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J Biol Chem, Vol. 274, Issue 26, 18582-18588, June 25, 1999
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From the Attachment of glycosylphosphatidylinositol (GPI)
is essential for the surface expression of many proteins. Biosynthesis
of glycosylphosphatidylinositol is initiated by the transfer of
N-acetylglucosamine from
UDP-N-acetylglucosamine to phosphatidylinositol. In
mammalian cells, this reaction is mediated by a complex of PIG-A,
PIG-H, PIG-C, and GPI1. This complexity may be relevant for regulation and for usage of a particular phosphatidylinositol. However, the functions of the respective components have been unclear. Here we
cloned the mouse GPI1 gene and disrupted it in F9 embryonal carcinoma cells. Disruption of the GPI1 gene caused a
severe but not complete defect in the generation of
glycosylphosphatidylinositol-anchored proteins, indicating some
residual biosynthetic activity. A complex of PIG-A, PIG-H, and PIG-C
decreased to a nearly undetectable level, whereas a complex of PIG-A
and PIG-H was easily detected. A lack of GPI1 also caused partial
decreases of PIG-C and PIG-H. Therefore, GPI1 stabilizes the enzyme by
tying up PIG-C with a complex of PIG-A and PIG-H.
Many eukaryotic cell surface proteins are post-translationally
anchored to the membrane by carboxyl-terminal linkage to
glycosylphosphatidylinositol (GPI)1 in the endoplasmic
reticulum (1, 2). The basic backbone structure of GPI that is conserved
in all GPI-anchored proteins found in protozoa, yeast, slime mold,
fish, and mammals is formulated as
EtN-P-6Man The synthesis of GPI is initiated by the transfer of
N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to
phosphatidylinositol (PI) to yield GlcNAc-PI (1, 11). Genes involved in
this step have been cloned by means of complementation of mutants
derived from mammalian cells and yeast Saccharomyces
cerevisiae: PIG-A, PIG-H, and
PIG-C from mammalian cells (12-14), and GPI1,
GPI2 and GPI3/SPT14/CWH6 from yeast (5, 15-17).
PIG-A and PIG-C are homologues of GPI3
and GPI2, respectively, whereas PIG-H and
GPI1 are not similar to each other (12, 16). A mammalian
GPI1 homologue has also been cloned, based on the sequence
homology (18). A PIG-H homologue is not found in the
S. cerevisiae genome. It was shown that PIG-A, PIG-H, PIG-C,
and mammalian GPI1 proteins form a complex in the endoplasmic reticulum
that has UDP-GlcNAc:PI GlcNAc transferase (GPI-GnT) activity in
vitro (18). Because PIG-A has homology to a bacterial GlcNAc
transferase and other glycosyltransferases, this protein is thought to
be a catalytic subunit (17, 19, 20). However, functions of three other proteins cannot be predicted from their sequences. Class A, C, and H
mutant cells, corresponding to PIG-A, PIG-H, and
PIG-C mutations, do not express GPI-anchored proteins on the
cell surface, and their membranes do not have GPI-GnT activity,
indicating that these three proteins are essential for the enzyme (21,
22). Similarly, S. cerevisiae gpi2 and gpi3
mutants are defective in GPI anchoring, indicating essential roles of
GPI2 and GPI3 in GPI synthesis (5). In contrast,
S. cerevisiae gpi1 is not essential for growth, and the
gpi1-disrupted mutant showed temperature-sensitive growth.
Moreover, the gpi1-disrupted cells incorporated a
significant amount of inositol to proteins and generated mature Gas1p,
a major GPI-anchored protein. Therefore, GPI1 may have a regulatory
function rather than a direct role in the enzymatic function (15).
As described above, human GPI1 (hGPI1) is contained in the GPI-GnT
complex (18). It has also been shown that hGPI1 physically associates
with three other subunits, suggesting a role for hGPI1 in complex
formation (18). More recently, it was reported that human and mouse
GPI1 cDNAs complemented S. cerevisiae gpi1
and Schizosaccharomyces pombe gpi1 mutants (23), indicating
that mammalian GPI1 is functionally homologous to yeast Gpi1p (24). To
clarify the function of GPI1 in GPI-GnT, in the present study, we
generated GPI1 knockout mouse cells and found that GPI1 is necessary for stable formation of the GPI-GnT complex and stable expression of the PIG-C and PIG-H proteins.
Cells and Culture--
Mouse embryonal carcinoma F9 cells were
obtained from the American Type Culture Collection and cultured in
Dulbecco's modified Eagle's medium supplemented with 15% fetal calf
serum on 0.1% gelatin-coated dishes. Mouse GPI-deficient mutant
lymphoma T1M1 (Thy-1 Plasmids--
To express mouse GPI, its cDNA was cloned to a
pMEEB expression vector. To express GST-tagged PIG-A, GST-tagged-PIG-H,
and FLAG-tagged-PIG-C at the same time, each fragment was cloned
between the PGK promoter and the PGK poly(A)
signal in pBS-PGKtk (7). Three constructs were tandemly blunt-ligated
in pBS-PGKzeo to introduce the zeocin resistance gene. This plasmid
restored the surface expression of GPI-anchored proteins on human
PIG-A-deficient JY5 cells, Pig-c-deficient T1M1
cells (Thy-1 Preparation of cDNA and Genomic Clones of mGPI1--
We
designed degenerate primers on the basis of hGPI1 and yeast Gpi1p amino
acid sequences to clone mGPI1 cDNA (15, 18). Two
primers, forward primer HF1 (5'-GTGCTCCCGCNGGNYTNAARATGAA-3') (N; A, T,
G, and C: Y; C and T: R; A and G) and reverse primer HR1
(5'-GGAYGGTGAAGAGNARNGTNCCDAT-3') (D; G, A, and T), and template DNA
derived from a mouse testis cDNA library were used for 35 cycles of
amplification reaction under the conditions of 94 °C (1 min),
55 °C (30 s), and 68 °C (1.5 min) for denaturation, annealing, and extension, respectively. A sequence of the PCR product was confirmed to be homologous to hGPI1. To obtain the 5' and 3'
regions, we screened 96 subpools of a cDNA library, each of which
contained 2 × 104 independent clones, with PCR using
the same primers. We selected four subpools showing a positive PCR
band. Using DNAs of these pools, forward vector primer, and HR1 primer
in PCR, fragments containing the 5' region were cloned from the
subpools. The longest clone was named pGH5c7. Using HF1 primer and
reverse vector primer, fragments containing the 3' region were cloned.
All subpools gave the same fragments. The longest 5' and 3' fragments
were ligated in the EcoRV site to generate full-length
cDNA. An analysis by 5' rapid amplification of cDNA ends with
the Marathon RACE kit (CLONTECH) using RNA from F9 cells confirmed that
this cDNA contained the longest 5' sequence.
To obtain genomic clones of mGPI1, we screened 1 × 106 plaque-forming units of lambda FIXII from a mouse
129/SvJ liver genomic library (Stratagene) using a 1.4-kb pGH5c7
fragment, which contained the 5' region of mGPI1 cDNA,
as a probe. We obtained eight positive clones.
Disruption of the mGPI1 Gene in F9 Embryonal Carcinoma
Cells--
A 9.4-kb HindIII genomic fragment of
mGPI1 was blunted and transferred to the SmaI
site of pBS, the EcoRI-XbaI fragment was then
transferred to pPNT (26), and the plasmid was named pPNT-18HdelF. A
2.1-kb XbaI-EcoRV fragment of mGPI1
containing exons 9 and 10 was blunted and transferred to pBS. Its
XhoI-NotI fragment and a 1.8-kb
XbaI-XhoI fragment derived from PGKpuro were
ligated in tandem in the XbaI-NotI site in
pPNT-18HdelF. This plasmid was then used as the first targeting vector.
A 3.0-kb EcoRV fragment containing a region extending from
the middle of exon 6 to exon 10 was transferred to the SmaI
site of pBS. Its XhoI-XbaI fragment and a 1.9-kb
XbaI-XhoI fragment derived from PGKneo were
ligated in the XbaI-NotI site of pPNT-18HdelF.
The generated plasmid was used as the second targeting vector.
F9 cells (2 × 107) were electroporated with 50 µg
of NotI-cut targeting vector at 230 V and 500 microfarads
using a GenePulser (Bio-Rad) and seeded on plates coated with 0.1%
gelatin. Positive selection was started the next day with 2 µg/ml
puromycin or 380 µg/ml G418. Three days later, negative selection
with 2 µM gancyclovir was performed. About 8-11 days
after transfection, we screened colonies for recombinants, using PCR
primers PGK-PA2970 (5'-CCTGAAGAACGAGATCAGCAGCCTCTGT-3') and MGPI-KO154
(5'-TGTTTTATCACAATCTTGAGCACTATGG-3') for the first targeting and
PGK3254 (5'-CTGAGCCCAGAAAGCGAAGGAGCAAAGC-3') and MGPI-KO154 for the
second targeting. PCRs were started at 93 °C (1 min) followed by
annealing at 62 °C (1 min) and extension at 68 °C (3 min) for 35 cycles using Long Template PCR enzyme and buffers (Roche Molecular
Biochemicals). Positive colonies were confirmed by Southern blot analysis.
Northern Blot Analysis--
For Northern blot analysis of mouse
Pig-c, poly(A) RNA (10 µg) prepared using Trizol (Life
Technologies, Inc.) and the Poly(A)Tract mRNA isolation system
(Promega) was separated on a 1% formaldehyde-agarose gel and then
transferred to a nylon membrane. The membrane was hybridized with a
Pig-c cDNA probe and rehybridized with a
EF1 Fluorescence-activated Cell-sorting Analysis--
To examine the
surface expression of GPI-anchored proteins, cells were stained with
biotinylated anti-Thy1.2 or anti-CD59 antibody and
phycoerythrin-conjugated streptavidin (Biomedica) and then subjected to
analysis in a FACScan (Becton Dickinson) (25).
In Vitro GPI-GnT Assay and Characterization of
Glycolipids--
Cell lysates (derived from 1 × 107
cells) and microsomes (200 µg of protein equivalents) were prepared
as described previously (18). They were labeled with 2 µCi of
UDP-[6-3H]GlcNAc (14 µCi/mol; American Radiolabeled
Chemicals) for 1 h at 37 °C as described, and the generated
glycolipids were separated by TLC on Kiesel Gel 60 (Merck) with a
solvent system of CHCl3:methanol:1 M
NH4OH (10:10:3) (18). To assay Dol-P-Glc synthase as a
control, cell lysates (106 cells) and microsomes (40 µg
of protein equivalents) were incubated with
UDP-[6-3H]glucose for 20 min at 37 °C in 50 mM HEPES/NaOH (pH 7.4), 25 mM KCl, 5 mM MgCl2 and 5 mM
MnCl2. The reaction was terminated by adding
CHCl3:methanol (2:1). The organic phase removed from the
mixture was washed two times with CHCl3:methanol
(2:1)-saturated water, evaporated, and separated by TLC with a solvent
system of CHCl3:methanol:water (10:10:3) (27). Amounts of
labeled lipids were quantitated by a Fuji Image Analyzer BAS1500 (Fuji
Film Co., Tokyo Japan). Treatments of lipids with Bacillus
thuringiensis PI-phospholipase C (PLC) (Funakoshi, Japan) and
acetylation of lipids were done as described previously (28). Mild
alkaline hydrolysis was done as described previously (21).
Immunoprecipitation and Western Blotting--
Transfectant cells
(2-4 × 108) were hypotonically lysed in water
containing 0.1 mM TLCK and 1 µg/ml leupeptin, mixed with
an equal volume of a lysate buffer (100 mM HEPES/NaOH (pH
7.4), 50 mM KCl, 10 mM MgCl2, 20%
glycerol, 0.1 mM TLCK, and 1 µg/ml leupeptin) and stored
at Fluorescence in Situ Hybridization--
Fluorescence in
situ hybridization was carried out with a biotinylated genomic DNA
probe for mGPI1 and a biotinylated cDNA probe for
hGPI1 prepared as described previously (10).
Cloning of mGPI1--
The full-length mGPI1 cDNA
contained 3062 nucleotides except a poly(A) tract, encoding a 581-amino
acid protein that had an 89% amino acid identity to hGPI1
(GenBank accession number for mGPI1 cDNA is AB008895.).
Using a mGPI1 cDNA probe, we obtained eight genomic
clones of mGPI1, mapped them, and sequenced around the exons
(GenBank accession numbers for the genomic sequences are AB008915 to
AB008921). Exon sequences were exactly the same as that of the
cDNA. The mGPI1 gene had 11 exons, containing a start
codon in exon 2 and a stop codon in exon 11 (Fig.
1A). This structure was
similar to a partial structure of hGPI1 determined by
analyzing a sequence obtained from GenBankTM (Fig.
1B). Their corresponding exons had the same lengths.
The amino acid sequence of mGPI1 was very different from
that published recently as mouse GPI1 (24) (GenBank
accession number AF030178), having only a 93% amino acid identity. We
think that the published sequence is not that of mouse GPI1
because: 1) our cDNA sequence and genomic exon sequence were
exactly the same, 2) many mouse expressed sequence tag (EST) nucleotide
sequences in the current databases matched exactly with our sequence,
3) the mGPI1 gene was successfully disrupted using our
mGPI1 DNA fragments, and 4) their sequence was simply that
of an EST clone and was not confirmed in mouse cDNA or DNA.
Disruption of mGPI1 in the F9 Embryonal Carcinoma Cell
Line--
We used the F9 cell line to generate
mGPI1-disrupted cells because it has a relatively high
frequency of homologous recombination and is easier to culture than
embryonic stem cells. We designed two targeting vectors: the vector for
the first targeting had the puromycin resistance gene in place of exon
2 to exon 8, and the vector for the second targeting had the neomycin
resistance gene in place of exon 2 to a part of exon 6 (Fig.
2A). Colonies grown after
antibiotic selection were screened with PCR (Fig. 2B) and
confirmed with genomic Southern blotting (Fig. 2C). Bands corresponding to the wild type allele decreased after the first targeting (lanes 2) and disappeared after the second
targeting with generation of the predicted targeted bands (lanes
3). After the second targeting, no 3.1-kb RNA band of
mGPI1 was seen in Northern blotting (Fig. 2D),
indicating that both alleles were disrupted.
Characterization of mGPI1 Knockout Mutants--
We first assessed
the surface expression of Thy-1, a GPI-anchored protein on
mGPI1-knockout F9 cells, and found that the expression decreased greatly but remained at a low level (Fig.
3A). The surface Thy-1
expression was restored upon transfection of mGPI1 cDNA but not upon transfection of an empty vector (Fig. 3B). To
confirm that mGPI1 knockout cells have some ability to
generate GPI-anchored proteins, we transfected a cDNA of CD59. CD59
was more efficiently expressed on the cell surface, whereas the
expression of endogeneous Thy-1 stayed very low (Fig. 3C).
These results showed that mGPI1 knockout cells still have
some GPI-GnT activity.
We next measured GPI-GnT activity in vitro using cell
lysates and microsomes of mGPI1 knockout cells (Fig.
4). In contrast to the significant
surface expression of GPI-anchored proteins on mGPI1
knockout cells, their lysates and microsomal membranes did not generate
GlcNAc-PI upon incubation with UDP-GlcNAc (lanes 2 in Fig.
4A and B), whereas they had comparable activities
of dolichol phosphate glucose (Dol-P-Glc) synthase. This
defect was restored by transfection of mGPI1 cDNA
(lanes 3) but not by transfection of an empty vector
(lanes 4). The lysates of mGPI1 knockout cells gave several radioactive spots, one of which had the same mobility as
GlcN-PI (lane 2), the second intermediate derived from
GlcNAc-PI by deacetylation. To determine whether it is GlcN-PI or a
non-relevant material, we first tested for the presence of PI by
treatment with PI-PLC (Fig.
5A). The spot was resistant to
PI-PLC (lanes 3 and 4), whereas the GlcN-PI
generated by the wild type cells was sensitive, as shown by a great
decrease of the intensity (lanes 1 and 2),
indicating that the spot did not contain PI. We next tested acetylation
that would convert GlcN-PI to GlcNAc-PI (Fig. 5B). GlcN-PI
generated by the wild type cells was acetylated to GlcNAc-PI, resulting
in a decrease of GlcN-PI and a slight increase of GlcNAc-PI
(lanes 1 and 2); however, the spot generated by
mGPI1 knockout cells did not shift to the GlcNAc-PI position
(lanes 3 and 4). As a final test, we treated the
spot with mild alkali (Fig. 5C). If it contained
diacylglycerol, then the acyl chains would be removed, causing a
mobility shift or disappearance from the lipid fraction. The spot was
resistant (lanes 3 and 4), whereas GlcN-PI and
GlcNAc-PI were sensitive (lanes 1 and 2). All of
these results indicated that the spot was not GlcN-PI. Other spots seen above and below the area corresponding to GlcNAc-PI and GlcN-PI should
also be non-relevant. They are not related to N-glycans because a 10× higher concentration of tunicamycin did not affect them
(data not shown). Therefore, the membranes of mGPI1 knockout cells did not have a detectable level of GPI-GnT activity in
vitro.
We thought that the weak expression of Thy-1 on mGPI
knockout cells (Fig. 3A) might be due to inefficient
recognition of GPI attachment signals of Thy-1 by GPI transamidase in
the presence of a limited amount of GPI. To test this, we replaced the
GPI attachment signal sequence of CD59 with that of Thy-1 and compared it with wild-type CD59. As shown in Fig.
6, CD59 bearing the GPI attachment signal
of Thy-1 was processed much less efficiently than CD59 bearing its
own signal.
GPI1 Is Required for the Stable Formation of GPI-GnT Complexes and
for the Full Expressions of PIG-C and PIG-H but not PIG-A--
The
above results demonstrated that a very low level of GPI-GnT activity
was expressed in the absence of mGPI1. A possible explanation for this
is that the GPI-GnT complex cannot be formed efficiently in the absence
of mGPI1. Another possible explanation is that a protein complex of
PIG-A, PIG-C, and PIG-H is formed even in the absence of mGPI1 but has
a very weak activity. To test the former possibility, we stably
transfected mGPI1 knockout cells with a vector bearing
cDNAs for FLAG-tagged PIG-C and GST-tagged PIG-A and PIG-H. A
vector for free GST was also cotransfected to the same cells. A clonal
cell population was isolated by limiting dilution and further
transfected with either mGPI1 cDNA or an empty vector.
Therefore, two cells would potentially express tagged PIG-C, PIG-A, and
PIG-H and free GST at the same levels, with the sole difference being
the presence or absence of mGPI1. From digitonin extracts of these
cells, we precipitated FLAG-tagged PIG-C with anti-FLAG beads, and
coprecipitations of GST-tagged PIG-A and PIG-H were assessed by Western
blotting with anti-GST (Fig.
7A). In the presence of mGPI1,
GST-tagged PIG-A and PIG-H were clearly coprecipitated (lane
1), indicating the formation of complexes of PIG-A, PIG-H, PIG-C,
and mGPI1. In contrast, almost no bands of GST-tagged PIG-H and PIG-A
were seen in the absence of mGPI1 (lane 2). These cells
expressed comparable levels of free GST in the cytoplasm (bottom
panel). Therefore, only a trace amount, if any, of the protein
complex was formed in the absence of mGPI1.
To evaluate the amounts of expressed FLAG-tagged PIG-C, we analyzed the
immunoprecipitates with anti-FLAG beads by Western blotting against
anti-FLAG antibody (Fig. 7A, lanes 3 and
4). The amount of FLAG-PIG-C was significantly lower in the
absence (lane 4) than in the presence (lane 3) of
mGPI1, whereas a nonspecific band (asterisk) and a light
chain of the antibody (L) showed comparable intensities. We
also evaluated the amounts of expressed GST-tagged PIG-A and PIG-H by
collecting them from Nonidet P-40 extracts with glutathione-Sepharose
beads followed by Western blotting against anti-GST antibody
(lanes 5 and 6). The amount of GST-tagged PIG-H
was significantly lower in the absence (lane 6) than in the
presence (lane 5) of mGPI1, whereas the amounts of
GST-tagged PIG-A were comparable. The intensities of these bands and
bands of free GST collected from the cytoplasm were quantitated by an image analyzer. Expression levels of PIG-C and PIG-H decreased to
one-third of the wild type levels in the absence of mGPI1, whereas
PIG-A and a control free GST were expressed at similar levels.
These results were from cloned cells. To eliminate the chance that an
unusual event might have occurred due to the use of cloned cells, we
used cells without limiting dilution. For this we transiently
cotransfected mGPI1 knockout cells with a vector bearing
cDNAs for FLAG-tagged PIG-C and GST-tagged PIG-A and PIG-H in
combination with mGPI1 cDNA or an empty vector and
assessed the complex formation and expressions of three tagged
proteins. We obtained results very similar to those with the cloned
cells (data not shown).
To test the association between PIG-A and PIG-H in the absence of GPI1,
we cotransfected GST-tagged PIG-H and FLAG-tagged PIG-A into
mGPI knockout cells (Fig. 7B, lane 1).
As a positive control, GST-tagged GPI1 and FLAG-tagged PIG-A were
cotransfected (lane 2). As negative controls, GST-tagged
ALDH (aldehyde dehydrogenase, a control endoplasmic reticulum membrane
protein (29)) and FLAG-tagged PIG-A (lane 3) and GST-tagged
PIG-H and FLAG-tagged ALDH (lane 4) were cotransfected,
respectively. FLAG-tagged PIG-A was efficiently coprecipitated with
GST-tagged PIG-H (lane 1), but not with GST-tagged ALDH
(lane 3). FLAG-tagged ALDH was not coprecipitated with
GST-tagged PIG-H (lane 4). Therefore, PIG-A and PIG-H
associated specifically and efficiently in the absence of GPI1. This
indicated that the efficient association of PIG-C with a complex of
PIG-A and PIG-H is dependent upon GPI1.
Because GP11 is an endoplasmic reticulum membrane protein (18), it is
unlikely that the GPI1 protein affects transcriptions of the
PIG-C and PIG-H genes. In fact, the expression
levels of endogenous mouse Pig-c mRNA assessed by
Northern blotting did not change between wild type and mGPI1
knockout cells (data not shown). It is likely that GPI1 affects the
stability of the PIG-C and PIG-H proteins. However, the decreased
levels of PIG-C and PIG-H were still 30% of the wild type levels,
which does not fully explain a nearly complete lack of complexes of
PIG-A, PIG-C, and PIG-H. Therefore, GPI1 must function in the
maintenance of the GPI-GnT complex itself.
To see whether the expression of GPI1 is conversely influenced by PIG-C
and PIG-H, we cotransfected FLAG-tagged hGPI1 and free GST, as a
control, into Pig-c-deficient TIM1 cells
(Thy-1 Chromosomal Localization of mGPI1 and hGPI1--
We investigated
the chromosomal localization of mGPI1 and hGPI1
using fluorescence in situ hybridization. With a 15.2-kb
genomic fragment from a lamda clone as a probe, we localized
mGPI1 to mouse chromosome 17B (Fig.
9A). Using a 2.9-kb
hGPI1 cDNA probe, we localized hGPI1 to human
chromosome 16p13.3 (Fig. 9B). These mouse and human
chromosome regions are syntenic (30). The autosomal location of
mammalian GPI1 is consistent with the notion that only
PIG-A is X-linked among all GPI-anchor synthesis genes
(1).
In this report, we present direct evidence that GPI1 is necessary
for the stable formation of GPI-GnT. We disrupted mouse GPI1
in F9 embryonal carcinoma cells and found that the generation of
GPI-anchored proteins was severely affected in the absence of GPI1.
This defect was accounted for by a decrease of complexes of PIG-C with
PIG-A and PIG-H to a nearly undetectable level, whereas an association
between PIG-A and PIG-H was still seen. In the absence of GPI1, the
levels of PIG-C and PIG-H were only one-third of the wild type levels,
indicating that GPI1 is needed to maintain normal levels of these
proteins. The level of PIG-A was not affected by the absence of GPI1.
These partial losses of PIG-C and PIG-H alone cannot fully account for
the nearly complete lack of the complex of these components. Therefore,
it is indicated that GPI1 is required for the efficient association of
PIG-C with a complex of PIG-A and PIG-H. This is consistent with our
previous report that GPI1 directly associates with each of the three
other proteins (18).
Among the four proteins participating in GPI-GnT, PIG-A, PIG-C, and
PIG-H are essential for activity because their mutant cells are
completely defective in GPI-GnT (21, 22), whereas GPI1 is not essential
because GPI1 knockout cells still had some ability to
generate GPI-anchored proteins. This phenotype of mGPI1 knockout F9 cells is similar to that of gpi1-disrupted
S. cerevisiae that was lethal at a higher temperature but
was viable and incorporated some inositol to proteins at 25 °C (15).
Expression of mammalian GPI1 in gpi1-disrupted S. cerevisiae restored growth at a higher temperature and synthesis
of GlcNAc-PI in vitro (24), indicating that mammalian and
yeast GPI1 proteins have conserved sites for association with other components.
In the absence of GPI1, proteins that are normally GPI-anchored would
compete for a limited amount of GPI. Proteins bearing different
carboxyl-terminal GPI attachment signals may have different affinities
for GPI transamidase that recognizes the GPI attachment signal and
replaces it with GPI (2). The mGPI1 knockout F9 cells
expressed a very low level of Thy-1 on the surface but expressed CD59
quite efficiently, indicating that the GPI attachment signal of CD59
functioned more efficiently than that of Thy-1. A possible reason for
this is that the signal sequence of CD59 contains asparagine at the We localized the mouse GPI1 gene to chromosome 17B and
localized human GPI1 to chromosome 16p13.3. This autosomal
location is consistent with the fact that two homologous recombinations were required to eliminate mGPI1. This is also consistent
with the observation that most, if not all, patients with paroxysmal nocturnal hemoglobinuria lost the ability for biosynthesis of GlcNAc-PI
in their affected blood cells due to somatic mutation of the
PIG-A gene in the hematopoietic stem cell (36). Because PIG-A is X-linked, a single somatic mutation should cause
the GPI-GnT deficiency, whereas two somatic mutations of
GPI1 should occur in the same cell to cause the mutant
phenotype. The latter probability would be extremely low. It is
difficult to predict the outcome of a hereditary lack of
GPI1. Although a complete lack of GPI synthesis causes
embryonic lethality (7, 8), no information is available about the
effects of a partial GPI deficiency.
We thank Dr. Junji Takeda for discussion and
Keiko Kinoshita for technical assistance.
*
This work was supported by grants from the Ministry of
Education, Science, Sports and Culture of Japan.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.
¶
To whom correspondence should be addressed: Dept. of
Immunoregulation, Research Institute for Microbial Diseases, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-8328; Fax:
81-6-6875-5233; E-mail tkinoshi{at}biken.osaka-u.ac.jp.
The abbreviations used are:
GPI, glycosylphosphatidylinositol;
GlcNAc, N-acetylglucosamine;
PI, phosphatidylinositol;
GnT, N-acetylglucosamine
transferase;
hGPI1, human GPI1;
mGPI1, mouse GPI1;
GST, glutathione
S- transferase;
PCR, polymerase chain reaction;
kb, kilobase pair(s);
PLC, phospholipase C;
TLCK, 1-chloro-3-tosylamido-7amino-2-heptanone;
ALDH, aldehyde
dehydrogenase.
Department of Immunoregulation,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,2Man1-6Man-1,4GlcN-1,6-inositol phospholipid (where EtN-P, Man, and GlcN are ethanolamine phosphate, mannose, and
glucosamine, respectively) (3). GPI anchor synthesis is essential for
yeasts (4, 5) and is probably also essential for protozoa (6). It is
essential for mouse embryogenesis (7, 8) and for the development of
skin (9). In affected hematopoietic cells from patients with the
acquired hemolytic disease paroxysmal nocturnal hemoglobinuria, the
first step of GPI anchor synthesis is defective due to somatic mutation
of the X-linked PIG-A gene (10).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
c) and S49 (Thy-1h)
cells were gifts from Dr. R. Hyman (Salk Institute, San Diego, CA) and
were cultured and transfected as described previously (25).
c), and Pig-h-deficient S49 cells
(Thy-1h). To express FLAG-tagged hGPI1, it was cloned
between the PGK promoter and the PGK poly(A)
signal in pBS-PGKtk and then ligated with pBS-PGKzeo to generate
pBS-FLAG-hGPI1/zeo. All of the other constructs used in this study were
described previously (25).
cDNA probe (a gift from Dr. S. Nagata). The
Pig-c cDNA probe was amplified with forward primer
5'-TGGGCTGTGGTATTTGAGTCCAG-3' and reverse primer
5'-TGGCTCCCACAGCACTGGAC-3' from a mouse cDNA library.
80 °C until used. After thawing on ice, the cell lysates were
further treated using a tight pestle Dounce homogenizer. Membranes and
cytosol fractions were divided by centrifugation at 100,000 × g for 1 h. The collected membranes were solubilized in
1% digitonin in 50 mM HEPES/NaOH (pH 7.4), 25 mM KCl, 5 mM MgCl2, 0.1 mM TLCK, and 1 µg/ml leupeptin or 1% Nonidet P-40 in 10 mM Tris·HCl (pH 7.7), 150 mM NaCl, 1 mM EDTA, 0.1 mM TLCK, and 1 µg/ml leupeptin.
Insoluble materials were removed by centrifugation at 100,000 × g for 1 h, and supernatants were used for
immunoprecipitation. For the cytosol fractions, Nonidet P-40 was added
at 1% and used for immunoprecipitation. Anti-FLAG M2 affinity gel
beads (Sigma) and glutathione-Sepharose beads (Amersham Pharmacia
Biotech) were used to collect FLAG- and GST-tagged proteins,
respectively. Proteins were separated by SDS-polyacrylamide gel
electrophoresis and analyzed by Western blotting. Western blotting was
carried out with biotinylated anti-FLAG monoclonal antibody M2 plus
horseradish peroxidase-conjugated streptavidin (Amersham Pharmacia
Biotech) or with anti-GST antibody (Amersham Pharmarcia Biotech) plus
horseradish peroxidase-conjugated anti-goat IgG antibody (Organon
Teknika) and visualized with chemiluminescence (Renaissance; DuPont).
The band intensities in Western blotting were quantitated by measuring
chemiluminescence with a Fuji Image analyzer LAS1000.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Genomic structures of mGPI1
and hGPI1. The genomic structure of mGPI1
(A) was determined from overlapped restriction maps of
genomic lambda clones and sequences of regions containing exons. The
genomic structure of hGPI1 (B) was determined by
analyzing nucleotide sequences from the GenBankTM.
GenBankTM accession numbers of genomic sequences of
hGPI1 are Z98881 and Z98883. 
and 
, noncoding and
coding exon regions, respectively. Exon numbers are indicated.
Restriction enzyme sites are as follows: Bm,
BamHI; E, EcoRI; H,
HindIII; RV, EcoRV; X,
XbaI; and Sp, SpeI.
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Fig. 2.
Disruption of mGPI1 in F9
cells. A, targeting vectors for homologous
recombination. The first and second targeting vectors were designed to
disrupt exon 2 to exon 8 and exon 2 to the middle of exon 6, respectively, and to contain the puromycin (puro) and
neomycin (neo) resistance genes, respectively. The HSV
thymidine kinase gene (tk) was included at the 5' end to
select against random integration. All selection marker genes were
driven by the PGK promoter and had a PGK poly(A)
signal. Restriction enzyme site: Xh, XhoI.
B, structures of disrupted mGPI1 after homologous
recombination. To screen homologous recombinants, PCR primers were used
as indicated (arrows). Probes A and B,
which were used for Southern blotting, are in bold.
C, Southern blotting of targeted mutants. Samples of genomic
DNA (5 µg) were cut with XbaI (left panel) and
SpeI (right panel) and probed with probes A and B
(see B), respectively. Lanes 1, F9 (wild type);
lanes 2, single knockout mutant; lanes
3, double knockout mutant. D, Northern blotting
of mGPI1 knockout mutant. Samples of total RNA (30 µg)
were separated in a 1% formaldehyde-agarose gel, transferred to a
nylon membrane, and probed with radiolabeled mGPI1 cDNA.
The membrane was reprobed with EF-1 cDNA as a
control. Lane 1, F9; lane 2, mGPI1
knockout mutant.
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Fig. 3.
Flow cytometric analysis of knockout mutants
of F9 cells. A, decrease in surface Thy-1 expression
after the disruption of mGPI1. Cells were stained with
biotinylated anti-Thy1 antibody (thick lines) or control
antibody (thin lines). Left panel, F9;
middle panel, single knockout mutant; right
panel, mGPI1 knockout mutant. B, restoration
of the surface Thy-1 expression with mGPI1 cDNA.
Left panel, mock transfectants of mGPI1 knockout
cells; right panel, mGPI1 stable transfectants of
mGPI1 knockout cells. C, transfection of
CD59 cDNA to mGPI1 knockout mutant.
Endogenous Thy-1 (left panel) and human CD59 (right
panel) were stained after stable transfection of CD59
cDNA into mGPI1 knockout cells. Thick lines,
anti-Thy1 (left panel) and anti-CD59 antibodies (right
panel); thin lines, control antibodies.
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Fig. 4.
In vitro GPI-GnT assay with cell
lysates and microsomes. Cell lysates (A) were prepared
from 1 × 107 cells and labeled with
UDP-[6-3H]GlcNAc for 1 h. In B,
microsomes (100 µg) were used under the same reaction conditions. The
same samples were also incubated with UDP-[6-3H]glucose
to measure Dol-P-Glc synthase to assess the amount of membranes
(lower panel). The radiolabeled lipids were extracted and
resolved in TLC. Lanes 1, F9; lanes 2,
mGPI1 knockout mutant; lanes 3,
mGPI1-transfected knockout cells; lanes 4,
mock-transfected knockout cells.
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Fig. 5.
Characterization of GlcNAc-labeled
products. Lysates (from 2 × 107 cells) of wild
type (lanes 1 and 2 in all panels) and
mGPI1 knockout (lanes 3 and 4 in all
panels) F9 cells were labeled with UDP-[6-3H]GlcNAc for
1 h. A, extracted radiolabeled lipids were treated with
PI-PLC (lanes 2 and 4) or a reaction buffer
(lanes 1 and 3), and reextracted lipids were
analyzed by TLC. B, the same samples as in A were
treated with acetic anhydride (lanes 2 and 4) or
a buffer (lanes 1 and 3). C, the same
samples as in A and B were treated with mild
alkali (lanes 2 and 4) or a neutral buffer
(lanes 1 and 3).
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Fig. 6.
GPI attachment signals influenced the surface
expressions of GPI-anchored proteins. MGPI1 knockout F9
cells were transiently transfected with cDNAs of CD59
(A) and CD59 bearing the GPI attachment signal from Thy-1
(B). The surface CD59 expressions were determined 2 days
after transfection. Thin lines, anti-CD59 antibodies;
thick lines, isotype-matched control antibodies.
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Fig. 7.
The effects of mGPI1
disruption on GPI-GnT complexes. A,
mGPI1 knockout F9 cells were stably transfected with
FLAG-tagged PIG-C, GST-tagged PIG-A, GST-tagged PIG-H, and free GST
simultaneously. The cloned transfectant was further transfected with
mGPI1 cDNA (lanes 1, 3, and 5) or
an empty vector (lanes 2, 4, and 6). Cytosol and
membrane fractions from lysed cells (2 × 108) were
separated. Proteins were immunoprecipitated from the solubilized
membranes with the indicated beads. The immunoprecipitates were
analyzed by Western blotting with anti-FLAG or anti-GST antibodies.
Lanes 1 and 2, Western analysis with anti-GST of
anti-FLAG immunoprecipitates; lanes 3 and 4,
Western analysis of expression levels of FLAG-tagged PIG-C; lanes
5 and 6, Western analysis of expression levels of
GST-tagged PIG-A and GST-tagged PIG-H. Size markers (in kDa) are shown
on the right. B, association of PIG-A and PIG-H
in the absence of GPI1. MGPI1 knockout mutants were
transfected with GST-tagged PIG-H and FLAG-tagged PIG-A (lane
1), GST-tagged GPI1 and FLAG-tagged PIG-A (lane 2),
GST-tagged ALDH and FLAG-tagged PIG-A (lane 3), and
GST-tagged PIG-H and FLAG-tagged ALDH (lane 4). GST-tagged
proteins were collected from digitonin-solubilized membrane fractions
with glutathione-Sepharose beads (top and middle
panels) and Western blotted with anti-GST antibodies (top
panel) or anti-FLAG antibodies (middle panel). After
precipitation with glutathione-Sepharose beads, FLAG-tagged proteins in
the supernatants were immunoprecipitated with anti-FLAG beads and
Western blotted with anti-FLAG antibodies (bottom
panel).
c) and Pig-h-deficient S49 cells
(Thy-1h), obtained the cloned cells, and transfected them
with PIG-C and PIG-H cDNA, respectively, or
with an empty vector. An analysis by fluorescence-activated cell
sorting confirmed that PIG-C-transfected T1M1
(Thy-1c) cells and PIG-H-transfected S49 cells
(Thy-1h) but not vector transfectants restored the
surface expression of Thy-1 proteins (data not shown). However, the
amounts of expressed FLAG-tagged GPI1 did not change significantly in
the presence (Fig. 8A, lane 1)
and absence (lane 2) of PIG-C or in the presence (Fig.
8B, lane 1) and absence (lane 2) of PIG-H.
Therefore, it seems that GPI1 influences the stability of PIG-C and
PIG-H, but not PIG-A, but that the stability of GPI1 is not influenced
by PIG-C and PIG-H.
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Fig. 8.
Lack of PIG-C or PIG-H had no effect on
GPI1. A, T1M1 (Thy-1 c) mutants were
transfected with FLAG-hGPI1 and GST simultaneously, cloned with
limiting dilution, and then retransfected with PIG-C
cDNA (lane 1) or an empty vector (lane 2).
Membrane fractions were immunoprecipitated with anti-FLAG beads and
blotted with anti-FLAG antibodies (top panel). GST proteins
in the cytosol fractions were detected by Western blotting
(bottom panel). B, S49 (Thy-1h)
mutants were transfected with FLAG-hGPI1and GST simultaneously, cloned
with limiting dilution, and then retransfected with PIG-H
cDNA (lane 1) or an empty vector (lane 2).
Membrane fractions were immunoprecipitated with anti-FLAG beads and
blotted with anti-FLAG antibodies (top panel). GST proteins
were detected in the cytosol fractions by Western blotting
(bottom panel).
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Fig. 9.
Chromosomal localization of mGPI1
and hGPI1. Fluorescence in situ
hybridization fluorescent spots were detected in mouse chromosome 17B
(A) and in human chromosome 16p13.3 (B). The
arrows and bars indicate positive signals of
probes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
site (31) to which GPI is linked, whereas the signal of Thy-1 has
cysteine at the site (32). There are reports that the asparagine
site functions more efficiently than the cysteine site in the
GPI attachment reaction (33-35).
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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