From the
UDP-N-acetyl-D-glucosamine:
The identification of the oligosaccharide ligands that interact
with cell adhesion proteins of the selectin family (1, 2) has lent direct experimental support to the model
that cell surface carbohydrates act as carriers of specific information
in cell interaction and recognition. Further evidence supporting this
model is provided by the observation that the binding properties of
neural cell adhesion molecule(3) , intercellular adhesion
molecule(4) , and myelin Po protein (5) appear to be
modulated by stage- or tissue-specific patterns of glycosylation. The
precise changes of cell surface carbohydrates that occur during
development, differentiation, and maturation (6) suggest that
the expression of the enzymes that act in the biosynthetic pathway of
complex carbohydrates is finely regulated and point to the need for
elucidation of the molecular mechanisms of this control. Moreover, in vivo disruption by gene targeting of the
The branching pattern of N-linked
complex carbohydrates is specified by the action of distinct N-acetylglucosaminyltransferases, each one of which catalyzes
the transfer in a specific glycosidic linkage of a GlcNAc residue from
the donor substrate UDP-GlcNAc to a mannose residue of the trimannosyl
core pentasaccharide(12) . The enzyme
UDP-N-acetyl-D-glucosamine:
As an initial step to
characterize the transcription unit encoded by the GnT II gene and
study the molecular mechanisms that regulate expression of the GnT II
enzyme, the cDNA coding for rat GnT II was cloned and characterized. In
this paper, the complete nucleotide sequence of overlapping cDNA clones
that encode the entire GnT II polypeptide is reported. Analysis of the
deduced primary structure indicates a domain architecture and a type II
transmembrane topology similar to that of other glycosyltransferases.
Transient expression in COS cells of the cDNA segment coding for the
C-terminal region of rat GnT II was shown to direct the synthesis of an
enzymatically active polypeptide, providing unambiguous verification of
the cloned rat GnT II cDNA.
The sample
from the third affinity chromatography step was loaded on a CM-Sephadex
C-50 column (1.4
Active fractions from
the pH-pulsed CM-Sephadex column were pooled and directly loaded on a
small hydroxylapatite column (Bio-Gel HTP, Bio-Rad, 1.4
Active fractions (12, 13, 14, 15) from the
hydroxylapatite column were pooled, dialyzed against buffer X, and
loaded on a column of reactive yellow 86-agarose (Sigma, product R
8504, 1.5
At this stage, the enzyme was essentially pure. It was subjected to
gel filtration on a second, smaller Sephadex G-200 (Pharmacia Biotech
Inc., Superfine) column (as in step 4, Ref. 13, except the column
dimensions were 1.5
Plasmid pR9 containing the EcoRI fragment of
The 5`-end probe was derived from plasmid p5R7 (Fig. 1) by digestion with EcoRI. The coding region
probe was generated by PCR amplification of plasmid pR9 (see above)
using as primers two GnT II-specific oligonucleotides denominated N15
and N16, which map at nucleotide positions 397-420 and
1417-1394, respectively, of the rat GnT II cDNA sequence (Fig. 3). The 3`-untranslated probe was generated by PCR
amplification of a rat GnT II genomic clone fragment,
(
To isolate cDNA clones extending
further in the 5`-direction than clones R7, R9, and R9.5, cDNA was
synthesized from rat liver poly(A)
The deduced amino acid sequence agrees with direct protein sequence
data in 58 out of 59 residues (underscored with a solidbox in Fig. 3), which were positively identified by
sequence analysis of the rat liver GnT II polypeptide ().
The three amino acid sequences that were experimentally determined are
encoded by distinct regions of the cloned cDNA, thus confirming that
they represent different regions of a single translation product. At
each one of these regions, the alignment between the deduced and
experimentally determined amino acid sequence does not require the
introduction of any gap, providing compelling evidence for colinearity.
Inspection of the deduced amino acid sequence indicates that the two
partially overlapping amino acid sequences observed in the sequence
analysis of the purified 42-kDa GnT II polypeptide apparently represent
the N-terminal amino acid sequences of two polypeptide species whose N
termini differ in length by three amino acids, probably starting at
residues Val-71 and Val-74. The N terminus of the tryptic peptide
starts after a basic residue (Arg-100), as expected, and the
unidentified amino acid residue at position 8 in the sequence of this
peptide (, experiment 3) corresponds to a tryptophan
residue (Trp-108), which can be chemically modified during
electrophoresis(19) . The amino acid sequence that became
predominant after the first four cycles of the Edman degradation of the
intact GnT II enzyme, which was purified by reversed-phase HPLC (, experiment 1) actually corresponds to a segment of the
C-terminal region of the GnT II translation product. Clearly, after
reduction and SDS-PAGE, this peptide was missed on the 10% gels that
were used, peptides of <10 kDa either running close behind or with
the stacking buffer off the end of the gel. Amino acid residues
representing the N terminus of the 42-kDa GnT II polypeptides were also
detected in the first four cycles of the Edman degradation of the
intact GnT II enzyme. As the 42-kDa GnT II polypeptide has two species
differing in length at the N terminus by three amino acids, and as
prolines, which tend to give lower yields during the Edman cleavage
step, were present near its N terminus, the sequence of the small
C-terminal peptide became predominant. Taken together, these data
indicate that two polypeptides comprise the purified, active GnT II
enzyme, either as a result of proteolysis during purification or as a
result of normal proteolytic processing in the endomembrane system, or
both. The small polypeptide of undetermined size, which encompasses the
C-terminal 64 amino acid residues, interacts strongly, possibly through
disulfide bridges, with the larger polypeptide of 42 kDa, which extends
from residues 71 to near 378 of the initial translation product. Both
polypeptides migrated together during reversed-phase HPLC under
conditions that may be assumed to be rather denaturing.
The deduced
amino acid sequence of the rat GnT II polypeptide chain contains two
potential N-linked glycosylation sites having the consensus
sequence Asn-Xaa-Ser/Thr at Asn-64 and Asn-81. N-Linked
glycosylation at Asn-64-Asp-Ser can probably be excluded, since it has
never been observed at Asn residues, which form part of a site where
Xaa is a Pro or Asp residue(12) . N-Linked
glycosylation could occur at Asn-81-Leu-Thr, as suggested by the
observation that Asn-81 was left unidentified by amino acid sequence
analysis of the 42-kDa GnT II polypeptide (). The
calculated molecular mass of the unglycosylated form of this
polypeptide, which includes Asn-81 ( and Fig. 3), is
36,276 daltons, 5.7 kDa lower than the molecular mass of 42 kDa
determined by SDS-PAGE.
Computer-assisted analysis by the algorithm
of Kyte and Doolittle (24) with a span setting of 9 revealed a
hydrophobic stretch of 20 amino acids occurring at residues 10-29 (underlined in Fig. 3). This is the only hydrophobic
region in the GnT II polypeptide sufficiently long enough to span the
membrane lipid bilayer. The structural features of this N-terminal
hydrophobic segment predict that it may represent a signal sequence
that remains uncleaved and functions as a stop transfer signal during
membrane insertion. The sequence that follows the hydrophobic core does
not agree with the consensus sequence context for a signal peptidase
recognition and cleavage site(34, 35) . Clusters of
charged amino acid residues flank the two sides of this hydrophobic
domain. Since the N-terminal side is more positive with respect to the
C-terminal side (
The cloned cDNA includes 105 bp of
5`- and 721 of 3`-flanking sequences in addition to the 1326 bp of
protein coding region. Within the 5`-untranslated region, no AUG codons
5` to the presumptive AUG initiator codon at position 106 are observed.
The 5`-untranslated sequence has a G + C content of 76% and
probably extends further upstream. Two transcription start sites, which
map at
Since the 3`-untranslated region of rat liver GnT II mRNA
appears to have structural features that could negatively affect its
stability or translational
proficiency(40, 41, 42) , this region was
removed from the rat liver GnT II cDNA and replaced in the expression
plasmids with the 3`-untranslated region of the rat pre-proinsulin II
gene, which provides an efficient polyadenylation signal(28) .
Furthermore, the 5`-untranslated region of the rat pre-proinsulin II
gene was placed between the long terminal repeats of the Rous sarcoma
virus (which act as a strong promoter) and the cDNA for the cleavable
signal sequence of the human IL-2 receptor to provide an additional
potential stabilizing element to the messages encoded by the hybrid
constructs.
COS-7 cells were transfected either with plasmid
pLJ268_R9 or pLJ269_R9 by liposome-mediated uptake. Cells and culture
media were collected at various times post-transfection and assayed for
GnT II activity in the presence or absence of the acceptor substrate
MGn(+F). Cells transfected with plasmid pLJ268_R9 showed at 48 h
post-transfection a 10-fold increase in GnT II activity above the
endogenous level of mock-transfected cells. No significant increase of
GnT II activity above the endogenous level was observed in COS-7 cells
transfected with plasmid pLJ269_R9 carrying the rat cDNA out of frame
(data not shown). The results reported in demonstrate
that COS-7 cells transfected with plasmid pLJ268_R9 synthesize and
release into the culture medium a polypeptide endowed with GnT II
activity. A consistent and progressive increase of GnT II activity was
observed at 48 and 72 h post-transfection in the culture medium of COS
cells transfected with plasmid pLJ268_R9 as compared with the
negligible level of GnT II activity present in the culture medium of
COS-7 cells transfected with plasmid pLJ269_R9. The enhancement of GnT
II activity at 48 and 72 h post-transfection was 71- and 77-fold,
respectively. Since the increase of GnT II activity in the culture
medium of COS cells depends on the correct reading frame of the
transfected rat cDNA, the possibility that it may result from
activation events of the endogenous GnT II gene induced or mediated
upon transfection by the rat cDNA or vector sequences can be excluded.
To positively identify the product of recombinant GnT II catalyis,
enough product (225 nmol) was purified, as described under
``Experimental Procedures,'' to obtain a
Two independent experimental data indicate that the cloned
rat cDNA encodes the GnT II enzyme. First, the colinearity between the
nucleotide sequence of the cloned cDNA and 58 non-overlapping amino
acid residues, which were identified by sequence analysis of the rat
GnT II polypeptide (), was consistent with the translation
frame (Fig. 3). Second, expression in COS cells of the rat cDNA
region coding for the C-terminal 389 amino acids of the GnT II
polypeptide linked in frame to a human cDNA segment encoding the
cleavable signal peptide sequence of the IL-2 receptor resulted in the
synthesis and release into the culture medium of a polypeptide endowed
with GnT II activity (). This was formally demonstrated by
Analysis of the
deduced amino acid sequence of the rat GnT II protein predicts a
functional domain architecture and type II transmembrane topology
typical of the glycosyltransferases that have been cloned to
date(21, 44, 45, 46, 47, 48, 49) .
The membrane-bound form of rat GnT II is comprised of a short (9 amino
acids) N-terminal cytoplasmic segment, a 20-amino acid hydrophobic
stretch, which may act as a membrane anchor and stop-transfer signal,
and a large C-terminal lumenal region where the substrate binding site
and the catalytic domain are located. This conclusion is consistent
with the amino acid sequence analysis of the purified form of rat GnT
II and the results of the expression studies. A protease-sensitive
segment of
The subcellular compartmentation of GnT
II as well as any signal(s) involved in its localization to Golgi
membranes remain to be elucidated. It has been thought that the GnT II
enzyme resides in the medial Golgi compartment, since analysis of Golgi
membrane fractions from Chinese hamster ovary cells showed that GnT II
activity co-migrates with the GnT I and
GenBank and SwissProt data base
searches did not reveal significant sequence similarity to known DNA or
protein sequences, including those of other previously cloned
glycosyltransferases. Moreover, an extensive computer-assisted
comparison did not reveal regions of significant sequence similarity to
other mammalian N-acetylglucosaminyltransferases, including
human (45) and rabbit (46) GnT I, human (47) and
rat (48) GnT III, and rat and murine GnT V(49) ,
indicating that these enzymes are encoded by unrelated genes. This is
especially surprising in the case of GnT I and GnT II, which both
catalyze the formation of a GlcNAc
The sequence similarity between
rat and human GnT II cDNA is 89% in the coding region (nucleotides
106-1434 in Fig. 3) and 86% in the 3`-untranslated region
(nucleotides 1435-2147).(
Multiple GnT II transcripts, which
differ in their total abundance, were detected in rat liver, brain,
thymus, and spleen by Northern blot analysis, using a coding
region-specific probe (Fig. 6, panelB). The
DNA sequences coding for GnT II are organized in a single exon not
interrupted by introns both in the rat
The
origin and functional significance of the two minor GnT II transcripts
of 2.1 and 1.7 kb in length remain to be elucidated. These transcripts
do not hybridize to the probe containing the terminal portion of the
3`-untranslated region of rat GnT II cDNA (Fig. 6, panelC). Although the possibility that these mRNAs represent
the transcription products of a similar but distinct gene or pseudo GnT
II gene cannot be excluded, it is intriguing to speculate that the two
minor GnT II transcripts of 2.1 and 1.7 kb may result from the
differential utilization of distinct poly(A) addition sites during
maturation of a primary transcript. According to our current knowledge
of the function of the 3`-untranslated region of eukaryotic
mRNAs(54, 55, 56) , the hypothesis can be
extended to suggest that the differential utilization of the poly(A)
addition sites likely mediated by specific factors may regulate
expression of the GnT II gene by generating mRNAs species that differ
in length and functional properties.
Characterization of the
transcription unit of the GnT II gene will aid in elucidating the
molecular mechanisms by which expression of the GnT II enzyme is
regulated during development and differentiation or altered in human
diseases. Studies toward the identification of the nature of the
genetic mutation(s) that cause(s) a defect of GnT II activity in HEMPAS
patients are in progress.
Sequence
data were obtained by N-terminal Edman degradation of the rat liver GnT
II enzyme (Exp. 1), the 42-kDa GnT II polypeptide after purification by
SDS-PAGE and electroblotting (Exp. 2), and a tryptic peptide of the
purified 42-kDa polypeptide (Exp. 3). The amino acid assignments at
each cycle of the Edman degradation are reported in the one letter
code. Upper case letters indicate firm assignments, whereas lower case
letters indicate tentative assignments or minor sequences. X denotes an unidentified amino acid. The sequence data used for the
synthesis of degenerate oligonucleotide probes are underlined.
Expression of GnT
II activity in the culture medium of COS-7 cells transfected with
plasmids carrying the rat GnT II cDNA linked in frame or out of frame
to a segment of human cDNA coding for the cleavable signal peptide
sequence of the IL-2 receptor is shown.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We express our gratitude to Dr. Carla Marusic for
advice in the sequence analysis of cDNA clones, to Roberto Biagini and
Maria Cappelletti for technical assistance, and to Luigi Grisorio for
artwork.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-6-D-mannoside
-1,2-N-acetylglucosaminyltransferase II (EC 2.4.1.143)
(GnT II) is a Golgi resident enzyme that catalyzes an essential step in
the biosynthetic pathway leading from high mannose to complex N-linked oligosaccharides. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis analysis of the enzyme
purified from rat liver revealed a polypeptide of 42 kDa. Amino acid
sequences were obtained from the N terminus and a tryptic peptide.
Overlapping cDNA clones coding for the full-length rat GnT II were
obtained. The complete nucleotide sequence revealed a 1326-base pair
open reading frame that codes for a polypeptide of 442 amino acids,
including a presumptive N-terminal membrane-anchoring domain. The
region of cDNA coding for the C-terminal 389 amino acids of rat GnT II
was linked in frame to a cDNA segment encoding the cleavable signal
sequence of the human interleukin-2 receptor and transiently expressed
in COS-7 cells. A 77-fold enhancement of GnT II activity over a control
carrying the GnT II cDNA out-of-frame was detected in the culture
medium at 72 h after transfection.
H-NMR spectroscopy
confirmed that the oligosaccharide synthesized in vitro by the
recombinant enzyme was the product of GnT II activity. These data
verify the identity of the cloned GnT II cDNA and demonstrate that the
C-terminal region of the protein includes the catalytic domain.
1,2-N-acetylglucosaminyltransferase I gene(7, 8) causes arrest of the development of mouse embryos at an
early post-implantation stage, indicating that the glycosylation
enzymes play a vital role in mammalian development. Consistent with
this notion, defects of the activity of glycosylation enzymes have been
implicated in a few rare human diseases, including I-cell
disease(9) , congenital dyserythropoietic anemia
HEMPAS(10) , and carbohydrate-deficient glycoprotein syndrome
type II(11) .
-6-D-mannoside
-1,2-N-acetylglucosaminyltransferase II (GnT II)
(
)catalyzes the transfer of a GlcNAc residue from the
donor substrate to the
6-linked mannose of the core
pentasaccharide, forming a
1,2 glycosidic
linkage(12, 13, 14) . This reaction, which
initiates the formation of biantennary N-linked
oligosaccharides, is an essential step in the biosynthetic pathway,
leading from high mannose to complex N-linked
oligosaccharides. In vitro enzymic studies indicate that the
activities of GnT V and GnT VI, which initiate formation of the
branches linked
-1,6 and
-1,4, respectively, to the
6-mannose of the trimannosyl core, require the prior action of GnT
II(12, 15) . The physiological acceptor substrate of GnT
II is generated by the prior action of Golgi
-mannosidase II,
which removes two
-linked mannoses to expose the
6-mannose of
the core pentasaccharide(12) . The activity of GnT II is
markedly reduced in the mononuclear cells from at least one HEMPAS
patient (10) and in cultured fibroblasts from two individuals
affected by the recently identified carbohydrate-deficient glycoprotein
syndrome type II(11) , as indicated by the results of enzyme
assays and structural analysis of N-linked oligosaccharides.
Distinct genetic mutations appear to occur in HEMPAS patients, as
defects in the activities of different enzymes involved in the
biosynthetic pathway of N-linked complex carbohydrates have
been observed in these patients(10) . A defect of
-mannosidase II activity that results from a reduced level of the
-mannosidase II mRNA has been described in one HEMPAS patient who
shows a normal level of GnT II activity(16) . The identification
of the nature and origin of the genetic mutations underlying defects of
GnT II activity in human diseases will contribute to the elucidation of
the molecular mechanisms that regulate expression of the
glycosyltransferases and processing enzymes involved in the
biosynthetic pathway and thereby determine the classes of complex
carbohydrates displayed at the cell surface.
Purification of Rat Liver GnT II
Polypeptide
The GnT II enzyme was purified from rat liver
homogenates with major modifications of a procedure previously
published (13) to increase yields. The procedure was scaled up
5-fold, using 3.75 kg of rat livers. It was found that step 3, using
hydroxylapatite(13) , was difficult to scale up due to
channeling in the column and the need to concentrate large volumes.
Step 3 was replaced with a large Affi-Gel Blue column (15 2
cm), which was loaded, washed, and eluted with buffers as previously
described in step 8 (excluding glycerol), in the same relative
proportions as compared with the column volume. Step 5 (affinity
chromatography on 5-Hg-UDP-GlcNAc-thiopropyl-Sepharose with NaCl
elution) was modified by loading and washing with buffer M, containing
25 mM NaCl instead of 50 mM. Step 6 (affinity
chromatography on 5-Hg-UDP-GlcNAc-thiopropyl-Sepharose with UDP-GlcNAc
elution) was eliminated, as was step 8. Steps 7 and 9 (EDTA elutions
from 5-Hg-UDP-GlcNAc-thiopropyl-Sepharose) were applied sequentially,
using 25 mM NaCl in buffer M, instead of 50 mM, and
adding MnCl
to 25 mM after the first elution with
EDTA to ensure excess MnCl
was present in the loading
buffer of the second column. Four additional chromatographic
separations were performed to isolate the pure protein.
4.0 cm), equilibrated in 25 mM NaCl,
10 mM EDTA, 20% glycerol, 0.02% sodium azide, 0.1% Triton
X-100, 25 mM MES, pH 6.8 (buffer X). Fractions (5.0 ml) were
collected, using siliconized glassware(13) . The column was
washed with 15 ml of the same buffer after loading, then with 100 ml of
a solution containing 10 mM EDTA, 0.1% Triton X-100, 0.02%
sodium azide, 20% glycerol, and 25 mM TAPS, pH 8.9. The column
was then washed with 50 ml of a solution having the same constituents
except that 50 mM CAPS, pH 10.2, instead of 25 mM TAPS, pH 8.9, was used. The enzyme eluted, essentially
quantitatively, at pH 8.9 in six 5.0-ml fractions. Additional protein
eluted at pH 10.2, but almost no GnT II activity was detected. This
step gave approximately a 2-fold purification.
3 cm,
washed with buffer X, described above). The column was washed with 15
ml of the same buffer, then with 50 ml of buffer G(13) , and 50
ml of buffer H(13) , both buffers having an additional 20%
(v/total v) glycerol, collecting 5.0-ml fractions. The enzyme eluted
with buffer G (0.1 M in phosphate) and could be monitored
using 1-µl aliquots diluted to 20 µl with a solution containing
125 mM MES, 25 mM MnCl
, and 1 mg/ml
bovine serum albumin, pH 6.5. This step gave a 1.5-fold purification.
3.0 cm, washed with 2.0 M NaCl, then with
buffer X). 5-ml fractions were collected. The column was washed with an
additional 40 ml of buffer X, then with 25 ml of the same buffer
containing 0.5 M NaCl. The enzyme did not bind strongly to the
reactive yellow-agarose but was retarded, eluting in the wash with
buffer X. Some additional protein came off in the 0.5 M NaCl
wash. This step gave a 1.3-fold purification of the active enzyme.
48 cm). A single peak of GnT II activity
was observed. SDS-PAGE analysis of all active fractions showed a
42-kDa, silver-stained band, which correlated in intensity with
enzymatic activity. Heavy loading of the gel showed a minor band of
approximately 34 kDa.
Micropreparative Isolation of Protein and Peptides
for Sequencing
Automated Edman degradation was performed
using Applied Biosystems sequencers (470A and 477A). For sequence
analysis of the intact protein, the GnT II enzyme was first desalted
and purified free of detergent contaminants by reversed-phase HPLC
through trace enrichment onto Brownlee RP-300 (30 4.6 mm and 30
2.1 mm) columns (17, 18). The concentrated sample was applied
directly onto a Polybrene-treated glass fiber filter. For sequencing
the 42-kDa polypeptide, the HPLC-purified enzyme was reduced
(dithiothreitol) and alkylated (iodoacetic acid). The 42-kDa band was
isolated by preparative SDS-PAGE (12% gel) and electroblotted onto a
polyvinylidene difluoride membrane, which was sandwiched between the
glass block and the Polybrene-treated glass fiber filter. For analysis
of internal sequences, the 42-kDa band was electroeluted using a
modified electroelution apparatus(18, 19) . The eluted
protein was digested with trypsin at an enzyme:substrate ratio of 1:20.
Resultant peptides were isolated by microbore reversed phase HPLC on a
Brownlee RP-300 (30
2.1 mm) column.
Construction and Screening of a Rat Liver cDNA
Library
A cDNA library was constructed in gt11 (20) from rat liver poly(A)
-enriched RNA, by
priming reverse transcription with oligo(dT), as previously
described(21) . 5 µg of poly(A)
RNA was
used for cDNA synthesis. The cDNA library contained
3.2
10
gt11 phages. Recombinant phages were propagated and
screened following procedures, which have been described(21) .
The hybridization was carried out at 38 or 65 °C, depending on
whether degenerate synthetic oligonucleotide or DNA fragments were used
as probes. DNA oligonucleotides were synthesized with an automated
synthesizer (Applied Biosystems, 381A) using the phosphoramidite
chemistry. The synthetic oligonucleotides were purified and end labeled
with [
-
P]ATP (Amersham Corp., >6000
Ci/mmol) and polynucleotide kinase, as previously
described(21) . DNA fragments were labeled with
[
-
P]dCTP (Amersham, > 3000 Ci/mmol) and
Klenow's fragment to a specific activity of >10
cpm/µg following the random priming procedure(20) .
5`-Extension of Rat GnT II
cDNA
Complementary DNA was synthesized from rat liver
poly(A)-enriched RNA by priming reverse transcription
with the rat GnT II-specific oligonucleotide
5`-CCGTCTTTATCGACATTCCTCAGC-3`, designated R9P1R, that is complementary
to a sequence in clone R9 (Fig. 2). 200 ng of poly(A)
RNA and a RNase H
reverse transcriptase
(Stratagene, StrataScript) were used for first-strand cDNA synthesis.
Following purification on a GlassMax spin column (Life Technologies,
Inc.), a homopolymer of deoxycytidilic acid was added to the 3`-end of
single-stranded cDNA, using terminal deoxynucleotidyl transferase. The
reaction (20 µl) was carried out in10 mM Tris-HCl, pH 8.4,
containing 25 mM KCl, 1.25 mM MgCl
, and
200 mM dCTP. The C-tailed cDNA was amplified by PCR using a
nested rat GnT II-specific oligonucleotide
5`-CAAAGTTCAACTGGTACACTAGGG-3`, designated R9P3R (Fig. 2), as a
3`-primer and a 36-mer DNA oligomer
5`-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3`, where I indicates
inosine, as a 5`-primer. This DNA oligomer, designated ``anchor
oligonucleotide,'' contains an adaptor region, which carries
multiple restriction sites (underlined) and a sequence complementary to
the homopolymeric tail. Inosine residues were introduced in the
homopolymeric tract of G to lower the T
of the DNA oligomer. The reaction (100 µl) was carried
out in 10 mM Tris-HCl, pH 8.3, containing 50 mM KCl,
1.5 mM MgCl
, 0.01% gelatin, 0.5 mM of
each of the four dNTP, 20 pmol of each oligonucleotide primer, 2.5
units of Taq DNA polymerase (AmpliTaq, Perkin-Elmer Corp.),
and
5 pg of C-tailed single-stranded cDNA for 30 cycles in a
thermo cycler (Perkin-Elmer, model 480). Cycle conditions were as
follows: 94 °C, 1 min; 56 °C, 2 min; and 72 °C, 2 min. PCR
amplification was also carried out on glass capillary plugs (
2.5
µl) of agarose gel (3% NuSieve and 1% Seakem, FTC) containing
size-selected cDNA molecules. In these reactions, a 20-mer
oligonucleotide 5`-GGCCACGCGTCGACTAGTAC-3` representing the adaptor
region of the anchor oligonucleotide was used as 5`-primer and either
the GnT II-specific oligonucleotide R9P3R or R9P4R (Fig. 2) as a
3`-primer. PCR amplification was for 25 cycles, with the following
conditions: 94 °C, 1 min; 60 °C, 2 min; and 72 °C, 2 min.
Figure 2:
5`-Extension of rat GnT II cDNA. The
nucleotide sequence of the 5`-end of the open reading frame in
gt11 cDNA clones R7, R9, and R9.5 (Fig. 1), including the
artificial EcoRI site (lowercaseletters), is reported in the upperpart of the figure. The arrows indicate the 5`-3` direction
and length of the three nested GnT II-specific oligonucleotides used as
3`-primers in first-strand cDNA synthesis and amplification. The
analysis of the two major cDNAs generated by priming reverse
transcription of rat liver poly(A)
RNA with the GnT
II-specific oligonucleotide R9P1R is illustrated in the lowerpart of the figure. The two cDNA species of
360 and
250 bp were size-selected by gel electrophoresis and amplified by PCR. LanesA and B, 360 bp of cDNA; lanesC and D, 250 bp of cDNA. The adaptor
oligonucleotide was used as a 5`-primer and either the GnT II-specific
oligonucleotide R9P3R or R9P4R as a 3`-primer. The PCR products were
resolved by electophoresis through a 4% agarose gel and visualized by
ethidium bromide staining and UV irradiation. A ladder of DNA
fragments, which differ in length by 126 bp (lanem),
was used as molecular size markers.
cDNA Subcloning and Nucleotide
Sequencing
Restriction fragments of cDNA inserts were
isolated from purified phage DNA and subcloned into the vectors
pBlueScript SK or pBSKS and their variants plus and minus (Stratagene)
by standard methods(20) . The PCR-amplified cDNA was cloned into
the pCRII vector (Invitrogen). Escherichia coli strains
XL1Blue or DH5Mcr (20) were used as plasmid carriers. DNA
sequence analyses were performed either on the
gt11 phage vector
or on the pBR322-based vectors by the dideoxy chain termination
method(22) , using
-[thio-
S]dATP (DuPont NEN,
1000-1500 Ci/mmol). Sequenase 2 (U. S. Biochemical Corp.) or Vent
Exo
(New England Biolabs) DNA polymerases were used
in the sequencing reactions, according to the manufacturer's
instructions. Sequences were determined on both strands, following the
strategy outlined in Fig. 1. Nucleotide sequence data were
analyzed on a DEC VAX/9000 computer, using the software developed by
the Genetic Computer Group (GCG, Madison, WI)(23) . The
hydropathy plot was generated using a GCG implementation of the Kyte
and Doolittle algorithm(24) . Sequence data base searches were
performed on the GenBank data base (release 84) and Protein sequence
data base (SwissProt, release 29), using a GCG implementation of the
Lipman and Pearson algorithm (25) or the BLAST
programs(26) .
Figure 1:
Restriction map and
sequencing strategy of rat GnT II cDNA. The sequence corresponding to
the coding region is indicated by the openbox. Only
restriction sites relevant to the sequence analysis are indicated. The
letter ``P'' denotes the position of the synthetic
oligomer used as a specific primer in cDNA synthesis. Horizontalarrows indicate direction and extent of sequence
determination. The arrows originating in a solidcircle represent sequences determined using the gt11
forward or reverse sequencing primers. The arrows originating
in a solidsquare represent sequences determined from
subcloned fragments using the KS, SK, T3, T7, or M13 reverse sequencing
primers. The arrows originating in a solidtriangle represent sequences determined using GnT
II-specific oligonucleotides.
Construction of Variable Reading Frame Expression
Plasmids
The vectors used for expressing the rat GnT II
cDNA were derived from pBC12BI (27) and kindly provided by
Jarema Kochan (Dept. of Genetics, Hoffman-LaRoche Inc., Nutley, NJ).
The 700-bp BamHI-SmaI fragment of pBC12BI containing
the coding sequences and the large intron of the rat preproinsulin II
gene (28) was replaced, by standard procedures(20) , with a
850-bp HinfI-NciI fragment of human cDNA for the IL-2
receptor, coding for the cleavable signal peptide sequence and the
N-terminal region of the mature polypeptide(29) . The resulting
plasmid denominated pLJ79 was digested with BstEII, blunt
ended by filling-in, and digested with SacI to remove a SacI-BstEII fragment containing the human cDNA coding
for the mature polypeptide of the IL-2 receptor. The large fragment of
pLJ79 was recircularized by ligation to each one of three partially
double-stranded synthetic DNA oligomers, whose 5`-ends are
complementary to a SacI 3`-overhang. The sequences of the
three DNA oligomers designated RF1, RF2, and RF3 are
5`-AGCTCCAGCTGAATGAATGA-3`, 5`-AGCTCGCAGCTGAATGAATGA-3`, and
5`-AGCTCAGCTGAATGAATGA-3`. These DNA oligomers contain a PvuII
restriction site (underlined), which allows the insertion of a DNA
fragment in all three reading frames, and is flanked by multiple
translation termination codons. The vectors containing the DNA
oligomers RF1, RF2, and RF3 were designated pLJ268, pLJ269, and pLJ267,
respectively.
gt11 clone R9 (Fig. 1) was digested with EcoRI and
treated with mung bean nuclease (New England Biolabs). The blunt-ended EcoRI fragment from plasmid pR9 was purified by
electroblotting onto and elution from a DEAE-membrane (Schleicher &
Schuell, NA45) and digested with XmnI to remove the
3`-untranslated region of rat GnT II cDNA (Fig. 1). The 1176-bp EcoRI-XmnI fragment containing the coding sequences
of rat GnT II cDNA, including the translation termination codon, was
purified as described above and cloned into the unique PvuII
site of vectors pLJ268 and pLJ269. The recombinant plasmids were
designated pLJ268_R9 and pLJ 269_R9, respectively, and their structure
was verified by digestion with several restriction enzymes and sequence
analysis using multiple GnT II-specific oligonucleotides as sequencing
primers. The sequence analysis using as a primer the rat GnT
II-specific oligonucleotide designated N5 that represent the complement
of a segment (nucleotide position 364-348 in Fig. 3) of the
rat GnT II cDNA confirmed that in plasmid pLJ 268_R9 the rat GnT II
cDNA is linked in frame to the cDNA for the signal sequence of the
human IL-2 receptor, whereas in plasmid pLJ269_R9 the rat GnTII cDNA is
out of frame, and a premature stop codon is generated (Fig. 4).
Figure 3:
cDNA and predicted protein sequence of rat
GnT II. Nucleotides and amino acids are numbered at the left of each line. The predicted protein sequence is
shown in the standard three letter code below the cDNA
sequence. Regions of the GnT II polypeptide from which direct protein
sequence data were obtained are underscored with a solidbox. Openareas denote unidentified
amino acids by protein sequencing. Asterisks indicate
potential sites for N-linked glycosylation. The proposed
membrane-anchoring sequence is underlined. The entire sequence
shown was derived independently from both strands of the cDNA, as
indicated in Fig. 1. Nucleotides 1-348 derive from clones p5R7,
p5R13, p5R16, p5R27, and p5R32; 268-2155 from clones R7 and R9;
342-2104 from clone R9.4; 678-2155 from clone R9.3; and 914-2155 from
clone R9.2. Where sequences from two or more clones overlapped, they
were identical. The sequence in clone R9 complementary to the synthetic
DNA oligomer used as a specific primer in the cDNA synthesis is underlined.
Figure 4:
Structure of expression plasmids. These
plasmids contain the SV40 origin of replication and a segment of human
cDNA coding for the cleavable signal peptide sequence of the IL-2
receptor (hum il2_R sps) under transcriptional control of the Rous
sarcoma virus long terminal repeat (RSVLTR). The rat
cDNA region coding for the C-terminal 389 amino acids of GnT II is
linked in frame (pLJ268_R9) or out of frame (pLJ269_R9) to the hum
il2_R sps cDNA. The rat GnT II cDNA is flanked on its 3`-end by the
3`-untranslated region of the rat preproinsulin II gene (rat ins-2
3`UTR). The region of the rat preproinsulin II gene containing the
first intron, including the donor and acceptor splice sites (rat ins-2
ivsA), flanks the 5`-end of the hum il2_R sps cDNA. The bacterial
origin of replication and the -lactamase gene of the pLJ vectors
are derived from pXF3 (27), a pBR322 derivative that lacks sequences
inhibitory to DNA replication in COS cells. The nucleotide sequence of
plasmids pLJ268_R9 and pLJ269_R9 at the junction between the hum il2_R
sps and the rat GnT II cDNA was verified as described under
``Experimental Procedures'' and is reported in the lowerpart of the figure. The deduced amino acid sequence is
shown in the three letter code below the nucleotide sequence.
The opentriangle indicates the cleavage site of the
signal peptidase.
Transfection of COS Cells
COS-7 cells
were kindly provided by Paolo Comoglio (University of Torino) and grown
in Dulbecco's modified Eagle's medium containing 10% (v/v)
fetal calf serum and 10 µg/ml gentamycin sulfate (complete medium),
in a humidified atmosphere containing 5% CO, at 37 °C.
Confluent cell monolayers were trypsinized, and 5
10
cell aliquots were plated in 60-mm dishes 24 h before
transfection. COS-7 cells (80% confluent) were transfected either with
plasmid pLJ268_R9 or pLJ269_R9, by liposome-mediated uptake, using
Lipofectin (Life Technologies, Inc.) as described by the manufacturer.
Culture media and cell monolayers were collected at 24, 48, and 72 h
post-transfection and stored at -80 °C. As a control for
transfection efficiency, COS-7 cells were transfected with
pSV
-neo and cultured in complete medium containing 600
µg/ml of G418 (Life Technologies, Inc., Geneticin). Colonies of
neo
cells were enumerated at 3 weeks after transfection.
The transfection efficiency was
10%.
In Vitro Assay for GnT II Activity
Assays
were carried out as previously described (13) with minor
modifications. Instead of a glycopeptide, the reducing oligosaccharide
having the structure
Man1-6(GlcNAc
1-2Man
1-3)Man
1-4GlcNAc
1-4(Fuc
1-6)GlcNAc,
referred to as MGn(+F) was used as an acceptor substrate. It was
prepared from bovine fibrin as previously described (30) with
two modifications. First, instead of direct treatment of the fibrin
with hydrazine, glycopeptides were first prepared with Pronase (1/50)
and were isolated on a Sephadex G-50 column. Second,
re-N-acetylation was carried out directly after hydrazine
treatment(31) . The
H-NMR spectrum of the substrate
is shown in panela of Fig. 5. Cell pellets
(
1
10
cells) were lysed in 80 µl of a
buffer containing 50 mM NaCl, 15 mM MnCl
,
1 mM sodium azide, 1% (v/v) Triton X-100, and 50 mM MES, pH 6.8. Culture media were diluted 4-fold with the above
buffer. 20-µl aliquots of a cell lysate or diluted culture medium
were used in each assay. Reactions (25 µl) were carried out in 50
mM MES, pH 6.8, containing 1 mM
UDP-[
C]GlcNAc, 465 µM MGn(+F),
40 mM GlcNAc, 15 mM MnCl
, 50 mM NaCl, 1 mM sodium azide, and 1% (v/v) Triton X-100. The
product was quantitated using small Dowex AG1-X8 columns(13) .
Each assay was carried out in duplicate.
Figure 5:
Identification of the product of
recombinant GnT II catalysis by H-NMR spectroscopy.
Structural reporter regions of 500 MHz
H-NMR spectra of (a) the oligosaccharide acceptor and (b) the product
catalyzed by recombinant GnT II secreted by COS-7 cells upon
transfection with plasmid pLJ268_R9 are shown. Structures are shown above each spectrum, respectively. Indicated on the spectra
are the anomeric proton signals (
= 4.4-5.3), the
mannose H-2 signals (
= 3.95-4.3), the fucose H-5 and
H-6 (methyl) signals, and the acetamido methyl signals (
=
2.0-2.1). Residues are numbered according to Vliegenthart et
al. (43). Acetamido methyl protons are shown at
20-30%
full intensity.
Identification of the Product of in Vitro Catalysis by
Samples containing the
culture medium of COS-7 cells transfected with plasmid pLJ268_R9 at 72
h post transfection were concentrated approximately 4-fold and
simultaneously dialyzed against a buffer containing 50 mM NaCl, 1.0% Triton X-100, 0.02% sodium azide, 15 mM
MnClH-NMR Spectroscopy
, and 50 mM MES, pH 6.8, on a Micro-ProDiCon
dialyzer (Bio-Molecular Dynamics), at 4 °C. This solution (2.0 ml)
was incubated with MGn(+F) (300 nmol) and
UDP-[
C]GlcNAc (1 mCi/mmol, 10 µmol) at 37
°C for 75 min. After dilution with 10 ml of water, the reaction
mixture was passed through two columns in tandem, followed by 5
40-ml washes with water, Dowex AG1-X8 (Cl
form,
100-200 mesh, 2.5
8.2 cm), and Bio-Beads SM-2 (Bio-Rad,
20-50 mesh, 2.5
5.1 cm). The eluate was rotary evaporated
and applied in a 3.0-ml volume to a Bio-Gel P-4 column (Bio-Rad,
200-400 mesh, 1.5
92 cm). Fractions (4 ml) were
collected, and aliquots (10 µl) were monitored for
C-labeled product. Two peaks were found, one centered at
fraction 32, which comigrated with GlcNAc (25 nmol, based on specific
activity) and one centered at fraction 21 (235 nmol). The high
molecular weight material was concentrated and chromatographed on a
Waters GlycoPak N HPLC column (7.8
300 mm), with
acetonitrile/water (75/25) at 1.0 ml/min, monitoring at 200 nm. A major
biphasic peak appeared at 125-145 min, which gave 225 mol of
product. The product was examined in D
O by 500 MHz
H-NMR spectroscopy at 300 K on a Bruker AM-500 spectrometer
(University of Washington). Chemical shifts were relative to internal
acetone at
= 2.225 ppm.
Northern Blot Analysis
Total RNA was
prepared from rat organs as previously described(21) .
Poly(A) RNA was selected by affinity chromatography on
OligoTex-dT (Quiagen) according to the manufacturer's
instructions. The RNA concentration of each preparation was determined
from the absorbance at 260 nm(20) . 5-µg aliquots of each
poly(A)
-enriched RNA preparation were subjected to
electrophoresis through an agarose gel containing 2.2 M formaldehyde(20) . RNAs of a defined length (Life
Technologies, Inc., types II and III) were used as molecular size
markers. Capillary transfer onto neutral nylon membranes (Stratagene,
Duralon) was in 3.0 M NaCl, 0.3 M trisodium citrate
(pH 7.0), for 16-18 h. The RNA blots were UV-irradiated at 1800
µJ to cross-link RNA to the membranes, which were prehybridized in
a solution containing 50% (v/v) formamide (Life Technologies, Inc.,
UltraPure), 10% dextran sulfate, 1% SDS, 0.9 M NaCl, and 200
µg/ml of sheared salmon sperm DNA as a carrier at 42 °C for 4
h. The hybridization was carried out at 42 °C for 16-18 h in
prehybridization solution containing a labeled DNA fragment at 2
10
cpm/ml. Membranes were washed three times at
room temperature in 0.3 M NaCl, 30 mM trisodium
citrate (pH 7.0), and 0.1% SDS and three additional times at 65 °C
in 1.5 mM NaCl, 0.15 mM trisodium citrate (pH 7.0),
and 0.1% SDS. Membranes were exposed to x-ray film (Kodak, XAR-5) at
-80 °C, using two intensifying screens, for 24-72 h.
Probes were stripped off a blot by washing the membranes once at 65
°C in water for 15 min and several times at 95 °C in 1.5 mM NaCl, 0.15 mM trisodium citrate (pH 7.0), and 0.1% SDS
for 30-45 min.
using
the T3 and T7 promoter primers. The human
-actin cDNA probe was a
2.0-kilobase pair BamHI fragment derived from plasmid
pHF
A-1(32) , kindly donated by Peter Gunning. Hybridization
with the human
-actin cDNA probe was carried out at 37 °C, and
membranes were washed at 50 °C.
Isolation of cDNA Clones for Rat GnT
II
The GnT II enzyme was purified over 50,000-fold from rat
liver homogenates. It was isolated in a quantity of approximately 100
µg in about 2.5% yield. The specific activity of the purified
enzyme was 12 µmol of GlcNAc transferred/min/mg of protein.
SDS-PAGE analysis of the purified enzyme under reducing conditions
revealed a major polypeptide having a M of
42
kDa. N-terminal sequence analysis of the unreduced protein after
reversed-phase HPLC yielded two sets of phenylthiohydantoins, in
approximately equal amounts, for the first four cycles. However, by
cycle five, a single major sequence of 33 residues became predominant,
the other sequence apparently becoming less abundant (,
experiment 1). Therefore, the protein was reduced and alkylated,
preparatively run on SDS-PAGE, and electroblotted onto a polyvinylidene
difluoride membrane. N-terminal sequence analysis of the 42-kDa GnT II
polypeptide yielded two amino acid sequences (, experiment
2), which indicated, based on a partial overlap that could be discerned
between the major and minor amino acid sequences, a ragged N terminus.
Sequence analysis of a tryptic peptide derived from the purified 42-kDa
GnT II polypeptide yielded a sequence of 21 residues (,
experiment 3).
)Two 8-fold degenerate DNA oligomers of 17
nucleotides representing distinct segments of the amino acid sequence
of the tryptic peptide of rat GnT II (underlined in ) were
synthesized and used as hybridization probes to screen the
oligo(dT)-primed rat liver cDNA library. The two oligonucleotide
probes, having the sequences
5`-GTIGAYAARGAYGGIAC-3` and
5`-ARIACIARYTCICCIGG-3`, where R denotes A
or G and Y denotes C or T, were designated P4 and P5,
respectively. Approximately 6.2
10
recombinant
phages were screened, using the DNA oligomer P4 as a hybridization
probe. Seven clones, denominated R1, R3, R7, R9, R15, R17, and R20,
were detected. Three clones, R1, R7, and R9, hybridized to both probes
P4 and P5 and were isolated by plaque purification. A detailed
restriction map was generated for each clone, and restriction fragments
were subcloned in pBlueScript-based plasmid vectors for DNA sequence
analysis. The clones R1, R7, and R9 contained cDNA inserts that were of
similar length (
1.9 kilobase pairs), showed a similar restriction
pattern, and cross-hybridized (data not shown). The nucleotide sequence
of the cDNA inserts from clones R7 and R9 was determined on both
strands, as outlined in Fig. 1. The nucleotide sequence of the
two cDNA clones was identical and is shown in Fig. 3, where it
extends from nucleotide 268 to the terminal artificial EcoRI
restriction site. Four additional cDNA clones, designated R9.2, R9.3,
R9.4, and R9.5, were obtained through screening of the oligo(dT)-primed
rat liver cDNA library, using the 826-bp EcoRI-HindIII fragment of clone R9 as a hybridization
probe (Fig. 1). Restriction mapping and sequence analysis showed
that none of these clones extend further in the 5`-direction than
clones R7 and R9, their 5`-ends mapping to different regions within the
nucleotide sequence of clones R7 and R9 (Fig. 1). The 3`-end of
cDNA clones R9.2, R9.3, and R9.5 is identical to the 3`-end of cDNA
clones R7 and R9, whereas the the 3`-end of cDNA clone R9.4 maps 115 bp
upstream of the terminal EcoRI site. Although a canonical
poly(A) addition signal (AATAAA) is located 16 bp upstream of the
artificial EcoRI site in clones R7, R9, R9.2, R9.3, and R9.5,
no poly(A) tract is present in these clones nor in clone R9.4.
Therefore, these cDNA clones may represent self-priming events that
occurred within the GnT II mRNA.
-enriched RNA by
priming reverse transcription with the GnT II-specific oligonucleotide
R9P1R (Fig. 2). Following addition of a poly(C) tract to the
3`-end, the single-stranded cDNA was amplified by PCR using a nested
GnT II-specific oligonucleotide designated R9P3R (Fig. 2) as a
3`-primer and an anchor oligonucleotide containing a poly(G) tract as a
5`-primer. Two major cDNA fragments of approximately 380 and 260 bp
were detected in an agarose gel stained with ethidium bromide. No
detectable DNA fragments were generated in a control reaction in which
reverse transcriptase had been omitted (data not shown). To verify the
authenticity of the two products of reverse transcription, the two cDNA
fragments of 380 and 260 bp in length were separated by gel
electrophoresis, and each fragment was further amplified by PCR using
the adaptor oligonucleotide as a 5`-primer and either the GnT
II-specific oligonucleotide R9P3R or R9P4R (Fig. 2) as a
3`-primer. As illustrated in Fig. 2, PCR amplification of each
cDNA fragment using the two nested GnT II-specific oligonucleotides
R9P3R and R9P4R generates two products that differ in size by
60
bp. This value closely matches the distance between the two
oligonucleotides along the sequence of rat GnT II cDNA (Fig. 2),
thus indicating that both cDNA species are reverse transcription
products of rat liver GnT II mRNA, the smaller fragment probably being
the result of pausing events during first-strand cDNA synthesis.
Therefore, the larger cDNA fragment of
380 bp in length was cloned
into a plasmid vector. The nucleotide sequence of the cDNA inserts of
five recombinant plasmids, designated p5R7, p5R13, p5R16, p5R27, and
p5R33, was determined on both strands, as outlined in Fig. 1. The
sequences of all of these cDNA clones where they overlapped were
identical and aligned perfectly to the sequence of the corresponding
genomic region.
The overlapping cDNA sequence of clones
p5R7, p5R13, p5R27, R7, R9, R9.2, R9.3, R9.4, and R9.5 is shown in Fig. 3.
Sequence Analysis of Rat GnT II cDNA
The
cDNA sequence predicts an open reading frame, which starts at the
presumptive AUG translation initiation codon (nucleotide position 106)
and continues for 1326 nucleotides to encode a polypeptide of 442 amino
acids. The sequence context of this AUG is favorable for translation
initiation. A purine is present at position -3. The presence of a
purine at this position is the most critical and conserved feature that
is observed in the flanking sequences of AUG codons utilized for
initiation of protein synthesis in higher eukaryotes(33) .
(C - N) = -3)(36) , a
type II membrane topology is possible either by the positive inside
rule (37) or the charge difference
model(36, 38) .
450 and 435 bases upstream of the presumptive AUG
translation initiation codon, were detected by RNase protection
experiments using restriction fragments of a rat GnT II genomic clone
encompassing 1.1 kilobase pairs of 5`-flanking DNA sequences.
Although at a low frequency, 5`-untranslated regions of
400-600 bases in length have been observed among eukaryotic
mRNAs(39) . The 3`-untranslated region of rat GnT II mRNA
contains three canonical polyadenylation signals, having the consensus
sequence AATAAA (bold in Fig. 3). A cluster of AUUUA
sequence motifs (40, 41) is found in the region between
the first and second polyadenylation signals. In addition, multiple
characteristic short sequence repeats, which have been implicated in
the turnover of the apolipoprotein II mRNA(42) , are found
dispersed along the region flanked by the first and third
polyadenylation signals.
Expression of Rat GnT II cDNA in COS-7
Cells
The identity of the cloned cDNA for rat GnT II was
unambiguously verified by expressing the cDNA segment coding for the
C-terminal region of the GnT II polypeptide in COS cells. An indication
that the substrate binding site and the catalytic domain of the GnT II
enzyme are located in the C-terminal region of the GnT II polypeptide
was provided by the observation that the membrane-solubilized form of
rat GnT II, which is enzymatically active(13) , encompasses the
C-terminal 373 amino acid residues ( and Fig. 3). The
structures of the plasmids used for transfection are illustrated in Fig. 4. In these plasmids, the rat cDNA sequence coding for the
C-terminal 389 amino acid residues of the GnT II polypeptide was linked
in frame (pLJ268_R9) or out of frame (pLJ269_R9) to the human cDNA
segment coding for the cleavable signal peptide sequence of the IL-2
receptor. Plasmid pLJ268_R9 carrying the rat GnT II cDNA in frame codes
for a hybrid protein that will be acted upon by the signal peptidase,
the mature polypeptide being transported across the endoplasmic
reticulum, through the Golgi apparatus, and released into the
extracellular fluid. This was intended to facilitate detection of GnT
II activity above the endogenous level. Indeed, cultured mammalian
cells, which lack GnT II activity, are not yet available. Conversely,
plasmid pLJ269_R9 carrying the rat GnT II cDNA out of frame encodes a
short fusion protein prematurely terminating 13 amino acid residues
after the signal peptidase cleavage site and was used as a negative
control.
H-NMR
spectrum, as shown in panelb of Fig. 5.
Characteristic of the product of GnT II catalysis was the major
downfield shift of the
1-6-linked mannose H-2 signal, as
compared with the acceptor substrate, from
= 3.972 to
4.107 upon substitution at the 2 position with a
-GlcNAc
residue(43) . In addition, the intensity of the signals
representing the
1-2-linked GlcNAc H-1 protons (doublet,
= 4.555, J
= 8.3 Hz) and N-acetamido methyl protons (singlet,
= 2.052) in
the product were increased 2-fold as compared with the substrate.
Expression of GnT II mRNA
To examine the
transcription product(s) of the rat GnT II gene,
poly(A)-enriched preparations of RNA from rat liver,
brain, thymus, and spleen were subjected to Northern blot analysis.
Three GnT II-specific probes containing the 5`-end, the coding
sequence, or the 3`-untranslated region of the cloned cDNA were used.
The results are illustrated in Fig. 6, where the same amount (5
µg) of poly(A)
-enriched RNA was used in each lane.
A major transcript of 2.8 kb in length that hybridized to all three GnT
II-specific probes was detected in these organs. In addition, two minor
transcripts of 2.1 and 1.7 kb in length that hybridized only to probes
containing the coding sequence and the 5`-untranslated region of GnT II
cDNA were detected in all rat organs examined (Fig. 6, panelsA and B). The lack of hybridization of the two
minor transcripts to the 3`-untranslated region probe (Fig. 6, panelC) indicates that they do not result from RNA
degradation. As a matter of fact, a single 2.1-kb mRNA species was
detected by probing the same blots at intermediate stringency
conditions with a human
-actin cDNA probe (data not shown). The
weak hybridization signals, which correspond to an RNA species of
5.0 kb, comigrate with 28 S ribosomal RNA and could be the result
of nonspecific hybridization. As illustrated in Fig. 6, the
overall levels of GnT II mRNA vary in different rat organs, being
higher in the thymus and spleen and progressively lower in the liver
and brain. The relative abundance of the major and two minor
transcripts showed no variation.
Figure 6:
Northern blot analysis. 5-µg aliquots
of poly(A)-enriched RNA from rat liver (L),
brain (B), thymus (T), and spleen (S) were
fractionated by electrophoresis through a denaturing 1.1% agarose gel.
Three distinct fragments of rat GnT II cDNA were used as hybridization
probes. PanelA, a 365-bp EcoRI fragment of
plasmid p5R7 (Fig. 1) containing the 5`-end of the cDNA (nucleotide
position 1-348 in Fig. 3); panelB, a 1021-bp
cDNA fragment containing the coding region and extending from
nucleotide 397 to 1417 of the rat GnT II cDNA sequence reported in Fig.
3; panelC, a 574-bp fragment of a rat GnT II genomic
clone
containing the terminal 448 nucleotides of the
3`-untranslated region of rat GnT II cDNA (nucleotide position
1699-2147 in Fig. 3) and 126 bp of 3`-flanking DNA sequence. The
autoradiographs illus-trated in panelsA and B refer to distinct blots derived from different portions of a gel.
The autoradiograph illustrated in panelC refers to a
blot derived from a separate gel. The migration of the two ribosomal
RNAs is indicated by the arrows on the leftside of the threepanels.
H-NMR spectroscopy of the product of in vitro catalysis by the enzyme (Fig. 5). The cDNA potentially codes
for the membrane-bound form of the enzyme. It contains an open reading
frame of 1326 nucleotides that encodes a polypeptide of 442 amino
acids, with a calculated molecular mass for the unglycosylated
polypeptide of 51,109 Da. In the absence of information on the
molecular size and N-terminal amino acid sequence of the membrane-bound
form of GnT II, the AUG codon at position 106 (Fig. 3) is assumed
to function as a translation initiation codon.
50 amino acids (residues 30-80 in Fig. 3)
enriched in proline, glycine, and hydrophilic residues appears to
separate the catalytic portion of GnT II from the membrane-anchoring
domain. As postulated for other
glycosyltransferases(21, 44) , this hydrophilic region
may represent a solvent-exposed region of the enzyme, which could act
as a flexible arm, enabling the catalytic domain to move relative to
the plane of the membrane.
-mannosidase II
activities(50) . However, recent immunocytochemical studies
indicate that the subcellular distribution of both GnT I (51) and
-mannosidase II (52), previously thought to be
medial Golgi resident enzymes, is less topographically restricted and
shows cell type-specific variations.
1-2Man product, both
utilize UDP-GlcNAc as a donor substrate, both require Mn
ions for catalysis, and both are encoded by a gene not
interrupted by introns(53) .
(
)It is
possible that distinct N-acetylglucosaminyltransferases may
have related three-dimensional structures and that only a limited
number of widely dispersed amino acids are conserved along the
otherwise variable peptide sequence.
)
Moreover, two
canonical polyadenylation sites whose relative positions correspond to
the first and third polyadenylation sites in the 3`-untranslated region
of rat GnT II cDNA (Fig. 3), have also been conserved in the
cloned human GnT II cDNA. The conserved sequence similarity of the
3`-untranslated region across mammalian species suggests a conserved
function of this region, probably in regulating GnT II mRNA stability
or translational proficiency.
and human
genome. Therefore, the occurrence of a transcript of 2.8 kb in
length indicates that the cloned cDNA may not include the complete
3`-untranslated region of rat GnT II mRNA. Considering that
transcription of the rat GnT II gene
starts at
450
bases upstream from the AUG translation initiation codon at position
106 of the cDNA (Fig. 3), a transcript of 2.8 kb in length can be
accounted for by a 1.1-kb 3`-untranslated region, which extends
0.4 kb further downstream of the 3`-end of the cloned rat GnT II
cDNA. This conclusion is consistent with the observation that none out
of the eight cDNA clones that were isolated contained a terminal
poly(A) tract, probably representing self-priming events during cDNA
synthesis due to the formation of local secondary structures in the
mRNA. The variation of the overall level of GnT II mRNAs observed in
rat organs could be the result of a transcriptional control mediated by
tissue-specific transcription factors that specifically recognize and
bind to sequence motifs, as suggested by sequence analysis of the
5`-untranslated region of the rat GnT II gene.
Table: Amino acid sequence analysis
/EMBL Data Bank with accession number(s) U21662.
-6-D-mannoside
-1,2-N-acetylglucosaminyltransferase II; GnT I,
UDP-N-acetylglucosamine:
-3-D-mannoside
-1,2-N-acetylglucosaminyltransferase I; GnT III,
UDP-N-acetylglucosamine:
-D-mannoside
-1,4-N-acetylglucosaminyltransferase III; GnT V,
UDP-N-acetylglucosamine:
-6-D-mannoside
-1,6-N-acetylglucosaminyltransferase V; GnT VI,
UDP-N-acetylglucosamine:
-6-D-mannoside
-1,4-N-acetylglucosaminyltransferase VI; IL-2,
interleukin 2; PAGE, polyacrylamide gel electrophoresis; HPLC, high
pressure liquid chromatography; MGn(+F),
Man
1-6(GlcNAc
1-2Man
1-3)Man
1-4GlcNAc
1-4(Fuc
1-6)Glc-NAc;
PCR, polymerase chain reaction; CAPS,
3-[cyclohexylamino]-1-propanesulfonic acid; MES,
2-(N-morpholino)ethanesulfonic acid; TAPS, N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic
acid; bp, base pair(s); kb, kilobase(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.