(Received for publication, October 30, 1996, and in revised form, March 5, 1997)
From the Department of Immunoregulation, Research Institute for
Microbial Diseases, Osaka University, Osaka 565, Japan and the
Division of Cancer Biology, Department of Radiation
Oncology and Department of Biochemistry, Emory University School of
Medicine, Atlanta, Georgia 30335
Many eukaryotic cell surface proteins are bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Several genes involved in GPI anchor biosynthesis have been cloned using complementation of mutant mammalian cell lines and yeasts that are defective in its biosynthesis pathway. However, the gene involved in the second step of this pathway, in which N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is N-deacetylated to form glucosaminyl (GlcN)-PI, has not been cloned. In this study, we established a GPI anchor-deficient mutant of Chinese hamster ovary (CHO) cells defective in the second step. Complementation analysis with the known GPI anchor mutant cells demonstrated that it belonged to the same complementation group as the CHO cell mutant G9PLAP.85. Using the new mutant, we cloned a rat gene termed PIG-L (for phosphatidylinositol glycan class L) that is involved in this step. PIG-L encodes a 252-amino acid, endoplasmic reticulum membrane protein, most of which is in the cytoplasmic side. This orientation of PIG-L protein is consistent with the notion that the second step of GPI anchor biosynthesis occurs on the cytoplasmic side of the endoplasmic reticulum.
Many eukaryotic cell surface proteins are bound to the membrane by a carboxyl-terminal linkage to a glycosylphosphatidylinositol (GPI)1 anchor (1, 2). The conserved core of this glycolipid consists of a lipid containing inositol (usually phosphatidylinositol (PI)), a glucosamine, three mannose residues and phosphoethanolamine. At least 50 GPI-anchored proteins with a wide variety of functions have been identified in mammals, including cell-surface hydrolytic enzymes, receptors, adhesion molecules, complement inhibitors, and antigens of unknown functions. GPI anchors are also frequently used in many protozoa and yeasts (3).
GPI anchor synthesis occurs in the endoplasmic reticulum (ER) and essentially consists of the sequential addition of sugar components and phosphoethanolamine to PI. Several mutant mammalian cell lines defective in this biosynthesis pathway have been very useful in studying that of mammals. Three complementing mutants (classes A, C, and H) are known in the first reaction, which is the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to PI to form GlcNAc-PI (4, 5). Genes for these mutants have been characterized: PIG-A (6), PIG-H (7), and PIG-C (8) for class A, H and C, respectively. In the second step, GlcNAc-PI is N-deacetylated to form GlcN-PI. Although two mutant cell lines are known in this step (9, 10), the genes involved have not been cloned. Following inositol acylation of GlcN-PI (11), three mannoses are added to the GPI core. All of these mannoses are derived from dolichol phosphomannose (12-14). The class B mutant is defective in the third mannosylation (5, 15), and the responsible gene, PIG-B, has been cloned (16). In the final step, phosphoethanolamine is transferred to the core, which contains three mannose residues (17). Class F is defective in this step (5, 18), and PIG-F (19, 20), which complements the class F mutant, has been cloned.
Mature GPI anchor precursors are post-translationally linked to proteins in the ER. Proteins that are to be GPI-anchored have at their carboxyl terminus a signal sequence that directs GPI anchor addition (2). The signal peptide is exchanged with the GPI anchor by transamidation, forming an amide linkage between the new carboxyl terminus and ethanolamine (21). Class K mutant cells synthesize the mature GPI anchor precursors but are defective in the transamidation step (9, 22). The gene for the class K mutation has not been cloned. The yeast Saccharomyces cerevisiae mutants gaa1 (23) and gpi8 (24) have similar defects to those of the mammalian class K mutant, indicating that at least two genes are involved in this step.
Most mammalian genes involved in GPI anchor biosynthesis have been cloned by means of expression cloning using mutant cell lines. To further elucidate the pathway, it is necessary to create new mutants. In this study, we established a mutant from Chinese hamster ovary (CHO) cells that were stably transfected with human decay-accelerating factor (DAF) and CD59 cDNAs to introduce known GPI-anchored proteins. This mutant was of the same complementation group as the mutant G9PLAP.85 (10), which is defective in the second step of the pathway, the deacetylation of GlcNAc-PI. The new mutant had no background expression of GPI-anchored protein and allowed us to clone a gene involved in this step.
The CHO-K1 cell mutant G9PLAP.85,
defective in the second step of GPI anchor biosynthesis, and its
wild-type G9PLAP (CHO cell transfected with human placental alkaline
phosphatase), were as described (10). CHO-K1 cells, their mutants and
transfectants were cultured in Ham's F-12 medium supplemented with
10% (v/v) fetal calf serum. The Thy-1-deficient murine lymphoma cell
lines BW5147(Thy-1a), S1A(Thy-1
b),
T1M1(Thy-1
c), BW5147(Thy-1
e),
EL4(Thy-1
f), and S49(Thy-1
h) (25) (all
provided by Dr. R. Hyman, Salk Institute, San Diego, CA) were cultured
in Dulbecco's modified Eagle's medium supplemented with 10% (v/v)
fetal calf serum.
A plasmid containing DAF and CD59 cDNA was constructed using
pME18sf (a gift from Dr. K. Maruyama) (26), pKP-CD59 and
SR
-DAF. CD59 cDNA (27) was subcloned into pKP3 (a gift from Dr.
H. Nojima), a modified vector of pcD2, to make pKP-CD59. DAF cDNA
provided by Dr. M. A. Davitz (28), was subcloned into
pME18sf
to make SR
-DAF. A 1500-bp
SfiI-SfiI fragment containing the neomycin
resistance gene was obtained from pKP-CD59 and ligated into the
SfiI site of pME18sf
to make pME-Neo. Next, a
450-bp fragment containing CD59 coding sequence was obtained also from
pKP-CD59, which was digested with Asp718, blunt-ended, then
digested with EcoRI. Plasmid pME-Neo-CD59 was constructed by
ligating this fragment with XbaI-digested, blunt-ended, and
EcoRI-digested pME-Neo vector. Finally, a 2900-bp HindIII-SmaI digested and blunt-ended fragment
containing Neo and CD59 was removed from pME-Neo-CD59 and ligated into
SmaI site of SR
-DAF. This construct was named pDNC (for
pME-DAF-Neo-CD59).
A rat cDNA library prepared from the rat C6 glioma in an expression vector pMEPyori (20) was a gift from Dr. Y. Maeda. This library was prepared from oligo(dT)-primed, size-selected (>1.5 kb) cDNA and consisted of about 6 × 105 independent clones.
A fragment containing PyT but lacking the polyoma origin of replication
was obtained from pdl3027 (29) by digestion with BamHI,
subcloned into the BamHI site of pBluescript II (pBSII) vector (Stratagene, La Jolla, CA), and then removed by
EcoRV/SpeI digestion. The fragment was ligated
into NruI/AvrII site of pcDNA I vector
(Invitrogen, NV Leek, The Netherlands) to prepare
pcDNA-PyT(ori).
Plasmids were transfected into 1.2 × 107 cells suspended in 0.8 ml of HEPES-buffered saline (30) by electroporation at 350 V and 960 microfarads using a Gene-Pulser (Bio-Rad).
Complementation AnalysisComplementation analysis with somatic cell fusion and immunofluorescence staining proceeded as described (31).
Cloning of PIG-L cDNAA sample of 440 µg of the
cDNA library plasmids was cotransfected with 440 µg of
pcDNA-PyT(ori) plasmids into M2S2 cells (see
"Results") (total 1.4 × 108 cells) by
electroporation. Two days later, transfected cells were double-stained
with anti-CD59 monoclonal antibody 5H8 (a gift from Drs. M. Tomita and
Y. Sugita, Showa University, Tokyo, Japan) in combination with
fluorescein-conjugated sheep anti-mouse IgG (Organon Teknika Corp, West
Chester, PA) and biotinylated anti-DAF monoclonal antibody IA10 (32) in
combination with phycoerythrin-conjugated streptavidin (Biomeda, Foster
City, CA), and sorted using FACS-Vantage (Becton Dickinson, Mountain
View, CA). From the 707 DAF- and CD59-positive cells sorted, 1350 independent plasmid clones were recovered as described by Hirt (33) and
by transformation into Escherichia coli MC1061. Pooled
plasmids were transfected again into M2S2 cells, and the cells were
analyzed for DAF and CD59 expression. DAF and CD59 expression was
restored in about 10% of the cells. These pooled plasmids were
transfected into M2S2 cells and selected again. Plasmids obtained from
the sorted cells were cloned into 96-well plates and screened as
described (16) to identify those that restore DAF/CD59 expression on
M2S2 cells.
The
3-kb SmaI-SmaI fragment containing SR promoter
and PIG-L cDNA was removed from pMEPyori-PIG-L and ligated into the
SmaI site of pBSII-PGK-Hyg (34) to make pBSII-PGK-Hyg-PIG-L.
This construct was transfected into M2S2 and G9PLAP.85 cells, which were selected with 200 µg/ml hygromycin. Finally, clones that permanently restored GPI anchor biosynthesis were analyzed.
Biosynthesis of GPI intermediates from UDP-[6-3H]GlcNAc (1 µCi) by microsomes was measured as described (10).
Northern Blot Analysis of PIG-LA rat PIG-L cDNA probe, the 0.7-kb SalI/XbaI fragment of pMEEB-FLAG-PIG-L (see below), was labeled with 32P by the random primer labeling method using High Prime (Boehringer Mannheim, Mannheim, Germany). To prepare a hamster PIG-L cDNA probe, we synthesized degenerated primers each corresponding to nucleotides 541-563 and complementary to nucleotides 820-842 of rat PIG-L cDNA and amplified a specific fragment of predicted size (302 bp) from reverse-transcribed CHO cell cDNA that was synthesized from total RNA of IIIB2A cells using oligo(dT) primer. This fragment was cloned, sequenced, and radiolabeled.
Expression and Analysis of PIG-L and Its FLAG-tagged ProteinsWe fused FLAG (Eastman Kodak Co.) as a tag to the amino
terminus of PIG-L as follows. We amplified the PIG-L gene
from pMEPyori-PIG-L by PCR using a 5 primer
(5
-TGTCGACGAAGTGGTGGGTCTCTTG-3
) containing a SalI site
instead of the start codon, and a 3
primer
(5
-AGCAGGTCAGTCAGCCCAGGTCT-3
). The PCR product was subcloned into an
EcoRV site of pBSII vector, digested with
SalI/XbaI to obtain a fragment containing PIG-L, which was then ligated into a SalI/XbaI site of
pMEEB-FLAG-PIG-A (26) to exchange PIG-A for PIG-L. We termed this
construct pMEEB-FLAG-PIG-L.
To fuse FLAG to the carboxyl terminus of PIG-L, we amplified
PIG-L gene from pMEPyori-PIG-L by PCR using a 5 primer
(5
-CAGTTTCAGCGGGTTCAGTTTTC-3
) and a 3
primer
(5
-TTTACTTGTCATCGTCGTCCTTGTAGTCTCGCGAGAGCAACTGCAGCGAGTT-3
), with which we inserted an NruI site and FLAG sequences
between the PIG-L carboxyl terminus and the stop codon. The PCR product was subcloned into the EcoRV site of pBSII vector, digested
with ClaI/XbaI to obtain the fragment containing
PIG-L-FLAG, which was then ligated into a
ClaI/XbaI site of pMEEB (26). We termed this
construct pMEEB-PIG-L-FLAG.
These constructs were transfected into M2S2 cells, which were selected with hygromycin to establish stable transfectants. FLAG-tagged PIG-L extracted from cells with 1% Nonidet P-40 were Western-blotted against biotinylated anti-FLAG (M2) antibody (Kodak) and alkaline phosphatase-conjugated streptavidin (Life Technologies, Inc.), then developed using the Western-Light chemiluminescent detection system (Tropix, Bedford, MA). Protein-disulfide isomerase (PDI) was detected by Western blotting with rabbit anti-PDI antibodies (a gift from Drs. R. Masaki and A. Yamamoto, Kansai Medical University, Osaka, Japan) and alkaline phosphatase-conjugated goat anti-rabbit IgG antibodies (Organon Teknika Corp). Blots were quantified using a model GS-525 Molecular Imager system (Bio-Rad).
Immunofluorescence Microscopic Analysis of PIG-LM2S2 cells stably transfected with pMEEB-PIG-L-FLAG or pMEEB-FLAG-PIG-L were cultured on 14-mm diameter glass coverslips in 24-well plates for 24 h, washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, and permeabilized with 0.1% Triton X-100 in PBS for 1 h. Thereafter, they were incubated with Block Ace (Dainippon Pharmaceutical, Osaka, Japan) for 1 h, then stained with anti-FLAG antibody (Kodak), Rhodamine-conjugated donkey anti-mouse IgG antibodies (Chemicon International, Temecula, CA), rabbit anti-PDI antibodies, and fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG antibodies (Chemicon International). They were mounted on glass slides with Gel/Mount (Biomeda) and studied under a fluorescence microscope (BX50-FLA; Olympus Corp., Tokyo, Japan).
Isolation of Subcellular FractionsM2S2 cells (4.8 × 107) stably expressing PIG-L-FLAG fusion proteins were
resuspended in a buffer containing 0.25 M sucrose, 10 mM HEPES-NaOH, pH 7.5, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and
0.1 mM
N-p-tosyl-L-lysine
chloromethyl ketone, disrupted by means of nitrogen cavitation (50 p.s.i. N2 pressure for 15 min) and a tight pestle Dounce
homogenizer, and digested with 2 units/ml DNase I for 20 min at
4 °C. After centrifugation at 105 × g for 15 min at 4 °C, the post-nuclear supernatants were fractionated by
discontinuous sucrose gradient centrifugation as described (16).
Microsomes of M2S2 cells (equivalent to 5 × 107 cells), stably expressing PIG-L-FLAG or FLAG-PIG-L were prepared by centrifugation of the post-nuclear supernatants at 105 × g for 1 h at 4 °C, followed by resuspension of the pellets in 1 ml of buffer containing 0.25 M sucrose and 10 mM HEPES-NaOH, pH 7.5. These microsomes were incubated with 500 µg/ml proteinase K at 4 °C for 30 min. The proteinase K was inactivated by phenylmethylsulfonyl fluoride (10 mM), and then the microsomes were solubilized with 1% Nonidet P-40. To confirm the susceptibility of the substrate proteins to the enzyme, the membranes were solubilized with Nonidet P-40 and incubated with proteinase K in parallel. After centrifugation at 105 × g for 10 min, supernatants were Western blotted with anti-FLAG (M2) antibody, rabbit anti-PIG-L peptide antibodies (against amino acids 111-125) (Peptide Institute, Inc., Osaka, Japan), and anti-PDI antibodies.
To
establish new mutant cells, we first transfected a plasmid containing
both DAF and CD59 cDNA (pDNC) into CHO-K1 cells. After selection in
600 µg/ml G418, cells stably expressing DAF and CD59 were isolated by
sorting twice with a cell sorter and cloned by limiting dilution. One
colony, designated IIIB2A, expressed high levels of both DAF and CD59
(Fig. 1A), so we selected it for further
study.
We mutagenized IIIB2A cells with ethyl methanesulfonate (100 µg/ml) for 24 h. After a 48-h recovery period, cells that no longer expressed DAF and CD59 on their surface were selected using a cell sorter. This sorting was repeated once more before cloning by limiting dilution. Forty-six clones were recovered and 13 were classified by fusion with GPI anchor-deficient murine lymphoma cells of various complementation groups and mutant CHO clone G9PLAP.85. All 13 clones were of the same complementation group as G9PLAP.85, the mutant with the GPI biosynthetic defect in the deacetylation of GlcNAc-PI. The rest of the mutant clones also seemed to be of the same group, because fusion of the bulk mutant cells with G9PLAP.85 did not complement GPI biosynthesis. We termed this complementation group class L (see "Discussion"). One of the clones, designated M2S2, was selected for subsequent study because it had no residual DAF and CD59 staining when analyzed by FACS (Fig. 1B).
Expression Cloning of PIG-L cDNAWe obtained a cDNA
that complements the deficiency of M2S2 cells by expression cloning. A
rat C6 glioma cDNA library constructed in a vector
bearing a polyoma origin of replication was cotransfected with
pcDNA-PyT, which lacks this origin of replication
(pcDNA-PyT(ori)) and screened for clones that
complement DAF and CD59 expression on the M2S2 cells. We obtained three
clones of essentially the same lengths and restriction profiles, which
were thought to be derived from the same clone. We chose one for
further studies. This clone restored the surface expression of DAF and
CD59 on M2S2 cells (Fig. 1, A-C) and human placental
alkaline phosphatase (PLAP) expression on G9PLAP.85 cells (Fig. 1,
D-F).
To confirm that cloned cDNA complements the deficient deacetylation
of GlcNAc-PI, M2S2 and G9PLAP.85 cells stably expressing the cDNA
were established and the synthesis of GlcNAc-PI, GlcN-PI, and
GlcN-PI(acyl) from UDP-[6-3H]GlcNAc was measured in
lysates prepared from these cells. As shown in Fig. 2,
GlcNAc-PI, GlcN-PI, and GlcN-PI(acyl) were synthesized in the wild-type
cell lines, G9PLAP and IIIB2A (lanes 1 and 4, respectively), while only GlcNAc-PI was synthesized in the mutant type
cell lines, G9PLAP.85 and M2S2 (lanes 2 and 5,
respectively), indicating a deficiency in GlcNAc-PI deacetylation.
Transfectants expressing the cDNA synthesized GlcN-PI and
GlcN-PI(acyl) as well as GlcNAc-PI similarly to the wild-type cells
(lanes 3 and 6). Therefore, deacetylation of
GlcNAc-PI was restored by expressing the cloned cDNA. Extent of
deacetylation in the transfectants (lanes 3 and
6) were higher than those in the wild type cells (lanes 1 and 4), indicating that overexpression
of PIG-L induced deacetylation activity higher than normal.
We thus cloned the target cDNA and termed the gene PIG-L for phosphatidylinositol glycan of complementation class L.
Characteristics of PIG-L cDNA and mRNAThe PIG-L
cDNA consisted of 1903 base pairs and the longest open reading
frame spanning nucleotides 391-1149, encoded a predicted protein of
252 amino acid residues (Fig. 3A)
(DDBJ/EMBL/GenBank accession No. [GenBank]). The sequence around the
initiation codon agrees well with the Kozak consensus sequence. A
hydrophobicity plot revealed an amino-terminal hydrophobic sequence
(Fig. 3B). This may be used as a signal peptide or
alternatively, to attach to the membrane (see below).
The Northern blot analysis of PIG-L mRNA in C6 glioma
cells demonstrated a major 1.9-1.7-kb message and a minor 4.9-kb
message (Fig. 4, lane 1). The cloned PIG-L
cDNA may correspond to the major message. To analyze PIG-L mRNA
in wild-type and mutant CHO cells, we amplified and cloned a fragment
of hamster PIG-L cDNA of 302 bp that encoded a part of hamster
PIG-L protein having 88% amino acid identity with rat PIG-L protein.
Using this cDNA as a probe, we analyzed wild-type IIIB2A and class
L M2S2 CHO cells with Northern blotting (Fig. 4, lanes 2 and
3). The wild-type cells had 3.6-, 2.4-, 1.9-, and 1.3-kb
messages (lane 2). The class L mutant lacked 3.6-, 1.9-, and
1.3-kb messages and expressed only a small amount of 2.4-kb messages
(lane 3). Therefore, defective deacetylation in the mutant
is due to PIG-L mRNA abnormality. Since four messages are
simultaneously affected in the mutant, it is likely that they are the
products of single PIG-L gene. For both C6
glioma and CHO cells, total RNA samples did not give a signal,
indicating that PIG-L mRNAs are minor components.
Comparison of PIG-L Sequence with Other Known Nucleotide and Protein Sequences
There was no overall homology between PIG-L and
known deacetylases such as acetylornithine deacetylase,
N-acetylglucosamine-6-phosphate deacetylase, and
glucosaminoglycan N-acetylglucosaminyl
N-deacetylase/N-sulfotransferase (35-42). A
Basic Local Alignment Search Tool (BLAST) search (43) revealed that an
S. cerevisiae open reading frame YM8021.07 (GenBank accession number: [GenBank]) (44) and a human EST [GenBank] (45) gave high
homology scores with PIG-L amino acid sequence (Fig. 5).
The S. cerevisiae open reading frame YM8021.07 encoded a
putative 304-amino acid protein with 24% amino acid identity with
PIG-L. Human EST [GenBank] gave a sequence of 87 amino acids that had 52%
amino acid identity with a segment of PIG-L near the amino terminus.
When the most similar portions were compared (56 amino acids), the
identity increased to 73%. They may be yeast and human homologues of
the PIG-L gene, respectively.
ER Localization and Membrane Orientation of PIG-L Protein
To
localize intracellular expression site of PIG-L, its amino or carboxyl
terminus was tagged by FLAG and the fusion constructs, FLAG-PIG-L and
PIG-L-FLAG, respectively, were stably expressed in M2S2 cells. The
fusion proteins were active as shown by complementation of the surface
expression of DAF and CD59 (data not shown). The cells transfected with
FLAG-PIG-L were fixed, permeabilized with Triton X-100, probed with
anti-FLAG antibody, and observed using indirect immunofluorescence.
Cell surface and nuclei were not stained, whereas the intracellular
compartment was reticularly or uniformly stained (Fig.
6A), suggesting that the ER was stained. The
staining profile of FLAG-PIG-L in red almost completely
coincided with that of the ER protein PDI in green (Fig.
6B). Therefore, PIG-L protein is localized in the ER. The
cells transfected with PIG-L-FLAG gave a similar result (data not
shown).
To further confirm this, cells expressing PIG-L-FLAG were disrupted and fractionated into the ER, Golgi, plasma membranes, and cytoplasm by sucrose density gradient centrifugation. The fusion proteins were detected as a 30-kDa band by Western blotting against anti-FLAG antibody mainly in fractions 3 and 4, which contained the ER, indicating that PIG-L protein is an ER membrane protein (Fig. 6, C and D).
We then investigated the orientation of PIG-L protein in the membrane.
Cells expressing FLAG-PIG-L and those expressing PIG-L-FLAG were
disrupted to obtain membrane vesicles bearing PIG-L-FLAG or FLAG-PIG-L.
These vesicles were digested with proteinase K, followed by Western
blotting using anti-FLAG antibody or anti-PIG-L peptide antibodies
against amino acids 111-125. As shown in Fig. 7,
PIG-L-FLAG and FLAG-PIG-L were not protected against proteinase K
digestion when visualized using an anti-FLAG antibody (lanes 2 and 4 in upper panel), whereas ER lumenal
protein PDI was protected (bottom panel). The results were
similar with the anti-PIG-L peptide antibodies (middle
panel). Therefore, most of the PIG-L protein is located on the
cytoplasmic side of the ER.
There was no difference in size between PIG-L-FLAG and FLAG-PIG-L when visualized using an anti-FLAG antibody (Fig. 7, upper panel). This indicated that the amino-terminal hydrophobic portion was not cleaved as a signal peptide, and is rather used for attachment to the membrane.
In the present study, we cloned and characterized a novel gene that is involved in the second step in GPI anchor biosynthesis. Since expression cloning approach has been useful for cloning mammalian GPI anchor biosynthesis genes (46), we prepared a new GPI anchor-deficient mutant line of CHO cells that is defective in the second step and cloned a rat cDNA that complemented the defect. This mutant belonged to the same complementation group as the CHO mutant G9PLAP.85 that is also defective in the second step (10). We termed this complementation group class L and the cloned gene PIG-L.
A human K562 erythroleukemic cell line that is defective in the second step in GPI anchor synthesis has been termed a class J mutant (9). A gene corresponding to class J has not been cloned. We assigned a new complementation class for our mutants that are defective in the same step as the class J mutant. This is because class J mutant is now not available for comparison with our class L mutants. Therefore, the assignment of two complementation groups at the same step does not mean that two genes are involved in that step.
The second step in GPI anchor biosynthesis is deacetylation of the first intermediate GlcNAc-PI to generate GlcN-PI. Microsomes prepared from class L mutants accumulated the first intermediate without generating GlcN-PI, while those prepared from PIG-L-transfected mutants generated the second intermediate. Therefore, PIG-L cDNA transfection induced GlcNAc-PI deacetylase activity in the microsomes and the activity induced by the overexpression of PIG-L was higher than normal. Moreover, PIG-L protein was expressed on the ER membrane. Thus, PIG-L protein may be GlcNAc-PI deacetylase, although enzymatic activity is yet to be demonstrated with purified PIG-L protein.
The deacetylation of GlcNAc-PI in mammalian cell-free systems has been shown to be stimulated by GTP hydrolysis (47). Defects in this reaction could, in principle, be due to mutations in either the deacetylase itself or the mediator of the GTP regulation. The PIG-L protein does not contain the main sequence elements of GTP-binding motifs as defined by Valencia et al. (48), suggesting that it does not bind this nucleotide. Microsomes from class L mutants transfected with PIG-L were capable of GlcNAc-PI deacetylation in the absence of GTP, and this activity was stimulated by this nucleotide (data not shown). While this similarity in the properties of this reaction in transfectants and wild-type cells does not conclusively exclude the possibility that PIG-L is the mediator of the GTP stimulation, it seems highly unlikely. Furthermore, if PIG-L is the deacetylase, the fact that it does not appear to bind GTP indicates that the enzyme and regulatory component are separate proteins.
Two groups of deacetylases that act on N-acetylglucosamine moiety, N-acetylglucosamine-6-phosphate deacetylases (38-40) and glucosaminoglycan N-acetylglucosaminyl N-deacetylase/N-sulfotransferase (41, 42), have been cloned. PIG-L has no sequence homology to these deacetylases or to acetylornithine deacetylase (35-37). Additionally, PIG-L has no homology to proteins with known functions. Therefore, the function of PIG-L cannot be predicted from its primary structure.
PIG-L encodes a predicted protein of 252 amino acids whose molecular mass is about 30 kDa. The GlcNAc-PI deacetylase of African trypanosomes has been partially purified (49). The most highly purified preparation contained several bands in the molecular mass range 45-60 kDa. The relationship between the trypanosomal proteins and PIG-L is not clear.
The topography of GPI anchor biosynthesis on the ER membrane is not completely understood. GlcNAc-PI and GlcN-PI are oriented toward the cytoplasm on the ER because they were sensitive to PI-specific phospholipase C when intact microsomes were exposed to the enzyme (50). Two proteins involved in synthesis of GlcNAc-PI, PIG-A and PIG-H, are also oriented toward the cytoplasm (26). Thus orientation of both biosynthesis proteins and the glycolipid intermediate indicates that the first step occurs on the cytoplasmic side of the ER. Consistent with orientation of the second intermediate, most of the PIG-L protein was located on the cytoplasmic side of the ER. The second reaction, therefore, also occurs on the cytoplasmic side.
The transfer of mature GPI anchor precursors to proteins is thought to occur on the lumenal side of the ER. Therefore, some intermediate or mature GPI anchor precursors should flip into the lumenal side from the cytoplasmic side. Vidugiriene and Menon (51) analyzed the orientation of later intermediates in microsomes prepared from Trypanosoma brucei and found that they are cytoplasmically oriented; hence, mature GPI precursors may flip. In contrast, analysis of the PIG-B protein that is involved in transferring the third mannose from dolichol phosphate-mannose to an intermediate bearing two mannoses demonstrated that its functional site resides on the lumenal side (16). This is consistent with the notion that dolichol phosphate-mannose serves as a mannosyl donor on the lumenal side for N-glycans and for yeast O-linked mannoses (52, 53). Another observation that supports the lumenal usage of dolichol phosphate-mannose is that the Lec 35 mutant of CHO cells synthesizes dolichol phosphate-mannose but does not use it for either N-glycan or the GPI anchor (54, 55). Although the exact step at which flipping occurs is yet to be determined, our present results further support the notion that it proceeds after the second step.
The GPI mannosyltransferases present in the lysates of blood stage Trypanosoma brucei transferred mannoses to GlcN-PI to generate later GPI intermediates when GlcN-PI was added as an exogenous substrate (56). However, the upstream intermediate GlcNAc-PI was a better substrate than GlcN-PI, suggesting a degree of substrate channeling via the GlcNAc-PI deacetylase enzyme to the mannosyltransferase (56). We metabolically labeled PIG-L-transfected and mock-transfected wild-type CHO cells with radioactive mannose to see if overexpression of PIG-L has any effect on later biosynthesis steps. Both cells generated similar amounts of mannose-containing GPI intermediates.2 It is not clear, therefore, if a similar substrate channeling exists in mammalian cells.
For expression cloning PIG-L using the CHO cell mutant, we
cotransfected a cDNA library containing Pyori with
pcDNA-PyT(ori) rather than with
pcDNA-PyT(ori+) (19) for the following reasons. The PyT
vector containing Pyori replicates in the cells and may interfere with
replication of the cDNA plasmids.3 In
addition, when PyT(ori+) was applied, rearranged plasmids
containing both the
-lactamase gene and the PyT gene
appeared and they overcame the cDNA plasmids after repeated sorting
(19). We used two cycles of sorting in PyT(ori
) system,
but we did not detect rearranged plasmids. Thus, when CHO cells are
used for cloning, we recommend origin-less polyoma large T DNA for
cotransfection with a cDNA library.
CHO cells have been useful for preparing various mutants. For example, mutants corresponding to various biosynthesis steps of N-glycan have been established and used to clone responsible genes (57). There are several reaction steps in GPI anchor synthesis at which mutants have not been obtained. We used CHO cells to prepare GPI anchor-deficient mutants for these steps. It appeared, however, that only class L mutants were generated after ethyl methane sulfonate treatment. During establishment of G9PLAP.85, several mutants of class L were obtained (10), suggesting that CHO cells have a "hot spot" of mutation caused by ethyl methane sulfonate within the PIG-L gene. We plan to stably transfect CHO cells with PIG-L cDNA before mutagenesis to generate mutants for other reaction steps.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D88364[GenBank].
We thank Drs. K. Ohishi and Y. Maeda for discussion and K. Nakamura, K. Kinoshita, and H. Zhang for technical assistance.