Current Drug Targets, 2009, 10, 477-482 477

Structure, Expression, and Regulation of UDP-GlcNAc: Dolichol

Phosphate GlcNAc-1-Phosphate Transferase (DPAGT1)

Roger K. Bretthauer*

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA

Abstract: Glycosylation of proteins on asparagine amino acids (N-linked) in proteins of eukaryotic cells is initiated by the
biosynthesis of dolichol-pyrophosphate-N-acetylglucosamine from dolichol-phosphate and UDP-N-acetylglucosamine.
The enzyme catalyzing this reaction, UDP-GlcNAc:Dolichol Phosphate GlcNAc-1-Phosphate Transferase (DPAGT1), has
been further characterized in several cell types with respect to its gene, gene products, membrane topology, functional
sites, lipid dependence, and metabolic regulation. This review summarizes these properties as an update from an earlier
detailed and critical review by Lehrman (Lehrman, M. A. (1991) Glycobiology, 1, 553-562).

INTRODUCTION ject of this review. Properties of the enzyme, UDP-Glc-

Glycosylation of proteins in eukaryotic cells is an impor-
tant, and often life-dependent, process affecting both struc-
ture and function of the resulting glycoproteins. Two major
covalent glycosylation types are referred to as N-
glycosylation and O-glycosylation, the first being character-
ized with N-acetylglucosamine (GlcNAc) covalently at-
tached to the side chain amide nitrogen of an asparagine
residue in the protein and the second being characterized
with a hexose covalently attached to the side chain hydroxyl
oxygen of a serine or threonine residue of the protein. The
covalently attached hexose residues in both types are gener-
ally further substituted with additional sugar residues to yield
the final protein-bound oligosaccharide (although O-linked
monosaccharide structures are more commonly found in
many glycoproteins). This review will concern only the N-
linked type of protein glycosylation and will detail only the
initial biosynthetic reaction leading to the final presence of a
covalently-bound GlcNAc residue on asparagine. As has
been well-established in many eukaryotic systems, the aspar-
agine-linked GlcNAc results from transfer to the protein of
an oligosaccharide containing fourteen sugar residues, with
GlcNAc being the reducing-end residue which becomes co-
valently attached to asparagine. This unique process is con-
sidered to be a cotranslational process, occurring in the
rough endoplasmic reticulum, and utilizes a lipid-linked oli-
gosaccharide, Glc3Man9GlcNAc-GlcNAc-P-P-dolichol, as
substrate oligosaccharide donor.
Several earlier review articles have documented the dis-
covery, biosynthesis, and involvement in protein glycosyla-
tion of dolichol-linked saccharides, and of other aspects (in-
hibitors, topography, subcellular location) of the enzymes
involved [1-10]. The dolichol cycle in the rough endoplasmic
reticulum, leading to the biosynthesis of the lipid-linked oli-
gosaccharide, is initiated with the synthesis of dolichol-P-P-
GlcNAc via an enzyme-catalyzed reaction which is the sub-
*Address correspondence to this author at the Department of Chemistry and
Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA;
E-mail: Roger.K.Bretthauer.1@nd.edu
NAc:dolichol phosphate N-acetylglucosamine-1-phosphate
transferase (GPT), from several biological sources have been
described, and leading references will be found in the fol-
lowing discussion. The reader is particularly directed to the
review article on the biosynthesis of dolichol-P-P-GlcNAc
that was published in 1991 by Lehrman [10]. Much of that
early information will not be repeated in this current review.
TUNICAMYCIN INHIBITION OF GPT
The unique feature of this enzyme to be specifically in-
hibited by the antibiotic tunicamycin has been of utmost im-
portance in characterizing the GPT gene and its expression
(transcription and translation) and in understanding the cellu-
lar functions of the GPT reaction products. Resistance to this
antibiotic has been utilized as a selection marker for GPT in
several different cell types [11-13]. Inhibition or deletion of
GPT in cells results in preventing synthesis of the lipid-
linked oligosaccharide that is required for glycosylation of
asparagine residues in proteins, and thus the numerous bio-
logical functions of N-linked ollgosaccharides will be altered
or prevented.
GPT GENE, GENE PRODUCTS, AND MEMBRANE
TOPOLOGY
Tunicamycin-resistant (200-fold compared to wild type)
CHO cells were shown to have an increased (15-fold) GPT
specific activity [12]. A clonal derivative of these cells ex-
hibited a 550-fold increased resistance to tunicamycin, a 15-
fold increased GPT specific activity, and some alteration in
the protein N-linked oligosaccharides [14]. The latter effect
was shown to be a probable result of a decreased cellular
level of dolichol-P-mannose (50-fold decrease compared to
wild type cells) that results from depletion of the cellular
dolichol-P pool due to increased levels of GPT. Increased
levels of GPT in this CHO clone was supported by demon-
strating increased levels of a cDNA that hybridized with the
yeast ALG7 gene, and contained an open reading frame sup-
porting a protein of 408 amino acids with a molecular mass

1389-4501/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.

478 Current Drug Targets, 2009, Vol. 10, No. 6 Roger K. Bretthauer

of 44.9 kDa (comparable to the ALG-7 gene of yeast) [15, ing at the 5’-noncoding sequence and containing the ATG
16]. initiation codon and the last exon containing the TGA termi-
nation codon and two polyadenylation sites. Correlation of
Other CHO cells that were 80-fold resistant to tunicamy-
the introns and exons with the amino acid sequence predicted
cin and containing a 10-fold increase in GPT activity were
utilized to identify a 6.6 kb segment of a gene that was ho- membrane spanning domains (using the 10 transmembrane
model) indicated that most introns were located between the
mologous to a segment of the yeast ALG7 gene [17]. DNA
exons coding for the various transmembrane domains (do-
sequencing revealed an amino acid sequence that was 92%
mains 1, 2, 7, and 10 being in individual exons, and exons 3,
conserved with the corresponding yeast sequence. Increased
4, and 6 each containing two domain sequences). A better
levels of transcribed RNA of 2.0 and 2.2 kb were also ob-
correlation of exon and intron sequences with the membrane
served. The complete sequence of the cDNA clone for the
CHO GPT [18] predicted a corresponding protein of 408 spanning segments was seen with the 8 transmembrane
model, having all transmembrane segments other than 3 and
amino acids with a molecular weight of 46,197. Features of
4 being in individual exons. Two potential dolichol recogni-
the amino acid composition and sequence predicted ten
tion sites were localized to transmembrane segment 2 and 7
membrane hydrophobic spanning segments, four N-glyco-
for the 10 transmembrane model or 2 and 6 for the 8 trans-
sylation sites (non-glycosylated), and two potential dolichol
membrane model. Other aspects of this study indicated the
recognition sites (PDRS). A model of the membrane-
spanning protein that includes these various predicted seg- presence of several transcription initiation sites, the most
prevalent being approximately 200 nucleotides upstream
ments is presented. Alignment with the yeast ALG7 amino
from the initiation codon. The only difference in the amino
acid sequence revealed close identity for many segments of
acid coding region between this genomic sequence and the
the protein. Expression of the cDNA in COS-1 cells resulted
cDNA sequence was at nucleotide 280, being G in the gene
in a 5- to 7-fold increase in membrane-associated GPT activ-
and A in the cDNA. This would result in cysteine in the gene
ity that was inhibited by tunicamycin. These results, along
with other experiments utilizing polyclonal antibodies di- protein product and tyrosine in the cDNA protein product.
The authors conclude this change is probably due to RNA
rected to the CHO protein [19] verified the cDNA did code
editing rather than genetic polymorphism.
for the GPT enzyme protein rather than for some other pro-
tein perhaps influencing the GPT reaction.
FUNCTIONAL SITES IN GPT
Unique topological features of the ER-associated hamster
GPT that distinguish it from the typical and well-documen- Functional aspects of the two dolichol recognition sites
were examined by generating mutations in the sites at amino
ted type II glycosyltransferases associated with Golgi mem-
acid residues 68 to 78 or 223 to 233 [20]. Expression in
branes have been further elucidated [22]. A variety of tech-
CHO cells of GPT containing either one of the two mutated
niques, including antibodies to, insertion of epitope tags into,
PDRS revealed no significant increase in in vitro microsomal
and mutagenesis of, proposed membrane-spanning loops,
GPT enzme activity, showing both sites are required for
yielded results consistent with the multiple membrane span-
ning model. Specifically, loops between membrane-spanning catalytic activity. However, expression of either mutated
GPT resulted in cellular tunicamycin resistance, implying the
segments 1 and 2 and between 9 and 10 were shown to be
catalytically-inactive GPT is still capable of binding the tu-
cytosolic, whereas the loop between spanning segments 6
nicamycin inhibitor.
and 7 was lumenal. Additionally, the carboxyl terminus pre-
viously described was lumenal. The cytosolic loop between Functional aspects of the carboxyl terminus of GPT (408
segments 9 and 10 was shown to contain at least two essen- amino acids) expressed in either CHO or COS cells showed
tial amino acid segments for expression of active enzyme the carboxyl terminal 11 amino acids were not required for
activity in COS and CHO cells, with arginine 303 being in- expression of enzyme activity but removal of three addi-
dividually essential. These mutated forms of catalytically tional amino acids (Phe-Ser-Ile) from the 397-amino acid
inactive GPT still expressed immunologically active GPT protein eliminated such protein expression (lack of enzyme
protein. activity) and immunoblot analysis but retaining mRNA syn-
Studies on the mouse GPT have been carried out by thesis) [21]. The specificity of this tripeptide sequence was
shown by lack of expression of the 397-amino acid protein
Vijay and coworkers [39]. The cDNA sequence, derived
containing the hydrophobic sequence Leu-Met-Trp at the
from mouse mammary gland mRNA, contained an open
carboxyl terminus. However, addition to this latter mutated
reading frame for a 410 amino acid protein of molecular
structure of the remaining normal 11 amino acids did restore
mass 46.472 kDa. Two dolichol recognition sequences and
enzyme expression, thus demonstrating a “built-in functional
four N-glycosylation sequences were present, with an overall
96% idendity in sequence to the hamster GPT sequence. The redundancy” of the carboxyl terminus that is located in
membrane spanning segment 10 of the proposed model of
GPT gene was assigned to mouse chromosome 17 by use of
membrane-associated GPT.
somatic mouse/hamster cell hybrids and PCR for a 3’- non-
coding region (194 bp) of the mouse GPT cDNA. Those cell
OLIGOMERIZATION AND LIPID-DEPENDENCE OF
hybrids lacking chromosome 17 also lacked this particular
FUNCTIONAL GPT
segment of the cDNA.
Possible oligomerization (dimerization) of the hamster
Further studies with the isolated mouse genomic DNA
GPT noted in topological experiments was supported by
revealed the GPT gene to span a 7.5 kb segment [41]. This
chemical cross-linking studies [23]. Treatment of CHO
segment contained 9 exons and 8 introns: the first exon start-
membranes with Bis(sulfosuccinimidyl)suberate, followed
Structure, Expression, and Regulation of UDP-GlcNAc
by monitoring with a GPT polyclonal antibody, revealed the
monomeric 34-kDa protein (migration in SDS electrophore-
sis of the normal 46-kDa GPT protein) and a 67-kDa protein
assumed to be a dimeric form. The in vitro formation of this
dimeric form was enhanced by the presence of neutral deter-
gent (Nonidet P-40) but was not affected by substrates of the
GPT reaction (dolichol-P and UDP-GlcNAc). Expression of
the catalytically inactive R303K mutant GPT, shown to exist
in dimeric form, resulted in inhibition of endogenous mem-
brane GPT activity and also of co-expressed normal GPT
activity. These experimental results were consistent with the
formation of heterodimeric forms of the active and inactive
monomeric forms of GPT, lending support to the chemical
crossslinking results. As the observation of dimeric forms of
these GPTs required detergent extraction of membranes, the
physiological significance of the dimeric form in in vivo en-
zyme activity and/or regulation of activity remained open to
further studies.
Radiation inactivation of GPT activity in rat lung micro-
somes was employed in attempts to determine the functional
molecular weight (radiation target size) of the membrane-
embedded enzyme [24]. The method utilized (see reference
25 for details) was based upon irradiation of standard puri-
fied enzymes and constructing a graph of the inactivation
rate constants versus the previously reported functional mo-
lecular weights (as determined by radiation inactivation).
From this standard curve, the inactivation rate constant of the
rat lung microsomal GPT corresponded to a functional mo-
lecular weight of 79,400 +/- 8000. From the reported value
of 46kDa from the gene sequence for the CHO enzyme, and
thus 92kDa for the dimeric form, the rat lung value could
represent activity of a dimeric GPT enzyme. This experimen-
tally determined functional molecular weight is open to
broad interpretation, mainly because the detailed method
utilized compared functionality (catalytic activity) of a puri-
fied protein in an aqueous environment to that of a protein
embedded in a native hydrophobic membrane. Dependence
of the in vitro rat lung GPT activity on specific phospholip-
ids has been demonstrated by delipidation of the membranes
resulting in complete loss of enzyme activity recoverable by
addition of phospholipids [26-28] and by demonstrating en-
hancement of enzyme activity in intact membrane vesicles
by specific phospholipids [29]. Thus the observed functional
molecular weight derived from radiation inactivation could
be the result of a membrane phospholipid-protein complex
being the catalytic component. As an example of supporting
evidence for this interpretation, radiation inactivation of
hyaluronan synthase in membranes of Streptococcus was
considered to yield a functional catalytic mass corresponding
to a monomeric protein plus 16 cardiolipin molecules [30].
Other studies were carried out with the vertebrate (frog)
hyaluronan synthase and the fusion synthase protein with
jellyfish green fluorescent protein, both expressed in yeast
[31]. Irradiation of the yeast membranes yielded results in-
terpreted as a functional synthase mass and a fusion syn-
thase-green fluorescent protein mass corresponding respec-
tively to a monomeric enzyme and the monomeric fusion
protein. In both cases, an additional mass of approximately
20 kDa was considered to result from functional phospholip-
ids.
Current Drug Targets, 2009, Vol. 10, No. 6 479
The solubilized and partially purified GPT from pig aorta
has been shown to be stabilized and activated by phospholip-
ids, phosphatidylglycerol being best. In addition, GDP-Man ,
Dol-P-Man, and a heat-stable factor activated enzyme activ-
ity, but presumably in independent ways as the effects were
additive to phospholipid stimulation [32].
Membrane orientation of GPT in microsomal vesicles
from livers of chick embryos was investigated by Kean [37,
38]. Susceptibility of GPT activity in sealed vesicles to tryp-
sin inactivation was based upon known luminal location of
mannose 6-phosphatase and thus its resistance to proteolytic
inactivation. In addition, the substrate dolichol phosphate
was added in the form of liposomes so GPT activity assays
could be carried out in the absence of detergents disruptive
of vesicle integrity. The observed loss of GPT activity upon
trypsin treatment of the vesicles was therefore concluded to
be a result of cytoplasmic orientation of the GPT catalytic
activity.
The cytoplasmic orientation of GPT was demonstrated in
sealed microsomal vesicles from rat liver [46]. Using luminal
mannose-6-phosphatase activity as the control for vesicle
intactness, activity of GPT was determined after vesicles
were permeabilized with detergents (Triton X-100 and
CHAPSO), treated with protease (subtilisin), or treated with
Staph. aureus
-toxin to induce membrane pores. In all
cases, dolichol phosphate substrate for the GPT enzyme as-
say was furnished in phosphatidylcholine liposomes in the
absence of detergent. A lack of correlation of GPT activity
with loss of mannose-6-phosphatase latency upon detergent
treatment, a loss of GPT activity upon protease treatment
with retention of mannose-6-phosphatase latency, and no
loss of GPT activity with loss of mannose-6-phosphatase
latency upon pore formation with
-toxin, all indicated a
cytoplasmic orientation of the GPT enzyme activity.
The human lung GPT has been expressed in S. cerevisiae
using techniques of heterologous complementation to sup-
press expression of the endogenous GPT [39]. Analysis of
the human lung GPT cDNA insert in a recombinant yeast
strain revealed a derived amino acid sequence of 400 amino
acids with a corresponding molecular weight of 44.7 kDa, a
42% sequence identify to the S. cerevisiae enzyme, and ap-
proximately 90% sequence identify to other mammalian
GPTs. Enzyme activity in isolated membranes was inhibited
by tunicamycin and yielded the expected acid-labile
GlcNAc-linked lipid.
GPT was purified from lactating bovine mammary glands
[40]. Enzyme activity of the purified protein was highly un-
stable but could be somewhat stabilized in the presence of
substrates, glycerol, and phosphatidylglycerol. SDS-PAGE
analysis revealed two proteins migrating as 46 kDa and 50
kDa oomponents and, after generation of antibodies to each
of these proteins, another 70 kDa protein was identified im-
munologically. The 70 kDa protein was suggested to be ei-
ther a biological precursor of the 46 kDa and 50 kDa pro-
teins, or was the native enzyme degraded by proteases during
purification to the two smaller proteins. Determination of the
molecular mass of the purified enzyme by chromatography
on a standardized Sephacryl S-300 column (in buffer con-
taining KCl and phosphatidylglycerol but lacking detergent)
480 Current Drug Targets, 2009, Vol. 10, No. 6
yielded a mass of 330-360 kDa,. This result was presumably
due to aggregation of the native protein(s).
REGULATION OF GPT ACTIVITY
Important aspects of the metabolic regulation of GPT
activity have been described by Kean for the enzyme in mi-
crosomes from embryonic chick retinas. Initial observations
on the stimulation of lipid-linked GlcNAc biosynthesis by
GDP-mannose [33] were later determined to result from the
synthesis of dolichol-P-mannose in the membranes and the
resulting dolichol-P-mannose being the activator of GPT
[34]. More detailed analyses of regulation of the GPT-
catalyzed reaction with chick retina microsomes (in vitro
reaction mixture containing showdomycin to specifically
inhibit the elongation of GlcNAc-P-P-dolichol to GlcNAc-
GlcNAc-P-P-dolichol) revealed inhibition of the synthetic
reaction by the product GlcNAc-P-P-dolichol [35]. This
product competitively inhibited toward UDP-GlcNAc and
noncompetitively toward dolichol-P. A further feed-back
type inhibition for GPT was observed by the second product,
GlcNAc-GlcNAc-P-P-dolichol, in this biosynthetic pathway
utilizing UDP-GlcNAc. In this case, inhibition was uncom-
petitive toward UDP-GlcNAc and competitive toward doli-
chol-P. The steady-state kinetics for the GPT-catalyzed reac-
tion (in chick retina microsomes) were concluded to be con-
sistent with a sequential bi-bi-mechanism with UDP-GlcNAc
binding first; the feed-back inhibition by GlcNAc-GlcNAc-
P-P-dolichol was proposed to result from its forming a dead-
end complex with the initial enzyme-UDP-GlcNAc complex.
The product of the GPT reaction, GlcNAc-P-P-dolichol, was
also shown to stimulate synthesis of Man-P-dolichol with
chick retina microsomes (and with a yeast Man-P-dolichol
synthase). As Man-P-dolichol is utilized as a mannosyl do-
nor in later reactions leading to synthesis of the final oligo-
saccharide-P-P-dolichol, the regulatory features observed for
the initial two reactions utilizing UDP-GlcNAc were sug-
gested as a means of controlling or modulating utilization of
dolichol-P as substrate for synthesis of GlcNAc-P-P-
dolichol, Man-P-dolichol, and Glc-P-dolichol.
Comparative aspects of the stimulation by Man-P-
dolichol and phosphatidylglycerol of GlcNAc-P-P-dolichol
synthesis in chick retina microsomes were carried out by
Kean [36]. In general, a 10-fold stimulation of initial reaction
rates with an increased Km for UDP-GlcNAc and a de-
creased Km for dolichol-P were observed for both activators.
However, a much higher concentration (200-fold) of phos-
phatidylglycerol, as compared to that for Man-P-dolichol,
was required for maximal stimulation. The effects of the two
activators on initial reaction rates were not additive as might
be expected if different sites were being affected. Different
sites were indicated by the observations that the substrate,
dolichol-P, repressed the stimulation by Man-P-dolichol but
not by phosphatidylglycerol.
EFFECTS OF GPT ACTIVITY AT THE CELL/
ORGAN LEVEL
Developmental and hormonal changes on the mammary
gland GPT enzyme were localized mainly to effects at the
RNA level [42]. Measurements were made of enzyme activ-
ity, of immunoreactive recognition against an 11-aminoacid
Roger K. Bretthauer
carboxy-terminal sequence of the enzyme, and of mRNA
with a cDNA probe against the entire coding region of the
enzyme. Each of these three parameters in mammary gland
tissues increased in parallel, from virgin animal levels, dur-
ing lactation, and subsequently decreased in post-lactation.
Hormonal effects were studied in explant cultures and pri-
mary epithelial cell cultures of mouse mammary glands. The
three parameters previously described were measured in cul-
tures containing individual hormones or combinations of
insulin, thryroxine, hydrocortisone, and prolactin. Maximal
stimulation, up to 6-to 7- fold, of each parameter was ob-
tained with the combination of insulin, hydrocortisone, and
prolactin. Comparison of the three parameters measured in
mammary gland microsomes or cell cultures, whether ex-
pressed on a tissue weight, protein, or DNA content, all indi-
cated that observed effects resulted from changes at the RNA
level.
Other studies were carried out with transient infections of
different GPT promoter/luciferase constructs into mouse
epithelial cells to identify negative regulatory sequences re-
sponsive to hormonal induction [44]. In particular, deletion
of a region of base pairs -1057 to -968 resulted in a 7-fold
enhancement of hormonal induction with no effect on basal
promoter activity. Two pentamer direct repeat sequences of
AGGAA and GAAAC were identified by DNase footprint-
ing to be within the regulatory region, and mutations within
these two sequences resulted in failure to compete for bind-
ing of nuclear proteins as seen for normal sequences. An
increased transcription of the promoter containing mutated
sequences led to the conclusion that hormonal induction of
GPT involves overcoming repression of the GPT gene by
cis-acting negative regulatory elements.
A requirement for functional GPT in implantation/devel-
opment of embryos in mice was demonstrated with use of a
deletion mutant GPT gene [45]. The GPT mutant was con-
structed using Cre-lox mutagenesis that deleted exons 1 and
2 of the GPT gene sequence that normally codes for tran-
scription start site, translation start site, and the first potential
dolichol phosphate recognition site. Chimeric mice contain-
ing this deletion were used to examine embrogenesis in off-
spring of various breeding crosses with wild type. Embry-
onic lethality was observed with the homozygous GPT mu-
tant gene, death resulting several days after uterine implanta-
tion and development through the morula and blastocyst
stages. Mice, heterozygous for the GPT gene deletion, de-
veloped normally, leading to the conclusion that the peri-
implantation embryonic lethality resulted from a recessive
gene deletion of GPT.
The severe effects of GPT mutations in humans has been
observed and characterized in a human subject [47]. The
female infant was clinically presented with hypotonia, sei-
zures, mental retardation, microcephaly, and exotropia. Re-
duced levels of sialylated serum transferrin of the patient
indicated a type-I congenital disorder of glycosylation
(CDG-I), which was confirmed by a greatly reduced labeling
with [2-3H]mannose of the lipid-linked oligosaccharide pool
in fibroblast cultures. The defect was localized to GPT activ-
ity in fibroblast microsomes from the patient being less than
10% of normal, and activity in paternal and maternal micro-
somes being reduced to intermediate levels between patients
Structure, Expression, and Regulation of UDP-GlcNAc
and controls. Analyses of DNA, cDNA, and RNA from fi-
broblasts revealed a point mutation of 660A>G (tyro-
sine>cysteine) for GPT in the paternal allele, and multiple
splicing forms in the maternal allele, of the patient.
Expression of the genes encoding the enzymes of the
dolichol pathway in yeast appears to be highly regulated by a
variety of mechanisms including transcriptional activation
[48]. The first enzyme in the pathway, GPT, appears to be
the major determinant of growth response [49]. Increased
levels of ALG7mRNA correlated with two control points in
the cell cycle. Reentry of cells into cell cycle from G0 upon
growth stimulation is accompanied by a rapid rise in the
mRNA ecoding ALG7 as well as other members of the path-
way. Additionally, in yeast cells arrested in late G1 by treat-
ment with
–factor exhibited a decline specifically in
ALG7mRNA, suggesting a possible connection between the
transferase and START, the control point for transition into
S phase. Hypomorphic mutants expressing low levels of
GPT have implicated the enzyme and, thus, the pathway in
mitochondrial biogenesis in yeast. Although the organelle is
present, it lacks mitochondrial DNA and respiratory activity
[50].
Other significant metabolic consequences of elevated
levels of GPT in CHO-K1 cells were reported by Lehrman
and coworker [51]. Overexpression of GPT in these cells, by
either gene amplification or cDNA transfection, resulted in
the previously reported accumulation of M5Gn2-P-P-Dol. But
the expected resulting alterations, because of excessive utili-
zation of Dol-P in the GPT reaction, in levels of fully glyco-
sylated dolichol-P-P-linked oligosaccharides (expected to
increase) and dolichol-P-linked monosaccharides (expected
to decrease) were not observed. Other experiments with
Lec35 mutants and overexpressers of GPT suggested that
excessive GPT may interfere with the function of the Lec35
protein in utilization of Dol-P-Man/Glc in further glycosyla-
tion of M5Gn2-P-P-Dol. The significance of these results is
discussed in relation to the human congenital disorder of
glycosylation CDG-If, suggesting pseudo-CDG-If pheno-
types could occur and particular specific screening assays
[52] would be required for detection.
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482 Current Drug Targets, 2009, Vol. 10, No. 6
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Accepted: August 20, 2008