From the Unité de Glycobiologie et
Signalisation Cellulaire, INSERM, U504, Bâtiment INSERM, 16 Avenue Paul Vaillant-Couturier, 94807 Villejuif, ¶ Biochimie A,
Hôpital Bichat, and
Généthon III, CNRS, URA
1923 1 Bis Rue de l'Internationale, 91002 Evry Cedex, and
** Hôpital Robert Debré, Assistance Publique
Hôpitaux de Paris, 75004 Paris, France
Received for publication, November 22, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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The underlying causes of type I
congenital disorders of glycosylation (CDG I) have been shown to be
mutations in genes encoding proteins involved in the biosynthesis of
the dolichyl-linked oligosaccharide (Glc3Man9GlcNAc2-PP-dolichyl)
that is required for protein glycosylation. Here we describe a CDG I
patient displaying gastrointestinal problems but no central nervous
system deficits. Fibroblasts from this patient accumulate mainly
Man9GlcNAc2-PP-dolichyl, but in the presence of
castanospermine, an endoplasmic reticulum glucosidase inhibitor
Glc1Man9GlcNAc2-PP-dolichyl
predominates, suggesting inefficient addition of the second glucose
residue onto lipid-linked oligosaccharide. Northern blot analysis
revealed the cells from the patient to possess only 10-20% normal
amounts of mRNA encoding the enzyme,
dolichyl-P-glucose:Glc1Man9GlcNAc2-PP-dolichyl
Type I congenital disorders of glycosylation (CDG
I)1 are often severe
multisystemic diseases characterized by the presence of
hypoglycosylated glycoproteins in the serum of affected individuals (1-5). Although glycoproteins play vital roles in many aspects of
human cellular physiology (6), the precise relationship between
glycoprotein hypoglycosylation and the clinical symptoms of these
diseases that include hypotonia, seizures, failure to thrive,
psychomotor retardation, and various dysmorphias is not understood
(2).
Hypoglycosylation of glycoproteins bearing
N-glycans is caused by either an insufficiency in the
biosynthesis of the lipid-linked oligosaccharide (LLO) precursor,
Glc3Man9GlcNAc2-PP-dolichyl, that
is required for protein glycosylation or inefficient transfer of the
sugar moiety of this LLO onto nascent glycoproteins in the lumen of the
endoplasmic reticulum (ER). Theoretically, mutations in any of the
genes encoding for the 30 or so proteins involved in this biosynthetic
pathway could lead to glycoprotein hypoglycosylation in CDG I patients.
However, mutations in only 7 of the genes encoding proteins of the
glycosylation pathway have so far been shown to underlie CDG I, and
these 7 cases have been classified as CDG I subtypes a-g (Ia,
phosphomannomutase 2 (7, 8); Ib, phosphomannose isomerase (9, 10); Ic,
dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl Here we report on a patient presenting clinical symptoms similar to
those observed in CDG Ib patients but whose PMI levels were found to be
normal. We demonstrate that cells derived from this patient accumulate
Glc0-1Man9GlcNAc2-PP-dolichyl and display dramatically reduced levels of mRNA encoding
dolichyl-P-glucose:Glc1Man9GlcNAc2-PP-dolichyl Western Blot--
Western blotting of serum transferrin was
performed as described previously (26) using a rabbit polyclonal
anti-transferrin antibody. Phosphomannomutase (27, 28) and
phosphomannose isomerase (29) activities were assayed as described previously.
Cells, Cell Culture, and Metabolic Radiolabeling of
Cells--
Skin biopsy fibroblasts, obtained from patient M. P. and a
patient diagnosed with CDG Ic (25), were grown in Dulbecco's modified
Eagle's medium containing 2 g/liter glucose, 10% fetal calf serum,
and 1% penicillin/streptomycin. Primary skin fibroblasts were
immortalized by Dr. Thierry Levade (INSERM U466), as reported previously (30), and cultivated as described above. EBV-transformed lymphoblasts were generated from peripheral blood mononuclear cells
isolated using a Ficoll-Paque Plus gradient and were grown in RPMI 1640 medium supplemented as described above. Confluent fibroblasts in
25-cm2 tissue culture flasks or EBV-transformed
lymphoblasts were pulse-radiolabeled for 30 min with 100 µCi of
[2-3H]mannose (23.9 Ci/mmol, PerkinElmer Life Sciences)
in 1 ml of glucose-free Dulbecco's modified Eagle's medium or RPMI
1640 supplemented with 0.5 mM glucose and 5% dialyzed
fetal calf serum. Where appropriate, the glycosidase inhibitors
castanospermine (CST, Cambridge Research Biochemicals, Northwich, UK)
and kifunensin (KIF, Toronto Research Chemicals Inc.) were added to the
cells 30 min prior to the onset of the radiolabeling period, at
concentrations of 2 mM and 100 µM, respectively.
Analysis of LLO Glucosylation in Streptolysin-O-permeabilized
Lymphoblasts--
EBV-transformed lymphoblasts (2 × 108 cells) were pulse-radiolabeled with 200 µCi of
[2-3H]mannose as described above. The cells were then
permeabilized with streptolysin-O (SLO, Bacto-Streptolysin O, product
reference 0482, BD Biosciences) as described previously (31). Briefly, cells were washed into cell permeabilization buffer (PB: 130 mM K+/glutamate, 10 mM NaCl, 2 mM EGTA, 1 mM CaCl2, 2 mM MgCl2, 5 mM HEPES/KOH, pH 7.3, containing 2 mg/ml bovine serum albumin and 1 mM
dithiothreitol) and incubated with 5 units of SLO (dissolved in PB) for
20 min at 4 °C. After washing twice with ice-cold PB, the cells were
warmed to 37 °C for 5 min and then placed on ice for 30 min. The
cells were again washed in PB prior to being resuspended in the same
buffer, dispatched into 1.5-ml centrifuge tubes, and incubated for
different times at 37 °C in the absence or presence of 2 mM UDP-glucose and/or 4 mM CST.
Isolation and Analysis of Lipid-linked and N-Linked
Oligosaccharides--
Radiolabeled cells and SLO-permeabilized cells
were extracted with organic solvents as described previously (19).
Briefly, cells were rinsed with ice-cold phosphate-buffered saline and then suspended in MeOH, 100 mM Tris/HCl, pH 7.4, containing
4 mM MgCl2, 2:1. Finally, an equal volume of
CHCl3 was added before vigorous shaking of the cell
suspension. After centrifugation, the lower (CHCl3) and
upper (methanolic) phases were removed and kept. The interphase
proteins were washed once with MeOH and then with H2O, and
then again with MeOH prior to being extracted twice with 2 ml of
CHCl3/MeOH/H2O, 10:10:3. The lower
(CHCl3) and 10:10:3 phases were dried, and hydrolyzed with
0.02 N HCl (32) in order to release oligosaccharides from
LLO. The 10:10:3-extracted protein pellet was dried and digested with
Pronase prior to being incubated with endo H in order to release
polymannose-type oligosaccharides from glycopeptides (33). After
desalting on AG-1/AG-50 columns, all oligosaccharide mixtures were
resolved by TLC on silica-coated plastic sheets (Merck) in
n-propyl alcohol/acetic acid/H2O, 3:3:2 for 36-48 h. Radioactive components were detected by fluorography after spraying the dried plates with En3hance®
(PerkinElmer Life Sciences). Standard oligosaccharides were generated
as follows: Man5GlcNAc2,
Man5GlcNAc2-P obtained from
[2-3H]mannose-labeled Thy Dolichyl-P-glucose Synthase Assay--
EBV-transformed
lymphoblasts from a normal individual and patient M. P. were
harvested, washed in phosphate-buffered saline, and resuspended in 50 mM Tris/HCl, pH 8.0 (buffer A), at 4 °C. The cells were
homogenized in buffer A by several passages through a syringe fitted
with a 24 × 0.5-mm needle. The homogenate was centrifuged at
1,500 × gAv for 5 min at 4 °C, and the
resulting supernatant was subjected to ultracentrifugation at
84,000 × gAv for 30 min at 4 °C. The
resulting pellet was resuspended in buffer A, and protein was
determined by the bicinchoninic acid (Sigma) method. Dolichyl-P-glucose
synthase was measured by incubating microsomal membranes in a buffer
containing 1 mM AMP, 25 mM Tris/HCl, pH 8.0, 5 mM MgCl2, 0.1% Triton X-100 and, where
appropriate, 1 µg of dolichyl phosphate, in a final volume of 50 µl. Finally, 0.5 µCi of UDP-[3H]glucose (11.5 Ci/mmol, PerkinElmer Life Sciences) was added to the mixtures which
were then incubated at 37 °C for 20 min. Reactions were terminated
by the addition of 150 µl of buffer, 400 µl of MeOH, and 600 µl
of CHCl3. After vigorous shaking the tubes were
centrifuged, and the upper and inter phases were discarded. The lower
CHCl3 phase, containing
dolichyl-P-[3H]glucose, was washed twice with new upper
phase prior to being assayed for radioactivity by scintillation counting.
Northern Blot--
Cells were rapidly disrupted in 4 M guanidine isothiocyanate, and total RNA was isolated
(35). RNA (20 µg) was denatured, electrophoresed, transferred onto
Positive TM Membrane (Appligene, Illkirch, France), and hybridized with
specific probes (see Table I) as described previously (36). In some
experiments cells were treated with the translation inhibitor emetine
(Sigma, product reference E-2375, dissolved in H2O) for
8 h before RNA extraction. 18 S rRNA was monitored using a
complementary oligonucleotide (37) or, where indicated, methylene blue
staining (38).
Mutation Analysis--
The different primers used for PCR,
sequencing, subcloning, and hybridization are listed in Table I. All
sequencing was performed on both strands and on two independent PCRs.
PCR products were purified with the QIAquick PCR purification kit
(Qiagen SA France) prior to automated sequencing. The genomic DNA from
the patient and parents was extracted with Trizol from
peripheral blood mononuclear cells isolated using a Ficoll-Paque Plus
gradient. Trizol and Ficoll-Paque were used according to the
manufacturer's instructions. DNA containing exon 4 from the
ALG8 gene from the patient was subcloned from genomic DNA
after PCR amplification using the primers described in Table I. The PCR
products obtained in this way were subcloned into the pCR3.1 plasmid
employing the Eukaryotic TA Expression Bi-directional kit (Invitrogen).
HIV-1-derived Lentiviral Vectors and Transduction of
Fibroblasts--
The hALG8 cDNA sequence was amplified
from the human expressed sequence tag (accession number AJ224875,
I.M.A.G.E. Clone ID 265361, obtained from the I.M.A.G.E. Consortium,
Livermore, CA (39)), using the primers indicated in Table I, and
subcloned into the pSIN.PW.eGFP HIV-1-derived transfer vector as
described previously (19). Transduction and radiolabeling of cells were performed as reported before (19).
A Patient with Gastrointestinal Problems but Without Central
Nervous System Deficits Presents with Hypoglycosylated Serum
Glycoproteins--
A girl, M. P., the first child of unrelated
healthy parents, was referred at 4 months of age for edemato-ascitic
syndrome related to severe hypoalbuminemia resulting from protein
losing enteropathy. Upon admission, she had no dysmorphic symptoms and normal psychomotor development, but had severe diarrhea and moderate hepatomegaly. Routine blood tests showed severe hypoalbuminemia (9 g/liter), normal aminotransferase activities, increased cephalin kaolin
time (3 N), and low factor XI (12%), protein C (21%), and antithrombin III (17%) levels. Abdominal ultrasonography,
echocardiography, and cerebral magnetic resonance imaging were normal,
but electroretinography showed slight anomalies. The combined presence
of coagulation factor anomalies and protein losing enteropathy was
suggestive of CDG. This diagnosis was confirmed upon investigation of
the glycosylation status of the serum glycoproteins from the patient by
Western blot as shown in Fig.
1A. Although the
electrophoretic profile of transferrin derived from serum of a normal
subject displays a single band, that from the patient reveals three
distinct components whose migration positions coincide with transferrin species observed to occur in patients previously diagnosed with type I
CDG (Fig. 1A). Initially, the girl required total parenteral nutrition and albumin infusions because of severe digestive
complications. Oral mannose treatment was ineffective, but digestive
indications improved with a low fat diet in association with essential
fatty acid supplementation. After 18 months of dietary treatment, the diarrhea and protein-losing enteropathy were resolved, but despite normal liver function tests there was mild hyperechogen hepatomegaly without portal hypertension, and coagulation anomalies persisted. Psychomotor development continued normally, and electroretinographic observations remained unchanged.
Accumulation of Hypoglucosylated Dolichyl-linked
Oligosaccharides in Skin Biopsy Fibroblasts Obtained from Patient
M. P.--
Further diagnosis was performed by assaying
phosphomannomutase and phosphomannose isomerase (PMI) enzyme activities
that are known to be deficient in CDG Ia and Ib, respectively. However, both these activities were found to be normal (phosphomannomutase, 4.1 units/g total protein (normal >3.4 units/g total protein), and PMI,
8.9 units/g total protein (normal > 5.5 units/g total protein)).
Next, skin biopsy fibroblasts from the patient were subjected to
metabolic radiolabeling with [2-3H]mannose in order to
examine LLO biosynthesis in these cells. After mild acid hydrolysis the
oligosaccharide moieties of LLO from the patient and normal cells were
resolved by TLC. A preliminary experiment revealed that although the
control cells yielded predominantly glucosylated LLO, accumulations of
LLO whose oligosaccharide structures comigrated with
Man9GlcNAc2, and to a lesser extent
Glc1Man9GlcNAc2, were apparent in
the cells from the patient (Fig. 1B). Similar results were
observed when cells from a CDG Ic patient (deficiency in
dolichyl-P-glucose:Man9GlcNAc2-PP-dolichyl
Glc1Man9GlcNAc2-PP-dolichyl
Accumulates upon Treatment of Fibroblasts from the Patient with the
Glucosidase Inhibitor Castanospermine--
When normal and M. P.
fibroblasts were radiolabeled in the presence of the ER glucosidase I
and II inhibitor CST, we noted that LLO profiles were different from
those generated in the absence of this agent. Thus, as demonstrated in
Fig. 2A, whereas the
CHCl3 phase derived from organic solvent extraction of
normal fibroblasts yields LLO containing mainly fully mannosylated
oligosaccharides bearing between zero and three glucose residues
(Glc0-3Man9GlcNAc2), the
chloroform/methanol/H2O (10:10:3) phase was observed to
comprise mainly an LLO possessing the fully glucosylated structure
(Glc3Man9GlcNAc2) that is known to
be efficiently transferred onto glycoprotein in the lumen of the ER.
Similar examination of the oligosaccharide species derived from LLO
recovered from CST-treated normal cells revealed the almost exclusive
appearance of fully glucosylated Glc3Man9GlcNAc2 in both organic
phases. By contrast, CST treatment of the cells from the patient
reduced the accumulation of Man9GlcNAc2 but
brought about substantial increases of
Glc1Man9GlcNAc2 as well as
Glc3Man9GlcNAc2 (Fig.
2B). When a similar experiment was conducted on fibroblasts
derived from a patient diagnosed as having CDG Ic (13, 14), the
glucosidase inhibitor had no effect on the accumulation of
Man9GlcNAc2, and furthermore, no
Glc1Man9GlcNAc2 was apparent under
these conditions (results not shown). These results can be explained by
the glucosyltransferase-glucosidase shuttle proposed by Spiro and
co-workers (40-42). According to this mechanism, represented in Fig.
2B, there are two ways in which
Man9GlcNAc2-PP-dolichyl can be formed in
mammalian cells. These authors showed that in addition to nascent
glucose-containing glycoproteins in the lumen of the ER, glucosylated
LLO are also susceptible to trimming by ER glucosidases, and it was
hypothesized that the ability to both add and remove glucose residues
from LLO allows the cell to regulate the pool size of mature
triglucosylated LLO. Thus, the accumulation of
Man9GlcNAc2-PP-dolichyl observed in patient
M. P. may be due to the deglucosylation, by ER glucosidase II, of a
pool of
Glc1Man9GlcNAc2-PP-dolichyl
that has arisen due to inefficient addition of the second glucose
residue by the hALG8 gene product
(dolichyl-P-glucose:Glc1Man9GlcNAc2-PP-dolichyl
glucosyltransferase, see Fig. 2B).
Examination of LLO Glucosylation in Intact and SLO-permeabilized
Lymphoblasts Derived from Patient M. P.--
Analysis of LLO
biosynthesis in EBV-transformed lymphoblasts from patient M. P.
revealed similar results to those obtained from fibroblasts, except
that the addition of CST to these cells caused a less dramatic
redistribution of glucosylated LLO species when compared with that
observed for fibroblasts (results not shown). Therefore,
EBV-transformed lymphoblasts from this subject were permeabilized with
the plasma membrane pore-forming reagent SLO in order to perform
in vitro analyses of LLO glucosylation. Permeabilized cells
were incubated in either the absence or presence of UDP-Glc as shown in
Fig. 3A. Under these
conditions there is no transfer of oligosaccharide from LLO onto
protein (43), and after these brief 10-min incubations less than 10%
of the LLO fraction is lost (probably as free oligosaccharides (43)).
After permeabilization, LLO in both the patient's and normal cells are found to be partially deglucosylated so that in both cases
Man9GlcNAc2-PP-dolichyl is a major species.
Upon incubation of permeabilized cells with UDP-Glc, LLO is
glucosylated such that the majority of LLO possesses three glucoses
after 5 min. However, it is evident that successful triglucosylation of
LLO is less efficient in the cells from the patient and that the
abnormality in LLO glucosylation is at the level of
Glc1Man9GlcNAc2-PP-dolichyl.
Crucially, these experiments revealed that the initial quantity of
Man9GlcNAc2-PP-dolichyl declines at the same
rate in both cell types, indicating that addition of the first glucose
residue is not limiting in M. P. cells. Furthermore, when the same
experiments were performed in the presence of CST, we were still able
to detect the
Glc1Man9GlcNAc2-PP-dolichyl intermediate, demonstrating that this component is not generated by ER
glucosidase I/II action on fully glucosylated LLO (Fig. 3B).
Finally, in both cell populations LLO glucosylation also occurs to a
lesser extent in the absence of UDP-Glc and is probably driven by an
endogenous pool of dol-P-Glc.
Reduced hALG8 mRNA Expression in Cells from Patient
M. P.--
In yeast, the ALG8 gene thought to
encode the
dolichyl-P-glucose:Glc1Man9GlcNAc2-PP-dolichyl
Identification of Two Mutations Leading to Premature Stop Codons in
the ALG8 Genomic DNA from the Patient--
The availability of the
hALG8 cDNA sequence allowed us to find five partial
genomic sequences from chromosome 11q14. By using these data, we were
able to define the structure of the gene that comprised 13 exons (Fig.
4B). All the exon/intron boundaries follow the AG/GT rule,
with the exception of intron 6 which starts with GC instead of GT. This
observation was confirmed in the four available genomic sequences
comprising exon 6 and the entire 276 bp of intron 6. By using the
hALG8 gene structure, we designed intronic primers (see
Table I) in order to amplify the
different exons. The ALG8 alleles from the patient were
sequenced and compared with those obtained from the genomic DNA from
the parents. As shown in Fig. 5A, two mutations were found
in exon 4 of the ALG8 sequence from the patient: the allele
originating from the father of the patient contained a deletion (413 del C) and that originating from the mother contained an insertion (396 ins A). In order to read the patient's sequence between these two
mutations, we subcloned this region from genomic DNA. Twenty
independent clones corresponding to either of the two alleles were
sequenced and found to occur in equal proportions, and additional
mutations in this region of the gene were not found. The 396 ins A and
413 del C mutations generated premature stop codons (underlined
bases in Fig. 5A) whose
translation is predicted to generate severely truncated polypeptides
(Fig. 5B). We also found a G665A variation in the sequence
of exon 6; the father was found to be heterozygous at this position,
but taking into account that both the mother and the patient possess
only A at this position, and that this variation occurs after the two
premature stop codons, we believe that this variation does not lead to
the hALG8p deficiency and may correspond to a polymorphism.
Effect of Translation Inhibition on the Quantity of ALG8 mRNA
Recovered from Normal and M. P. Lymphoblasts--
It is known that
the presence of premature stop codons can lead to a type of mRNA
degradation that is accomplished by a translation-dependent process known as nonsense-mediated mRNA decay (NMD) (46, 47). In
order to examine the possibility that the low expression of ALG8 mRNA in cells from patient M. P. is the result of
such a process, we treated the cells from the patient and the control with different concentrations of the translation inhibitor emetine, as
has been described previously (48). As shown in Fig.
6, Northern blot analysis reveals that
emetine provokes a concentration-dependent increase of
ALG8 mRNA in both control and M. P. lymphoblasts
suggesting that the message is stabilized in both cell lines. However,
whereas the message is only stabilized 3.5-fold in the control cells
treated with 100 µg/ml emetine, a 20-fold increase is observed in the cells from the patient.
Transduction of M. P. Fibroblasts with hALG8 cDNA--
In
order to demonstrate unambiguously that a deficiency in hALG8p is the
cause of the accumulation of underglucosylated LLO in cells from
patient M. P., we have transduced immortalized M. P. fibroblasts with
hALG8 cDNA using HIV-1-derived lentiviral transfer
vectors. Results presented in Fig. 7 show
that whereas a vector harboring GFP cDNA alone had little effect on
the distribution of LLO in CST-treated M. P. fibroblasts, a vector
containing both hALG8 and GFP cDNA markedly reduced the
appearance of underglucosylated LLO in the cells from the patient and
restored the distribution of LLO to a pattern similar to that observed
in normal, GFP-transduced, fibroblasts treated with CST. In this
experiment it was noted that the distribution of LLO between the
CHCl3 and 10:10:3 organic phases was different from that
usually observed. Although incubation of cells with CST favors the
recovery of triglucosylated LLO in the 10:10:3 phase (see Fig.
2A), we noted much reduced quantities of LLO in the
CHCl3 organic phase in this experiment. At present the
reason for this is not clear, but it is noteworthy that there is
significant variation in the distribution of LLO between the two
organic phases depending on the cell type and treatment (M. P. cells
transduced with GFP; 31% total LLO in CHCl3 phase, M. P.
cells transduced with GFP/ALG8; 10% total LLO in CHCl3
phase).
Transfer of Underglucosylated Oligosaccharides from LLO onto
Glycoprotein in M. P. Fibroblasts--
Finally, we examined the
nature of oligosaccharides that are transferred from LLO onto
polypeptides in the lumen of the ER. Cells were treated with CST and
KIF, an inhibitor of ER mannosidase I and Golgi mannosidase. When
fibroblasts were pulse-radiolabeled with [2-3H]mannose
under these conditions, N-linked oligosaccharides remain untrimmed and may reflect the structures that are transferred from LLO onto polypeptides. Accordingly, whereas in uninhibited control
fibroblasts the predominant N-linked oligosaccharides are
Glc0-1Man9GlcNAc and Man8GlcNAc,
in inhibited cells Glc3Man9GlcNAc is the
predominant species detected (Fig. 8, A and B).
Similar observations were made for the cells from the patient, but, in
inhibited cells, we noted a modest increase in the proportion of
an oligosaccharide migrating as Glc1Man9GlcNAc, when compared with that occurring in CST + KIF-treated control cells,
indicating that either this structure is transferred directly from LLO onto polypeptide or that Man9GlcNAc is
transferred onto polypeptide prior to being post-translationally
monoglucosylated by UDP-glucose:glycoprotein glucosyl transferase (UGGT).
In the present work we demonstrate that cells from a child
displaying serum glycoprotein hypoglycosylation, and some clinical symptoms suggestive of type I CDG, reveal an inefficiency in their ability to add the second glucose residue onto LLO. Under normal radiolabeling conditions the predominant LLO is
Man9GlcNAc2-PP-dolichyl. However, we were
unable to detect any changes in the expression, or mutations, in the
gene that encodes
dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl glucosyltransferase, nor were we able to detect a reduction in dolichyl-P-glucose synthase activity in the cells from this patient. In
fact, striking accumulations of monoglucosylated LLO only became apparent when M. P. fibroblasts were treated with CST, the ER glucosidase I and II inhibitor. Although the LLO species recovered from
the 10:10:3 organic phase are generally more heavily glucosylated than
those species recovered from the CHCl3 fraction, our
experience with primary human fibroblasts has shown that the proportion
of total LLO that is glucosylated is quite variable. At present the factors that lead to this variability are not understood. However, where control pulse-radiolabeling experiments do yield large quantities of Man9GlcNAc2-PP-dolichyl, parallel
incubations conducted in the presence of CST lead to a block in the
appearance of this structure and a concomitant appearance of
triglucosylated LLO, suggesting that the
glucosyltransferase/glucosidase shuttle functions under certain
cellular growth/stress conditions. Indeed it has been shown that the
deglucosylating reactions occur when tissue slices or cells are
deprived of oxygen (41), and it has been suggested that this stress
leads to inefficient glucosylation of LLO due to reduced availability
of dolichyl-P-glucose (41). More evidence for an ALG8p deficiency in
M. P. cells was obtained by examining LLO glucosylation in
SLO-permeabilized lymphoblasts. In this in vitro system we
were able to demonstrate that whereas M. P. cells could efficiently
add the first glucose to LLO, the addition of the second glucose
residue was slow when compared with that observed in control cells. It
was noted that in the absence of exogenously added UDP-Glc, the cells
from the patient accumulated predominantly
Man9GlcNAc2-PP-dolichyl, whereas in the
presence of the sugar nucleotide
Glc1Man9GlcNAc2-PP-dolichyl was
more abundant than Man9GlcNAc2-PP-dolichyl.
Thus, successful triglucosylation of LLO in cells from the patient may
be particularly sensitive to cellular UDP-Glc levels that are known to
fluctuate during hypoxia (49) and glucose insufficiency (50).
Interestingly, examination of LLO biosynthesis in a yeast strain
deficient in ALG8p reveals the accumulation of
Glc1Man9GlcNAc2-PP-dolichyl and not
Man9GlcNAc2-PP-dolichyl as has been observed
here in M. P. fibroblasts (51). It is not clear why yeast and
mammalian cells deficient in ALG8p should behave differently in this
respect, but although mammalian glucosidases have been shown to be
active toward glucosylated LLO (52, 53), we have been unable to find any data in the literature indicating that the yeast glucosidases are
active toward this substrate. In fact, in a yeast strain deficient in
both ALG8p and ER glucosidase I, the structure
Glc2Man9GlcNAc2 was detected
N-linked to protein (51). In that study it was proposed that
the elevated levels of
Glc1Man9GlcNAc2-PP-dolichyl, caused
on the one hand by the absence of ALG8p and on the other by the slow
transfer of Glc1Man9GlcNAc2 onto
protein, allowed ALG10p to "cap" the monoglucosylated LLO with an
Whatever the significance of the glucosyltransferase/glucosidase
shuttle, it is apparent that treatment of cells from the patient with
CST gave us an early clue as to the underlying defect in this patient.
Indeed, incubations performed in the presence of the glucosidase
inhibitor allowed us to identify
Glc1Man9GlcNAc2-PP-dolichyl as the
primary accumulating LLO intermediate in this case, and it is likely
that, in other CDG Ix cases where
Man9GlcNAc2-PP-dolichyl is seen to accumulate,
the use of CST will help pinpoint which of the three
glucosyltransferases (hALG6p, hALG8p, or hALG10p) is at fault.
We went on to demonstrate that in cells derived from this patient,
there is a dramatically reduced expression of the gene (hALG8) which encodes the enzyme that attaches the second
glucose onto growing LLO. First, the patient possesses very low levels of hALG8 mRNA, and second, the gene from the patient
contains two mutations in exon 4 which lead to PSCs. The reduced
expression of both the ALG8 alleles from the patient could
result from either a reduced transcription rate or a decrease in
message stability. NMD is responsible for the degradation of mRNA
containing PSCs, but this process only takes place if the PSC is ~50
bp from the last intron/exon junction of the gene in question (46, 47), which is the case for both the PSCs found in the patient. As NMD is
known to be a translation-dependent process, we treated
EBV-transformed lymphoblasts from patient M. P. with emetine, a
translation inhibitor known to stabilize certain mRNA transcripts
containing PSCs. Indeed, our results show that this reagent stabilizes
the patient's ALG8 transcript such that its level is the
same as that of its normal counterpart found in control cells treated
with the same concentration of the drug. We noted that emetine also
increases the expression of ALG8 mRNA in normal cells.
In fact, in yeast many of the ALG gene mRNAs behave like
the transcripts of "early growth-response" genes that are known to
be stabilized in the presence of protein synthesis inhibitors (54-56).
To date, this is the first time that NMD has been shown to be operative
in cells from patients with CDG. In other diseases it has been shown
that when NMD of PSC-containing mRNA is operational, the symptoms
are less severe than in those patients for which the mutant mRNA is
stable (46, 47). This is probably due to the fact that NMD can clear
the cell of PSC containing open reading frames, which, if translated
would lead to the accumulation of potentially deleterious, dominant
negative, truncated proteins (46, 47). If any mRNA is translated in patient M. P., it is apparent that only the first ~20% of the protein, containing only two potential transmembrane regions, is
produced rendering it highly unlikely that these two alleles could give
rise to active proteins. The dolichyl-P-monosaccharide requiring
mannosyltransferases and glucosyltransferases of the LLO pathway are
extremely hydrophobic enzymes comprising 10-14 transmembrane regions
(45). The importance of the transmembrane regions for enzyme function
is attested to by the observation that several of the disease causing
mutations in CDG I are found in or near the membrane spanning regions
that occur along the entire length of the defective
glycosyltransferases. By taking into account the paucity of
hALG8 mRNA, the above observations strongly suggest that
patient M. P. has little or no ALG8p activity.
The paradox raised by our work is that despite the probable
low leakiness of the hALG8 mutations presented here, the
patient's disease presents as a CDG with a less severe clinical
picture than that generally associated with CDG Ic in which a deficit in LLO glucosylation is also the underlying cause. It is clear that
cells from this patient manage to synthesize substantial quantities of
fully glucosylated LLO. In fact, incorporation of [2-3H]mannose into glycoproteins extracted from M. P.
fibroblasts was as efficient as that observed in control fibroblasts.
Furthermore, although substantial amounts of underglucosylated LLO are
generated when M. P. cells are treated with CST, the specificity of
oligosaccharyltransferase ensures that fully glucosylated
oligosaccharides are preferentially transferred from LLO onto
polypeptides in the lumen of the ER (Fig.
8). Surprisingly, although we noted that
when the fibroblasts from the patient are treated with CST there are
increased amounts of triglucosylated LLO, we were unable to show that
this allowed greater transfer of radioactive oligosaccharides from LLO
onto glycoprotein (results not shown).
3-glucosyltransferase (hALG8p), which catalyzes this reaction.
Sequencing of hALG8 genomic DNA revealed exon 4 to
contain a base deletion in one allele and a base insertion in the
other. Both mutations give rise to premature stop codons predicted to
generate severely truncated proteins, but because the translation
inhibitor emetine was shown to stabilize the hALG8 mRNA
from the patient to normal levels, it is likely that both transcripts
undergo nonsense-mediated mRNA decay. As the cells from the patient
were successfully complemented with wild type hALG8
cDNA, we conclude that these mutations are the underlying cause of
this new CDG I subtype that we propose be called CDG Ih.
INTRODUCTION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-glucosyltransferase (11-13); Id,
dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl
3-mannosyltransferase (14); Ie, dolichol-P-Man synthase I (15, 16);
If, the MPDU1 gene product known to facilitate
dolichyl-P-Glc and dolichyl-P-Man utilization (17, 18); and Ig,
dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl
6-mannosyltransferase (19-21)). Although there are too few patients representing each subtype of the disease to draw precise
genotype/phenotype relationships, CDG Ib (PMI deficiency) generally
presents as a disease in which central nervous system defects are
absent (9, 10). Often, PMI deficiency leads to a less severe form of
the disease because as well as the PMI-catalyzed conversion of fructose 6-phosphate to mannose 6-phosphate the cell possesses an alternative route for the generation of the latter intermediate. In fact, serum
mannose can be taken up by cells (22, 23) and phosphorylated by
hexokinase to yield mannose 6-phosphate. In PMI deficiency, the flux
through this alternative metabolic route can be augmented by giving
patients oral mannose, a treatment that has been shown to reverse serum
glycoprotein hypoglycosylation and alleviate the symptoms of this form
of the disease (24, 25).
3-glucosyltransferase (hALG8p). Sequencing of the hALG8
genomic DNA from the patient revealed each allele to possess mutations that generate premature stop codons.
EXPERIMENTAL PROCEDURES
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INTRODUCTION
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DISCUSSION
REFERENCES
1 mouse
lymphocytes was treated with alkaline phosphatase (34); a mixture
of Glc1-3Man9GlcNAc2
oligosaccharides was obtained by mild acid hydrolysis of LLO
recovered from [2-3H]mannose-labeled HepG2
hepatocellular carcinoma cells;
Man9-8GlcNAc2 oligosaccharides were
obtained from the cytosolic fraction of SLO-permeabilized HepG2
hepatocellular carcinoma cells as described previously (31).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Biochemical diagnostics performed on serum
and skin biopsy fibroblasts from patient M. P. A,
plasma samples from a normal subject (N), a CDG Ia patient
(Ia), a CDG Ic patient (Ic), and patient M. P.
(MP) were subjected to SDS-PAGE and Western blot analysis.
The numbers on the right-hand side of the
electrophoretogram indicate the migration positions of transferrin
species bearing 0, 1, and 2 N-glycan chains.
B, fibroblasts from a normal subject (N),
patient M. P. (MP), and a patient diagnosed with CDG Ic
(Ic) were pulse-radiolabeled with
[2-3H]mannose and extracted with organic solvents. LLOs
recovered from the CHCl3 phase were treated with mild acid,
and the released oligosaccharides were analyzed by TLC as described
under "Experimental Procedures." Radioactive components were
visualized by fluorography, and the migration positions of standard
oligosaccharides are indicated: M5,
Man5GlcNAc2; M9,
Man9GlcNAc2;
G1M9,
Glc1Man9GlcNAc2;
G2M9,
Glc2Man9GlcNAc2;
G3M9,
Glc3Man9GlcNAc2. C,
lymphoblasts from a normal subject (N) and patient M. P.
(MP) were assayed for dolichyl-P-glucose synthase activity,
in both the absence ( Dol-P) and presence
(+Dol-P) of dolichyl phosphate as described under
"Experimental Procedures."
3-glucosyltransferase) were examined, but in these fibroblasts the
monoglucosylated structure was less apparent (Fig. 1B).
These observations suggested that in patient M. P. there is
inefficient addition of glucose residues onto the growing LLO in the
lumen of the ER. However, enzymic assay of dolichyl-P-glucose synthase
(hALG5p) revealed that this enzyme that is responsible for the
synthesis of the glucose donor molecules required for LLO glucosylation
was not impaired in the fibroblasts from the patient (Fig.
1C). Finally, the
dolichyl-P-glucose:Man9GlcNAc2-PP-dolichyl glucosyltransferase (hALG6) gene from the patient was
sequenced, but no mutations were found.
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Fig. 2.
LLO glucosylation in M. P. cells and the
glucosyltransferase/glucosidase shuttle. A, normal
(N) and M. P. (MP) fibroblasts were
pulse-radiolabeled with [2-3H]mannose in either the
absence ( ) or presence (+) of CST. Cells were extracted with organic
solvents as described under "Experimental Procedures," and LLO were
recovered from both the CHCl3 (chloroform), and
chloroform/methanol/H2O, 10:10:3 (10:10:3),
phases. Subsequent to mild acid hydrolysis of the lipid-linked species,
the released oligosaccharides were resolved by TLC. The migration
positions of standard oligosaccharides are indicated, and the
abbreviations are defined in the legend to Fig. 1B.
B, the glucosyltransferase/glucosidase shuttle which
has been proposed by Spiro and co-workers (40-42) to operate in
mammalian cells. In human cells the biosynthetic steps are thought to
be carried out by the three glucosyltransferases which are now known to
be encoded by the human orthologs of the yeast ALG6,
ALG8, and ALG10 loci. The degradative steps have been
proposed to be catalyzed by ER glucosidases I and II (GLS1,
and -2 gene products, respectively).
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Fig. 3.
Analysis of LLO glucosylation in
SLO-permeabilized lymphoblasts derived from patient M. P.
A, EBV-transformed lymphoblasts from a normal person
(N) and the patient (MP) were pulse-radiolabeled
with [2-3H]mannose prior to being permeabilized with SLO
as described under "Experimental Procedures." The permeabilized
cells were then incubated in either the absence or presence of UDP-Glc
for the indicated times at 37 °C. Subsequently, the cells were
extracted with organic solvents, and LLO recovered from the
CHCl3, and 10:10:3 phases were pooled. 40,000 cpm of the
oligosaccharides liberated from LLO by mild acid hydrolysis were
resolved by thin layer chromatography as described in the legend to
Fig. 1. The abbreviations used are as described for Fig. 1.
B, a similar experiment was performed in either the
presence of UDP-Glc alone (left panel) or UDP-Glc and 4 mM CST (right panel). After resolution of
oligosaccharides released from LLO, the indicated radioactive species
were eluted from the thin layer chromatography plate and quantitated by
scintillation counting. The recovery of each oligosaccharide is
expressed as the % of the total (M9 + G1M9 + G2M9 + G3M9). Open circles,
cells from control subject; closed circles, cells from
patient M. P.
3-glucosyltransferase has been cloned (44), and the complete
sequence of the putative human ortholog of this gene (EMBL:
BC001133 and AJ224875) is available (45). This information allowed us
to create a probe in order to examine hALG8 mRNA
in the cells from the patient by Northern blot. As demonstrated in Fig.
4A, there was a dramatic
reduction in the quantity of the hALG8 message in the cells
from the patient when compared with that observed in normal
fibroblasts. By contrast, the level of the hALG6 message was
similar in the two cell populations. As the quantity of the
hALG8 message in the cells from the patient was less than
10% of that observed in control cells, it is apparent that both of the
transcripts of the alleles from the patient at this locus are affected
(Fig. 4A).
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Fig. 4.
Examination of hALG8 mRNA expression in
fibroblasts from patient M. P. and determination of hALG8
genomic organization. A, Northern blot analysis of
hALG6 and hALG8 from control (N) and M. P. (MP)
fibroblasts. Total RNA was extracted from pre-confluent fibroblasts.
Aliquots of the same total RNA preparation were loaded in duplicate
on the gel. After blotting, the membrane was cut in two. One membrane
was hybridized with the ALG8 probe and the other with the
ALG6 probe. Whereas the membrane that was hybridized with
the ALG8 probe was stripped and rehybridized with the 18 S
probe, the membrane that was hybridized with the ALG6 probe was stained
with methylene blue. The blot revealed identical coloring of the 18 S
region for all the lanes (not shown). The probes used for visualizing
hALG6 and hALG8 mRNA were generated by
32P labeling of the PCR products amplified by primer couple
6S/6AS or 1BisS/7TerAS (see Table I), respectively. B,
genomic organization of the human ALG8 gene was determined
by LFASTA (61) comparison of two mRNA sequences (BC001133 and
AJ224875) with 5 partial genomic sequences from chromosome 11q14
(GenBankTM accession numbers AP001805, AC023532, AP002520,
AP000571, and AP001447). Exon (uppercase letters) and intron
(lowercase letters) boundaries follow the ag/gt (in
bold) rule except for intron 6. The numbering of the
cDNA sequence starts with ATG.
Primers used in this study
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Fig. 5.
The genomic DNA from the patient contains two
mutations that lead to premature stop codons in the hALG8
gene. A, the region of hALG8 exon 4 in
which two mutations were found (numbering of the cDNA sequence
starts with ATG). The mutated allele from the father contained a
deletion (413 del C), and the mutant allele from the mother contained
an insertion (396 ins A). Both mutations induced frame
shifts that led to premature stop codons (underlined).
B, amino acid sequences were generated from normal
hALG8, and the two mutant alleles and Kyte-Doolittle (62)
hydropathy plots were derived. A hydropathy index greater than 2 often
indicates the presence of a series of amino acids capable of forming a
transmembrane region. New peptide sequences between the frameshift and
the premature stop codon are underlined.
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Fig. 6.
The effect of protein synthesis inhibition on
ALG8 transcript levels in M. P. lymphoblasts. A,
EBV-transformed lymphoblasts obtained from the patient (MP)
or a normal subject (N) were treated with the indicated
concentrations of emetine for 8 h. Total RNA was then extracted
from the cells and subjected to Northern blot analysis as described for
Fig. 4. B, ALG8 mRNA was quantitated by densitometric
scanning, and the results were normalized with respect to the quantity
of 18 S ribosomal RNA detected in the same gel lanes.
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Fig. 7.
Transduction of immortalized M. P.
fibroblasts with wild type hALG8 cDNA. HIV-1-derived
lentiviral vectors containing cDNA encoding either GFP
(GFP) alone or GFP and wild type hALG8 in a bicistronic
arrangement were constructed as described under "Experimental
Procedures." Fibroblasts from patient M. P. (MP),
immortalized with a plasmid encoding the simian virus 40 large T
antigen, or fibroblasts from a control subject (N) were
incubated with 100 multiplicities of infection of the transfer vectors.
Two days later, the cells were pulse-radiolabeled with
[2-3H]mannose in the presence of 2 mM CST for
30 min as described under "Experimental Procedures." The cells were
subsequently extracted with organic solvents to yield LLO that
partitioned in the CHCl3 phase and in the
CHCl3/MeOH/H2O, 10:10:3, phase (10).
Oligosaccharides were liberated from LLO and resolved by thin layer
chromatography. The migration positions of standard oligosaccharides
are shown to the left of the chromatogram; the abbreviations
are as described in the legend to Fig. 1. The indicated
oligosaccharides were eluted from the chromatography plates and
quantitated by scintillation counting, and the recovery of each
oligosaccharide is expressed as the percent of the total
(M9 + G1M9
+ G3M9).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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2-linked glucose residue. Apparently then, in yeast and mammalian
cells accumulated Glc1Man9GlcNAc2-PP-dolichyl may
have different fates, and in the latter cell type, under certain
conditions, this structure can be deglucosylated, whereas in yeast it
can be glucosylated by ALG10p.
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Fig. 8.
Thin layer chromatographic examination of
oligosaccharides found N-linked to glycoproteins
isolated from normal and M. P. fibroblasts. A,
fibroblasts from the patient (MP) and normal (N)
fibroblasts were pulse-radiolabeled with
[2-3H]mannose in either the absence ( ), or
presence (+) of the glycosidase inhibitors castanospermine and
kifunensin (C+K). The cellular glycoproteins were
sequentially treated with Pronase and endo H, and the released
oligosaccharides were resolved by thin layer chromatography as
described under "Experimental Procedures." B, after
visualization by fluorography, the oligosaccharide components derived
from the glycosidase-inhibited (C+K) cells were eluted from
the chromatography plate and quantitated by scintillation counting. The
recovery of the individual components is expressed as a percentage of
the total amount of oligosaccharide recovered for each of the two cell
populations. The abbreviations used are: M8,
Man8GlcNAc; M9, Man9GlcNAc;
G1M9,
Glc1Man9GlcNAc;
G3M8,
Glc3Man8GlcNAc;
G3M9,
Glc3Man9GlcNAc.
The fact that fibroblasts and lymphoblasts from patient
M. P. are able to synthesize triglucosylated LLO suggests that one or
both of the alleles can give rise to an active transferase, or an as
yet unidentified glucosyltransferase, with little or no similarity to
already described glucosyltransferases, is capable of carrying out this
reaction. Alternatively, when the
Glc1Man9GlcNAc2-PP-dolichyl pool is
elevated (sufficient endogenous UDP-Glc, or addition of CST to cells),
this monoglucosylated LLO may be glucosylated again by either ALG6p or
ALG10p. Should this be the explanation, it is more likely that the
second glucose is added by ALG6p. First, biochemically, it has not been
possible to distinguish the enzymes (ALG6p and ALG8p) that add the
first two 3-linked glucoses onto LLO (40, 57, 58), suggesting that
they may have very similar properties. Second, ALG10p adds an
2-linked glucose to the LLO, and this enzyme has been shown to have
distinct biochemical properties to those of ALG6p and ALG8p. Third,
analysis of the amino acid sequences of the three glucosyltransferases
reveals that ALG6p and ALG8p are more closely related to each other
(59) than either of them are related to ALG10p (45, 60).
In conclusion, we have identified a new subtype of CDG I, which we
suggest be called CDG Ih, in which there is a deficiency in hALG8p, the
enzyme which adds the second glucose onto growing LLO. The gene
encoding this enzyme was found to contain mutations that generate
premature stop codons in both of the alleles from the patient. Further
work is underway in order to understand how cells from this patient
synthesize fully glucosylated LLO despite possessing such apparently
drastic mutations, and why the clinical picture of this patient is so
mild when compared with that observed in patients with CDG I subtypes a
and c-g.
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ACKNOWLEDGEMENTS |
---|
We thank M. P. and family for cooperating with this study. Dr. Thierry Levade (INSERM U466, 1 Avenue Jean Poulhes, 31403 Toulouse, France) is thanked for the immortalization of fibroblasts. Recombinant lentiviral vectors were provided by the Gene Vector Production Network (Généthon, Evry, France), which is funded by the Association Française Contre les Myopathies.
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FOOTNOTES |
---|
* This work was supported by institutional funding from INSERM, an INSERM-AFM Research Network Grant "Réseau de Recherche sur les CDG," and by a grant from the Association Vaincre Les Maladies Lysosomales. This work was presented at the Euroglycan Meeting, April 18-20, 2002, Sitges, Spain (2).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.
§ Members of the French CDG Research Network (INSERM-AFM).
To whom correspondence should be addressed: INSERM U504,
Bâtiment INSERM, 16 Ave. Paul Vaillant-Couturier, 94807 Villejuif Cedex, France. Tel.: 33-1-45-59-50-47; Fax: 33-1-46-77-02-33; E-mail:
moore@vjf.inserm.fr.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M211950200
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ABBREVIATIONS |
---|
The abbreviations used are:
CDG, congenital
disorders of glycosylation;
LLO, lipid-linked oligosaccharide;
ER, endoplasmic reticulum;
endo H, endo--D-N-acetylglucosaminidase H;
PSC, premature stop codon;
NMD, nonsense-mediated mRNA decay;
SLO, streptolysin O;
EBV, Epstein-Barr virus;
CST, castanospermine;
KIF, kifunensin;
GFP, green fluorescent protein;
PMI, phosphomannose
isomerase;
HIV, human immunodeficiency virus.
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