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Journal ArticleDOI

Addition of glucose to dolichyl diphosphate oligosaccharide and transfer to protein.

01 Apr 1980-FEBS Journal (Federation of European Biochemical Societies)-Vol. 105, Iss: 2, pp 275-278

TL;DR: The glycosylation of asparagine residues in proteins is known to occur by transfer from a dolichyl diphosphate oligosaccharide containing glucose, but here the compound with three glucoses was used as donor in the transfer to protein.

AbstractThe glycosylation of asparagine residues in proteins is known to occur by transfer from a dolichyl diphosphate oligosaccharide containing glucose. Paper chromatography allowed the separation of oligosaccharides (obtained by acid hydrolysis of the dolichyl diphosphate derivative) containing 1, 2 and 3 glucose residues. Using this procedure it was found that the addition of all three glucoses to the dolichyl diphosphate oligosaccharide occur with dolichyl phosphate glucose as donor. Furthermore only the compound with three glucoses was used as donor in the transfer to protein. The addition of glucose to exogenous dolichyldiphosphate oligosaccharide labelled by transfer from radioactive guanosine diphosphate mannose was detected.

Summary (1 min read)

Jump to: [MATERIALS AND METHODS][RESULTS] and [DISCUSSION]

MATERIALS AND METHODS

  • Rat liver microsomes were prepared as described previously [4] .
  • Dol-P was isolated and purified up to the DEAE-cellulose step [5] .
  • Dol-P-P-Glc-oligosaccharide labelled in the glucose or in the mannose was obtained by incubation of microsomes with UDP-['4C]G1~ [7].
  • The glucose-containing oligosaccharides with three, two and one glucose residues are referred to as Glc3, Glcz and Glcl oligosaccharides.

RESULTS

  • A similar preparation has now been found to contain four substances when analyzed under the new conditions (Fig. 3 ).
  • Although it was shown some time ago [6] that Dol-P-Glc can serve as donor for the formation of Dol-P-P oligosaccharide it had not been established whether all the glucoses are transferred by that mechanism.
  • To decide this point, labelled Dol-P-Glc was incubated with liver microsomes and the Dol-P-P-Glc-oligosaccharide formed was hydrolysed and the oligosaccharides were chromatographed as described before.
  • Presumably there occurs a competition between UDP-Glc and GDP-Man for the Dol-P available.

DISCUSSION

  • The solvent for paper chromatography described in this paper is nearly the same as the one used by Huber et al. [17] for thin-layer chromatography.
  • The neat separation of the different glucose-containing oligosaccharides has led to the clarification of several details of their transformations.
  • Their rate of transfer is slower and may involve endogenous glucose donors.
  • The formation of Dol-P-P-Glc oligosaccharide has been carried out before by transfer from Dol-P-Glc to an endogenous acceptor present in the microsomal preparations [6] .

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Eur.
J.
Biochem.
105,
275-278 (1980)
Addition of Glucose to Dolichyl Diphosphate Oligosaccharide
and Transfer to Protein
Roberto
J.
STANELONI, Rodolfo A. UGALDE, and Luis F. LELOIR
Instituto de Investigaciones Bioquimicas “Fundacion Campomar” and Facultad de Ciencias Exactas
y
Naturales, Buenos Aires
(Received August
31,
1979)
The glycosylation of asparagine residues in proteins is known to occur by transfer from a dolichyl
diphosphate oligosaccharide containing glucose. Paper chromatography allowed the separation of
oligosaccharides (obtained by acid hydrolysis of the dolichyl diphosphate derivative) containing 1,
2 and
3
glucose residues. Using this procedure it was found that the addition of all three glucoses
to the dolichyl diphosphate oligosaccharide occur with dolichyl phosphate glucose as donor. Further-
more only the compound with three glucoses was used as donor in the transfer
to
protein. The
addition of glucose to exogenous dolichyldiphosphate oligosaccharide labelled by transfer from radio-
active guanosine diphosphate mannose was detected.
There is evidence showing that the donor for the
glycosylation of asparagine residues in proteins is a
dolichyl diphosphate oligosaccharide
[l
-
21. The evi-
dence shows that this oligosaccharide, whose structure
is known [3], is built up by the transfer of acetyl-
glucosamine phosphate to dolichyl phosphate fol-
lowed by the addition of one more acetylglucosamine,
nine mannoses and one to three glucoses. The oligo-
saccharide is then transferred to protein and the
glucose residues are removed. The complex oligo-
saccharides may then be formed by removal of six
mannoses and addition of acetylglucosamine, galac-
tose and sialic acid. Another pathway consisting in
removal or addition
of
mannose residues would lead
to the formation of the high-mannose-type oligo-
saccharides.
This paper reports studies on some aspects of the
process of glucose addition to the oligosaccharide and
of transfer
of
the latter to protein.
MATERIALS AND METHODS
Rat liver microsomes were prepared as described
previously [4]. Dol-P was isolated and purified up to
the DEAE-cellulose step [5]. Do~-P-[~~C]G~C was
prepared by incubation of UDP-[14C]Glc with liver
microsomes and purified by DEAE-cellulose chro-
matography [5,6].
Dol-P-P-Glc-oligosaccharide
la-
belled in the glucose or in the mannose was obtained
by incubation of microsomes with UDP-[’4C]G1~ [7]
Abbreviations.
The glucose-containing oligosaccharides with
three, two and one glucose residues are referred
to
as Glc3, Glcz
and Glcl oligosaccharides.
Dol,
dolichyl; Glc, glucose; Man,
mannose;
P,
phosphate.
or GDP-[’4C]Man [8] respectively. Dol-P-P-Glc-oligo-
saccharide labelled in mannose and glucose was ob-
tained from oviduct slices incubated with [‘4C]-man-
nose [9,10]. Free oligosaccharides were obtained from
the dolichyl diphosphate derivatives by mild acid
hydrolysis [lo].
Paper chromatography of the oligosaccharides was
carried out with the following solvent: 1-propanol/
nitromethane/water
(5
:
2
:
4). This mixture is referred
to as the propanol-nitromethane solvent.
RESULTS
Preparation
of
Dolichyl-Diphosphate-Glucose-Con-
taining Oligosaccharides. Incubation
of
UDP-[14C]G1~
with liver microsomes leads to the formation of la-
belled Dol-P-P oligosaccharides. Mild acid hydrolysis
of these compounds results in the liberation of free
oligosaccharides which can be separated by paper
chromatography into three different compounds. This
separation was first obtained by Kornfeld et al. [ll]
with
3
-butanol/pyridine/water (4:
3
:
4) as solvent in
a 16-day run. With the propanol/nitromethane solvent
the separation can
be
obtained in
7
days (Fig. 1).
The relative amounts of the different glucose-
containing oligosaccharides vary with the conditions
used for the synthesis. The changes at various incuba-
tion times are shown in Fig.
2.
The Glc3-oligosaccharide
increases up to 20min and then decreases progressively.
The Glc2-oligosaccharide follows a similar course but
does not decrease as fast while Glcl-oligosaccharide
remains constant. When Glcl and Glc2 oligosacchari-
des had to be prepared an incubation time of
80
min
was used. Under these conditions separation by paper

216
Dolichyl Diphosphate Oligosaccharide: Glucosylation and Transfer
.~
2
I0
G3 G2
G1
I
0
5
10
15 20 25
30
Distance
(cm)
Fig.
1.
Paper chromatogruphy
qJ'
the glucose-containing oligosac-
charides.
The compounds labelled in the glucose were obtained as
described in
[7].
(A) 1-Propanol-nitromethane solvent for
7
days,
(B)
I-Butanol/pyridine/water
(4:
3:
4)
for 20 days
Fig. 2.
Changes
in
the
dij'irent
glucose-conluining oligosaccharide
at different incubation times.
The incubation mixture contained
:
0.1
M Tris-maleate pH
7.7,
0.01
M
Mg-EDTA,
0.01
M MgC12,
0.02 M 2-mercaptoethanol, liver microsomes (about 10 mg of
protein), and 300000 counts/min of UDP-['4C]glucose (268 Ci/
mol). Final volume
250
PI.
The reaction mixture was incubated for
the indicated times at 30 "C. Methanol (2 ml), MgCh
4mM
(0.75
ml)
and chloroform (3.0ml) were then added and the isolation
of
Dol-P-P-Glc-oligosaccharides
was carried out as described pre-
viously [6]. The glucose-containing oligosaccharides obtained by
mild acid hydrolysis were chromatographed with propanol/nitro-
methane solvent for
7
days. The radioactive peaks were located with
a scanner and the corresponding pieces
of
paper were counted with
a toluene-based scintillation fluid, in a scintillation counter
chromatography was better because the amount of
Glc3-oligosaccharide was lower. The decrease in
glucose-containing oligosaccharides is brought about
by the glucosidases present in the microsome fraction
[I21
but a part may be due to transfer to protein.
Chromatography
of
a Glucose-Containing Oligosac-
charide Labelled
in
Glucose and Mannose.
Chromatog-
raphy with the propanol-nitromethane solvent has
allowed the separation of some products which were
thought to be homogenous. Preparations of glucose-
containing oligosaccharide obtained from tissue slices
incubated with radioactive glucose or mannose gave
only one peak when chromatographed on paper [lo,
131.
A
similar preparation has now been found to
contain four substances when analyzed under the new
conditions (Fig. 3). Information on their identity was
obtained using specific liver microsomal glucosidases
separated by gel filtration
[14].
The substance of peak
G3 when treated with the glucosidase specific for Gk3-
oligosaccharide yielded glucose and a product that
chromatographed like G2. When the substance of
peak G2 was treated with the glucosidase that acts
on Glc2 and Glcl oligosaccharides the products were
glucose and a substance which ran like peak
Go
in
Fig. 3 which was probably the glucose-free oligo-
saccharide.
A
substance with a similar mobility was
obtained on incubation of microsomes with GDP-
['4C]Man.
Transfer from Dolichyl Monophosphate Glucose.
Although it was shown some time ago
[6]
that Dol-
P-Glc can serve as donor for the formation of Dol-
P-P oligosaccharide it had not been established
whether all the glucoses are transferred by that mecha-
nism. There remained the possibility that some glu-
coses could be transferred directly from UDP-Glc or
some other donor. To decide this point, labelled Dol-
P-Glc was incubated with liver microsomes and the
Dol-P-P-Glc-oligosaccharide
formed was hydrolysed
and the oligosaccharides were chromatographed as
described before. Fig.4 shows that the three com-
pounds became labelled. These compounds had the
mobility in paper chromatography, with 1-butanol/
pyridine/water (4
:
3
:
4),
corresponding to Glc3, Glc2
and Glcl oligosaccharides. Treatment of peak G3
after elution from the paper with a glucosidase specific
for Glc3-oligosaccharide [14] led to the liberation of
radioactive glucose. The substances from peak GZ or
G1 also yielded glucose when treated with a glucosidase
specific for Glc2 and Glcl oligosaccharides.
The fact that the three glucose-containing oligo-
saccharides were labelled indicates that their glucoses
were transferred directly from Dol-P-Glc.
Transfer to Protein.
Previous work showed that
Dol-P-P-Glc-oligosaccharides
are transferred faster to
endogenous protein than glucose-free compounds
[15].
However, it was not established which of the
glucose-containing oligosaccharides was transferred.
A
Dol-P-P-Glc-oligosaccharide
preparation contain-
ing the Glcl, Glc2 and Glc3 components was incubated
with liver microsomes with and without Mn2+ ions
which are known to be required for transfer to protein.
The Dol-P-P-Glc-oligosaccharides were then extracted,
hydrolysed and chromatographed on paper.
As
shown
in Fig.
5
in the presence of Mn2
+,
when the saccharide
is transferred to protein there was a preferential dis-
appearance of the Glc3-oligosaccharide. This is also
shown in quantitative data (Fig.5). The decrease in
radioactivity in Glc3-oligosaccharide was compensated
by a similar increase in glycoprotein.

R.
J.
Staneloni,
R.
A.
Ugalde, and
L.
F.
Leloir
211
The
Transfer of Glucose to
a
Dolichyl-Diphosphate-
Glucose-Containing Oligosaccharide Precursor. The
formation of
Dol-P-P-Glc-oligosaccharide
has been,
in most studies, carried out by incubating labelled
UDP-Glc with microsomes. These provided an endog-
enous precursor believed
to
contain Man9GlcNAcz
joined to dolichyl diphosphate.
The addition of glucose to a precursor labelled in
the mannose has been tested repeatedly in this labora-
tory but without clear results. Presumably there occurs
a competition between UDP-Glc and GDP-Man for
the Dol-P available.
It was shown by Robbins et al. [16] that incuba-
tion of particulate enzyme preparation of fibroblasts
with GDP-[14C]Man led to the formation of Dol-P-P
oligosaccharides and that these became larger if UDP-
Glc was added in addition. The number of glucose
added was not determined.
Experiments have now been carried out using an
exogenous labelled acceptor. For this purpose liver
microsomes were incubated with GDP-[14C]Man and
the products having the solubility of Dol-P-P-Glc
oligosaccharide were isolated. The major oligosac-
charide formed was found to have a mobility slightly
higher than the Glcl-oligosaccharide. As shown in
Fig. 6 reincubation
of
the compound with microsomes
and UDP-Glc led to the formation of a lipid linked
oligosaccharide whose sugar moiety had the mobility
of Glc3 and Glcz oligosaccharides.
DISCUSSION
The solvent for paper chromatography described
in this paper is nearly the same as the one used by
Huber et al. [17] for thin-layer chromatography.
It
only differs in having slightly more water. With this
solvent maltooligosaccharides of up to 16 units can
be separated in 7days. The neat separation of the
different glucose-containing oligosaccharides has led
to
the clarification of several details of their trans-
formations.
Although Dol-P-P-Glc oligosaccharide was known
to become labelled
on
incubation with radioactive
Dol-P-Glc it remained to be proved
if
the three glu-
coses are introduced by the same mechanism. This
has now been proved. As to the transfer of glucose-
containing oligosaccharide from its Dol-P-P derivative
it had been detected long ago, but at that time the
existence of compounds differing in glucose content
was not known. It has now been found that only the
oligosaccharide containing three glucoses is trans-
ferred to protein. If the dolichyl diphosphate deriva-
tives of Glcz and
Glcl
oligosaccharides are also sub-
strates, the process is
so
slow that it was not detectable
in our experiments. The results complement those of
Turco and Robbins
[18] who isolated the glycopeptide
formed immediately after transfer from Dol-P-P-Glc
-
0
5 10
15
20
25
30
Distance
(cm)
Fig. 3.
Paper chromatography
of
oligosaccharides labelled in the
glucose
and
mannose.
The
preparation was carried out with oviduct
slices
as
described in [9,10]. Chromatography with
the
propanol/
nitromethane solvent for
7
days
1
5
10
15
20
25
30
Distance
(cm)
Fig.
4.
Tramfir
of
glucose
fioni
clolichyl
phosphate
[14CJglucose
to
endogenous acceptors.
The transfer reaction was carried out by
incubation of: 0.1
M
Tris-maleate buffer of pH
7.7,
0.6% Triton
X-100,
10
mM
Na-EDTA,
20 mM 2-mercaptoethano1,
Dol-P-
['4C]Glc
(4000 counts/min dried before the addition of the other
components). Total volume 50
PI,
After 10 min
at
30 "C chloroform
and methanol were added and fractionation
was
carried out as
described in [6]. The oligosaccharides obtained
by
mild acid hydro-
lysis were separated by paper chromatography with the solvent
I-butanol/pyridine/water
(4:3
:4)
for 20 days
oligosaccharide to protein and found that its size cor-
responded to the Gk3-containing compound. Transfer
of mannose-labelled oligosaccharides presumably not
containing glucose has been reported
[8,19].
Their rate
of transfer is slower and may involve endogenous
glucose donors. Further work would be required in
order to find out if the glucose-containing oligo-
saccharides are obligate intermediates in the glycosyla-
tion pathway.
The formation of Dol-P-P-Glc oligosaccharide
has been carried out before by transfer from Dol-P-Glc
to an endogenous acceptor present in the microsomal
preparations [6]. In the experiments reported here an
exogenous acceptor labelled in the mannose was
prepared by incubation of liver microsomes with
GDP[14C]Man followed by solvent extraction.
In-

278
R.
J.
Staneloni,
R.
A. Ugalde, and
L.
F. Leloir: Dolichyl Diphosphate Oligosaccharide: Glucosylation and Transfer
Distance
(crn)
Fig. 5.
Transjer
of
the
gluc~o.cc~-c~r~rr/oirii~i,~
oligosuccharides to protein.
The
Dol-P-P-Glc-oligosaccharides
‘“C-labclled in the glucose
(15000 counts/min) were dried and the following components were
added: 80 mM Tris-maleate buffer (pH 7.7), 2% Triton X-100 and
10
mg of microsomal protein in a total volume of 250
pl.
Where
indicated 8 mM MnCI2 was added. After
10
min at 30
“C
the mix-
ture was processed as described in the legend of Fig. 2. The glucose-
containing oligosaccharides were chromatographed on paper and
the protein precipitate was washed with methanol dissolved in
0.5 ml of Protosol and counted by scintillation. The numbers
indicate the radioactivity is counts/min in each peak. The radio-
activity in the protein (in counts/min) was: minus Mn2+: 900,
plus Mn2+: 4500. (A)
No
addition, (B) plus Mn2+
cubation of the compound with UDP-Glc led
to
the
formation
of
Dol-P-P-Glc oligosaccharides. The trans-
fer
of
glucose to an exogenous acceptor had
not
been
reported before.
It
is
a
necessary step for the study of
the corresponding enzyme and may be useful
in
deter-
mining the structure of the glucose acceptor which
is
somewhat uncertain. Thus Hubbard and Robbins
[20]
presented evidence suggesting that it may contain
eight
and
not nine mannose residues
as
would be
expected.
The authors express their gratitude to Marcel0 Tolmasky and
Claudio Petriella for their valuable collaboration. R.
S.
is a career
investigator
of
the
Consejo Nacional de Investigaciones Cientiji’cas
y
Ticnicas
(Argentina).
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Obligado 2490, RA-1428 Buenos Aires, Argentina
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Journal ArticleDOI
TL;DR: The structure of the saccharides linked to the cell surface giycoprotein and the role of glycopeptides in this study have been studied to provide an understanding of their role in inflammation.
Abstract: III. Structure of the saccharides linked to the cell surface giycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 A. Sulfated high molecular weight saccliaride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 B. Sulfated low molecular weight saccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 C. Nonsulfated glycopeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

109 citations


Journal ArticleDOI
TL;DR: In vitro enzyme assays demonstrated that the PHAR2.7 cell line is deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the oligosaccharides of newly glycosylated proteins.
Abstract: Glycosylation of asparagine residues of glycoproteins occurs by the transfer of a glucose3mannose9N-acetylglucosamine2 (Glc3Man9GlcNAc2) oligosaccharide from a lipid carrier to the nascent protein. Normally, this transfer is quickly followed by the stepwise removal of the glucose residues which are arranged in the sequence: Glc1 leads to 2Glc1 leads to 3Glc1 leads to 3Man. We now report studies which demonstrate that a lectin-resistant mutant of the BW5147 mouse lymphoma cell line is deficient in the enzyme which removes the two inner glucose residues. This cell line (PHAR2.7) was selected for resistance to the cytotoxic effects of Phaseolus vulgaris leukoagglutinating lectin (Trowbridge, I. S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Glycopeptides prepared from cells equilibrium-labeled with either [2-3H]mannose or [6-3H]galactose were characterized using lectin affinity chromatography, treatment with specific endo- and exoglycosidases, sizing by paper chromatography, and methylation analysis. Approximately 50% of the radioactivity in [3H]mannose-labeled glycopeptides from the mutant cells is present as glucosylated high mannose-type oligosaccharides whereas parent cell glycopeptides labeled under similar conditions lack detectable amounts of these species. Using [3H]galactose labeling, the major glucosylated oligosaccharides were identified as Glc2Man9GlcNAc2 and Glc2Man8GlcNAc2. In vitro enzyme assays demonstrated that the mutant cells cannot remove either of the two inner 1 leads to 3-linked glucose residues. Removal of the outer 1 leads to 3-linked glucose is normal. We conclude from these data that the PHAR2.7 cell line is deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the oligosaccharides of newly glycosylated proteins.

106 citations


Journal ArticleDOI
TL;DR: This new synthetic phenotype made it possible to isolate the ALG8 locus, encoding a potential glucosyltransferase of the endoplasmic reticulum, which resulted in underglycosylation of secreted proteins in Saccharomyces cerevisiae.
Abstract: Glc3Man9GlcNAc2 is the preferred substrate of the oligosaccharyltransferase of N-linked glycosylation of proteins, but nonglucosylated oligosaccharides can be transferred to proteins in Saccharomyces cerevisiae. Mutations affecting the addition of the three terminal glucose residues lead to accumulation of Man9GlcNAc2 or Glc1Man9GlcNAc2 in vivo but do not show any detectable growth defect. When these mutations were introduced into a strain with reduced oligosaccharyltransferase activity (due to the wbp1-1 mutation), a severe growth defect was observed: accumulation of suboptimal lipid-linked oligosaccharide and reduced oligosaccharyltransferase activity resulted in a severe underglycosylation of secreted proteins. This new synthetic phenotype made it possible to isolate the ALG8 locus, encoding a potential glucosyltransferase of the endoplasmic reticulum. The ALG8 protein is a 63.5-kDa hydrophobic protein that is not essential for the vegetative growth of yeast. However, the lack of this protein resulted in underglycosylation of secreted proteins.

91 citations


References
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Journal ArticleDOI
TL;DR: The synthesis of the complex-type oligosaccharide unit of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosACcharide from a lipid carrier to the nascent polypeptide.
Abstract: The synthesis of the complex-type oligosaccharide unit of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosaccharide from a lipid carrier to the nascent polypeptide Following transfer the oligosaccharide is "processed" by removal of glucose and mannose residues and the sugars that constitute the outer branches of the complex-type oligosaccharide are added The structure of the oligosaccharide moiety of the lipid-linked precursor has been elucidated in order to further define the steps involved in processing Since it was not feasible to obtain adequate amounts of material for standard structural studies, most of the structural studies were performed on radiolabeled material, with radioactivity incorporated differentially into glucose, mannose, and N-acetylglucosamine Based on endo-beta-N-acetylglucosaminidase CII digestion, alpha-mannosidase digestion, acetolysis, Smith periodate degradation, methylation analysis, and periodate oxidation, we propose the following structure for the oligosaccharide moiety of the lipid-linked oligosaccharide

397 citations



Journal ArticleDOI
TL;DR: To elucidate the sequence of processing, pulse-chase experiments were performed with virus-infected Chinese hamster ovary cells using labeled mannose, glucosamine, and galactose and demonstrated that processing is initiated by the rapid removal of 2 of the 3 glucose residues of the precursor oligosaccharide.
Abstract: The synthesis of the complex-type oligosaccharide units of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosaccharide from a lipid carrier to the nascent polypeptide. Following transfer, the oligosaccharide precursor, a branched molecule containing 3 glucose, 9 mannose, and 2 N-acetylglucosamine residues (Li, E., Tabas, I., and Kornfeld, S. (1978) J. Biol. Chem. 253, 7762-7770) is processed to give rise to the mature complex-type oligosaccharide. To elucidate the sequence of processing, pulse-chase experiments were performed with virus-infected Chinese hamster ovary cells using labeled mannose, glucosamine, and galactose. The processing intermediates were isolated and characterized. At 20 to 30 min after labeling, the major processing intermediates are (Glc)l(Man)e(GlcNAc)z and (Man)s(GlcNAc)z. Small amounts of (Man)s(GlcNAc)z and (Man)T(GlcNAc)z were detected along with trace amounts of (Glc)z(Man)s(GlcNAc)z and (Man)s(GlcNAc)z. In addition, by 20 to 30 min completely processed oligosaccharides containing only 3 mannose residues were also present. These data demonstrate that processing is initiated by the rapid removal of 2 of the 3 glucose residues of the precursor oligosaccharide. At 20 to 30 min the last glucose residue is removed and processing proceeds rapidly with the ultimate removal of 6 of the 9 mannose residues and the addition of the outer branch sugars. Clone 15B cells, a line deficient in UDP-GlcNAc: glycoprotein N-acetylglucosaminyltransferase I, accumulate an intermediate with the structure Mancvl+ 3 (Manal--+ 6) Manal--+ 6 (Mancul -+ 3) Ma@1 += 4 GlcNAc fil+ 4 GlcNAc. This indicates that normal processing requires the transfer of a Nacetylglucosamine residue to the mannose residue linked cwl + 3 to the P-linked core mannose. Following transfer of the N-acetylglucosamine residue, an Ly-mannosidase removes the 2 mannose residues linked to the mannose which is linked cul + 6 to the core mannose. Direct evidence for this reaction is presented in the following paper (Tabas, I., and Kornfeld, S. (1978) J. Biol. Chern. 253, 7779-7786). Complex oligosaccharide biosynthesis is then completed by the addition of the final outer Wacetylglucosamine residues and the galactose, sialic acid, and fucose residues.

331 citations



Journal ArticleDOI
TL;DR: The microsomal fraction of liver has been found to catalyze glucose transfer from UDPG to a lipid acceptor which appears to be identical to the compound obtained by chemical phosphorylation of dolichol.
Abstract: The microsomal fraction of liver has been found to catalyze glucose transfer from UDPG to a lipid acceptor which appears to be identical to the compound obtained by chemical phosphorylation of dolichol. The substance formed (dolichol monophosphate glucose) is acid labile and yields 1,6-anhydroglucosan by alkaline treatment. It can be used as substrate by the enzyme system yielding a glucoprotein which is subsequently hydrolyzed to glucose.

215 citations