linked sugar chains are also phosphorylated (43), MNN6 may also affect the O-linked oligosaccharide
phosphorylation.
Phosphorylated oligosaccharides are also found as a component of lipid
glycoconjugate, lipophosphoglycan, on the cell surface of the
protozoan, Leishmania (13, 44). Phosphoglycan polymer contains the repeating disaccharide unit
(Gal(1,4)Man
1-PO4-6) attached to a conserved core,
which in turn is linked to an unusual phosphatidylinositol-lipid
anchor. The repeating units are synthesized by the alternating transfer
of mannose 1-phosphate and galactose from GDP-mannose and
UDP-galactose, respectively (13). Since these reactions involve
transfer of mannose 1-phosphate from GDP-mannose, this study may also
contribute to the understanding of other mannosylphosphate transferases, such as those in Leishmania.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U43922.
We are grateful to T. Odani for the construction of strain TO3-6D, K. Ichikawa for the establishment of enzyme assay conditions, and Dr. T. Nishida for the construction of pET351 plasmid vector. X.-H. Wang thanks Mr. T. Odani and Dr. J. L. Horecka for helpful suggestions and discussion.