From the Department of Structural Biology and
Biochemistry, The Hospital for Sick Children, Toronto, Ontario M5G 1X8,
Canada, the § Department of Biochemistry, University of
Toronto, Toronto, Ontario M5S 1A8 Canada, and the ¶ Department of
Molecular and Medical Genetics, University of Toronto,
Toronto, Ontario M5S 1A8, Canada
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ABSTRACT |
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UDP-N-acetylglucosamine: UDP-N-acetylglucosamine: These studies indicate that although complex N-glycans are
not essential for the growth of cells in tissue culture, they play critical roles in mammalian morphogenesis. Complex N-glycans
are absent from bacteria (15) and yeast (16) and are present in very
small amounts, if at all, in protozoa (Trypanosoma cruzi (17), Leishmania (18), and Plasmodium (19)) and
Dictyostelium discoideum (20). All of the above organisms
except bacteria are capable of making N-glycans of the
oligomannose type. Complex N-glycans are present in most of
the multicellular invertebrate and vertebrate animals that have been
analyzed (nematodes (21-23), schistosomes (24-26), molluscs (27, 28),
insects (29), fish (30), birds (31-34), and mammals) and in plants
(35). However, a mutant Arabidopsis plant, which lacks GnT I
and is unable to synthesize complex N-glycans, shows no
apparent phenotype (36, 37), suggesting that complex
N-glycans do not play an essential role in plant
development. The data indicate that complex N-glycans appeared in evolution just prior to the appearance of multicellular organisms and that, at least in mammals, they play important roles in
the interactions between a cell and its cellular and fluid environment.
Because of the complexities encountered in the study of mammalian
development, we have initiated studies on the role of complex N-glycans in the development of a simpler organism, the
nematode worm Caenorhabditis elegans. Over 80% of the
C. elegans genome has been sequenced, and detailed
information is available on the morphology, development, and physiology
of this worm. The GnT I gene (MGAT1) has been cloned from
several mammalian and nonmammalian species (38). A computer search of
the C. elegans genomic DNA sequence data base for sequences
similar to the rabbit GnT I protein sequence using the BLASTP algorithm
(39) revealed three homologous sequences, the products of predicted
genes F48E3.1, B0416.6, and M01F1.1
(40). We report in this paper the sequences of the cDNAs of
these three genes which we have designated gly-12,
gly-13, and gly-14, respectively; all C. elegans glycosylation-related genes are named gly (41).
We present an analysis of the spatial and temporal pattern of gene
expression during C. elegans development and the enzyme
activities the genes encode when expressed in insect cells and in
transgenic worms. Preliminary reports of this work have appeared
(42-45).
Molecular Biology Procedures
Unless otherwise stated, standard molecular biology procedures
were used (46, 47). Oligonucleotides were synthesized on a Pharmacia
DNA synthesizer and purified by the cartridge method (Hospital for Sick
Children-Amersham Pharmacia Biotech Center, Toronto, Canada). All
cDNAs and DNA constructs were sequenced in both directions by the
double strand dideoxy method (48) using the Amersham Pharmacia Biotech
T7 Sequencing Kit.
Cloning of gly-13 cDNA by Phage Library Screening
The polymerase chain reaction (PCR) was used to prepare three
gly-13 gene-specific probes (based on the genomic sequence; GenBankTM accession number U23516), as follows (see Table I
for PCR primer sequences): probe A (142 nt), primers CEBF1 and CEBR2; probe B (145 nt), primers CEBF3 and CEBR4; probe C (168 nt), primers CEBF5 and CEBR6. PCR products were purified by electrophoresis on a 1%
agarose gel. DNA probes were labeled with [ Subcloning and Sequencing of gly-13 cDNA
Since there is an EcoRI site in the open reading
frame of gly-13 near the 3'-end, the full-length
gly-13 cDNA was obtained by partial digestion of the
Cloning of gly-14 cDNA by RT-PCR
The gly-14 cDNA was cloned by an RT-PCR approach
using the Marathon cDNA Amplification Kit
(CLONTECH) as recommended by the manufacturer.
Adaptor-ligated double-stranded cDNA was synthesized by reverse
transcription of adult C. elegans total RNA followed by
second strand synthesis and ligation of Marathon cDNA adaptor to
both ends of the double-stranded cDNA. The Marathon cDNA
adaptor has two primer binding sites: AP1 (outer) and AP2 (inner) (see Table I). PCR was then carried out three times in succession using the
adaptor-ligated cDNA as template and the following primer pairs
(Table I; gene-specific primers based on the genomic sequence, GenBankTM accession number Z46381): CEMR5-AP1 (once)
followed by CEMF1-CEMR6 (twice). PCR was also carried out three times
in succession using the following primer pairs (Table I): CEMF7-AP1
(once) followed by CEMF8-CEMR4 (twice). The PCR products could be
visualized by ethidium bromide staining of agarose gels only after the
third round of PCR. Fusion of the two PCR products was carried out by PCR using Vent DNA polymerase (New England Biolabs) and the primer pair
CEMF1-CEMR4 (Table I) to yield a cDNA fragment encoding the GLY-14
protein sequence containing the STOP codon but lacking 30 amino acids
at the amino terminus, including the putative transmembrane domain.
This truncated cDNA was subcloned into the NotI and
KpnI sites of the baculovirus transfer vector pVT-Bac-His
(kindly donated by Dr. David Joziasse, Vrije Universiteit, Amsterdam)
(38) and sequenced. Some of the missing 5'-sequence of
gly-14 cDNA was obtained by PCR using the AP2-CEMR10
primer pair (Table I) and adaptor-ligated cDNA as template.
Attempts to determine the remainder of the 3'-end of the cDNA by
3'-RACE were not successful.
Cloning of gly-12 cDNA by Phage Library Screening
A 1.2-kb gly-12 hybridization probe was made by PCR
using the Marathon adaptor-ligated C. elegans cDNA
(described above) as template and primers CEFF2-CEFR3 (Table I;
gene-specific primers based on the genomic sequence,
GenBankTM accession number U28735). The C. elegans cDNA library was screened with this probe, as
described above for gly-13. After three rounds of screening
with the same probe, nine positive plaques were identified. Phage DNA
from five of these plaques was prepared, and inserts were excised with
EcoRI, subcloned into pGEM7Zf(+), and sequenced. A portion
of the 3'-end of the gly-12 cDNA (88 nt) was obtained by
PCR using adaptor-ligated cDNA as template and three successive PCR
reactions with primer pairs CEFF1-AP1, CEFF2-AP2, and CEFF5-CEFR7,
respectively (Table I). Attempts to determine the remainder of the
3'-end of the cDNA by 3'-RACE were not successful.
Determination of 5'-Ends of gly-12, gly-13, and gly-14
cDNAs
Total cDNA was prepared by RT-PCR using as substrate total
RNA prepared from the L2 larval stage. The following PCR reactions were
carried out using this cDNA as template and the SL1 primer (Table
I) as the forward primer and reverse primers as shown in Table I.
gly-12--
PCR was carried out using CEFR15 as the reverse
primer. The solution was reamplified using the nested primer CEFR4. A
PCR product of the expected size was observed and further amplified with reverse primer CEFR10.
gly-13--
PCR was carried out using CEBR4 as the reverse
primer. A PCR product of the expected size was seen and reamplified
with the nested primer CEBR2.
gly-14--
A procedure similar to that used for
gly-12 was carried out using CEMR6, CEMR10, and CEMR2,
respectively, as reverse primers. The three final PCR products were
sequenced and all three messages showed trans-splicing to SL1.
Expression of GnT I in the Baculovirus/Sf9 Insect Cell
System
C. elegans GnT I was expressed in the
baculovirus/Sf9 insect cell system as described previously (38,
50, 51). DNA fragments encoding truncated GLY-12, GLY-13, and GLY-14
GnT I proteins lacking the amino-terminal cytoplasmic and transmembrane
domains and parts of the stem region were synthesized by PCR
amplification using Vent DNA polymerase and GnT I cDNAs as
templates (43, 39, and 31 amino acids were removed from the
amino-terminal end, respectively). The primer pairs used for
gly-13 and gly-14 are shown in Table I. The PCR
products were subcloned into the baculovirus transfer vector
pVT-Bac-His (38) downstream from and in frame with the ATG start site
of the plasmid using restriction enzyme sites introduced by the primers
(Table I). This vector encodes a cleavable signal sequence for
secretion from the Sf9 cells.
Full-length gly-12 cDNA was excised from plasmid
p78F-Myc (see below) and subcloned into pBlueBacHis C (Invitrogen)
downstream from and in frame with the ATG start site of the plasmid to
create a recombinant transfer vector encoding full-length GLY-12
(51).
Recombinant plasmids were sequenced and co-transfected with BaculoGold
linearized baculovirus DNA (PharMingen) to produce recombinant
baculovirus by homologous recombination in Sf9 cells (50).
Sf9 cells were infected with baculovirus, and after 5-6 days,
cells were sedimented and lysed in 0.5 ml of 25 mM MES, pH
6.5, 0.1% Triton X-100, 0.02% sodium azide.
GnT I Enzyme Assays
Sf9 cell lysates and culture medium and worm lysates were
assayed for GnT I activity using as acceptor substrate 0.5 mM Man Northern Analysis of C. elegans mRNA
Total (~20 µg) and poly(A)+ RNA (~2.5 µg)
from a mixed stage population of N2 hermaphrodites and total RNA from
staged synchronous populations was subjected to Northern analysis by
electrophoresis in a denaturing 1.0% agarose gel (10% formaldehyde)
(55). DNA size markers were prepared by digestion of Quantitation of gly-12 and gly-14 Messages by Competitive
RT-PCR
We were able to detect gly-14 mRNA only by
RT-PCR, not by Northern blot analysis, suggesting that
gly-14 is expressed at lower levels than gly-12
and gly-13. We used competitive quantitative RT-PCR to
estimate the relative abundance of both gly-12 and
gly-14 mRNA during development. Total cDNA was
obtained by oligo(dT)-primed reverse transcription of total RNA from
all six developmental stages in the presence of 1 µCi of
[ Preparation of DNA Constructs for Promoter Analysis
We used transcriptional fusion of GnT I genomic DNA to the
lacZ reporter gene to examine the spatial pattern of GnT I
expression during C. elegans development. Plasmids encoding
lacZ were provided by Dr. A. Fire, Carnegie Institute of
Washington (Baltimore,
MD).2
gly-13--
Cosmid B0416 (40) was grown overnight in 500 ml of
LB medium containing 50 µg/ml of ampicillin. DNA was prepared by
using the Qiagen Maxi-prep kit, and two gene fragments were cut out using SalI and PstI, respectively. A DNA fragment
(1008 nt) containing the putative promoter region immediately upstream
of the first exon, the complete 30-nt 5'-UTR, and the first 8 nucleotides of the open reading frame (the initiation codon ATG was
mutated to TTG) was obtained by PCR using the SalI fragment
from cosmid B0416 as a template and primers PRBF53 and PRBR1040 (Table
I). The PCR product was subcloned into the SalI and
BamHI sites of plasmid pPD95.11 upstream of the
lacZ gene creating plasmid p11B/prom. A genomic DNA fragment
(3428 nt) containing the remainder of the open reading frame and the
complete 3'-UTR of gly-13 was obtained by PCR using the
PstI fragment from cosmid B0416 as a template and primers
PRBF618 and PRBR4027 (Table I). The PCR product was subcloned into the
SpeI and AflII sites of plasmid p11B/prom, downstream of the lacZ gene, creating plasmid
p11B/prom-ORF.
gly-14--
Cosmid M01F1 (40) DNA was prepared as above. A DNA
fragment (3.4 kb) containing the complete open reading frame except for the first nine nucleotides and the 3'-UTR was amplified by PCR from
cosmid M01F1 using primers PRMF1158 and PRMR4530 (Table I) and
subcloned into the SpeI and AflII sites of
plasmid pPD95.11 downstream of the lacZ gene to create
plasmid p11M/ORF. A DNA fragment (2.6 kb) containing the putative
promoter region immediately upstream of the first exon, the complete
5'-UTR, and the first nine nucleotides of the open reading frame (the
initiation codon ATG was mutated to ACG) was amplified by PCR from
cosmid M01F1 DNA with primers PRMF33 and PRMR2664 (Table I) and
subcloned into the SalI and BamHI sites of
plasmid p11M/ORF upstream of the lacZ gene to create plasmid
p11M/prom-ORF.
Plasmid p57M/prom2.6, containing the same 2.6-kb putative promoter
region as p11M/prom-ORF but in which the region downstream of the
lacZ gene was replaced with the 3'-UTR of the
unc-54 gene, was constructed by inserting the 2.6-kb PCR
product from the promoter region (see above) into the SalI
and BamHI sites of pPD95.57. Deletion of ~1.3- and
~2.0-kb fragments from the 5'-end of p57M/prom2.6 with
EcoRV and NheI resulted in plasmids p57M/prom1.3
and p57M/prom0.6, respectively.
gly-12--
Cosmid F48E3 (40) DNA was prepared as above. A DNA
fragment (1740 nt) containing the putative promoter region immediately upstream of the first exon, the complete 5'-UTR, and four nucleotides from the open reading frame (the initiation codon ATG was mutated to
AAG) was amplified by PCR from the cosmid DNA with primers PRFF5 and
PRFR6 (Table I) and subcloned into the SalI and
BamHI sites of pPD95.57 upstream of the lacZ gene
to create plasmid p57F/prom.
DNA Constructs for Heat Shock-induced Expression of GnT I by
Transgenic Worms
gly-13 Expression Construct--
A NotI DNA fragment
was excised from the gly-13 baculovirus transfer plasmid
(see above) encoding the open reading frame downstream of the
transmembrane domain (nucleotides 114-1350 relative to the initiation
ATG codon at +1), blunt-ended, and digested with ApaLI. A
DNA fragment covering the first exon, first intron, and second exon was
amplified by PCR from cosmid B0416 using primers BEXF1 and BEXR2 (Table
I) and digested with NheI and ApaLI. These two
fragments were then subcloned into the NheI and
EcoRV sites of plasmids pPD49.78 and pPD49.83 downstream of
the heat shock promoter (57), producing plasmids p78B and p83B,
respectively. Myc spacer was amplified from plasmid
AS#13093 using primers SC#001
and SC#002 (Table I). This fragment contains sequences encoding a Myc
epitope tag (underlined) and 18 amino acids from the FEM-1 amino
terminus (italics)
(MAAEQKLISEEDLGRTPNGHHFRTVIYNAAAVGGMH). The Myc
spacer was subcloned into plasmids p78B and p83B immediately upstream
of the gly-13 sequence, producing plasmids p78B-Myc and p83B-Myc, respectively.
gly-14 Expression Construct--
A KpnI DNA fragment
was excised from the gly-14 baculovirus transfer plasmid
(see above) encoding the open reading frame downstream of the
transmembrane domain (nucleotides 90-1314 relative to the initiation
ATG codon at +1), blunt-ended and digested with HindIII. A
DNA fragment covering the first three exons and introns, exon 4, and
part of intron 4 was amplified by PCR from cosmid M01F1 using primers
MEXF1 and MEXR2 (Table I) followed by digestion with NheI
and HindIII. These two fragments were then subcloned into
the NheI and EcoRV sites of plasmids pPD49.78 and
pPD49.83 downstream of the heat shock promoter, producing plasmids p78M and p83M, respectively. Myc spacer was subcloned into these two plasmids, as described above, immediately upstream of the
gly-14 sequence to produce plasmids p78M-Myc and p83M-Myc, respectively.
gly-12 Expression Construct--
The amino terminus of the
gly-12 open reading frame was amplified by PCR using as
template one of the gly-12 cDNA clones isolated from the
cDNA library (see above) and primers FEXF1 (Table I) and CEFR4
(Table I). An NsiI site was introduced near the ATG start
codon. The carboxyl terminus of gly-12 was similarly
amplified by PCR using primers CEFF5 (Table I) and FEXR2 (Table I). The middle of the gly-12 cDNA was obtained directly from the
cDNA clone. These three fragments and Myc spacer were subcloned
into the NheI and SacI sites of pPD49.78 and
pPD49.83, producing plasmids p78F-Myc and p83F-Myc, respectively.
Preparation of Transgenic Worms
Promoter Analysis--
DNA injection into the C. elegans germ line was carried out as described by Mello et
al. (57, 58). Transgenic lines were established from F2
descendants of animals injected with 10 or 50 ng/µl of GnT
I::lac Z constructs and 50 ng/µl of plasmid pRF4, which carries the dominant rol-6 allele rol-6
(su1006) that serves as a transformation marker. The total DNA
concentration of the injection mixture was adjusted to 100 ng/µl by
the addition of pBlueScript SK( Heat Shock-induced Overexpression--
Heat shock plasmids (see
above) were injected in the following mixtures: (i) p78B/p83B mixture
(p78B (10 ng/µl), p83B (10 ng/µl), pRF4 (50 ng/µl), pBlueScript
II( C. elegans Culture, Heat Shock, and Worm Lysis
The standard laboratory wild type strain N2 or a
smg-1 (e1228) mutant derived from N2 nematodes was grown on
MYOB (62) agar plates seeded with Escherichia coli strain
OP50 (a leaky uracil-requiring strain). To examine the consequences of
overexpressing GnT I, gravid adults were allowed to lay eggs for 2-3 h
at 20 °C. Eggs were incubated for a further 6 h at 20 °C and
subjected to heat shock treatment at 33 °C for 1 h at 12-h
intervals until the animals reached adulthood.
To measure the activity of overexpressed enzyme, heat shock was carried
out at 33 °C for 2 h followed by recovery at 20 °C for a
further 2 h. Worms from 10-15 agar plates were harvested by
washing off the plates with M9 buffer, pelleted by centrifugation at
700 rpm for 2 min, washed with 10 ml of M9 buffer and suspended in 5 ml
of ice-cold M9 buffer. An equal volume of ice-cold 60% (w/w) sucrose
was added, and the suspension was mixed by inversion and centrifuged at
700 rpm for 5 min to remove bacteria. Worms were collected; washed
twice with 10 ml of M9 buffer; resuspended in 1 ml of buffer containing
20 mM Tris-HCl (pH 7.5), 250 mM sucrose, and
protease inhibitor mixture (Boehringer); and stored at Immunolocalization of Overexpressed C. elegans GnT I in
Heat-shocked Transgenic Worms
Heat-shocked transgenic worms were prepared as described above.
Worms were fixed and stained as described by Finney and Ruvkun (63).
Mouse monoclonal anti-Myc antibody 9E10 (64) was used at 1:5 dilution.
Fluorescein isothiocyanate-labeled secondary antibody (goat anti-mouse
IgG, Jackson ImmunoResearch Laboratories, Inc.) was used at 1:50
dilution. The worms were also co-stained with 4,6- diamidino-2-phenylindole.
Western Blot Analysis
Worm lysates were subjected to SDS-polyacrylamide gel
electrophoresis (10%) (65) followed by Western blot analysis with anti-Myc antibody 9E10 (1:50 dilution). Horseradish peroxidase-labeled secondary antibody (donkey anti-mouse IgG, Jackson ImmunoResearch Laboratories, Inc.) was used at 1:20,000 dilution. Proteins were detected by the ECL system kit (Amersham Pharmacia Biotech).
Cloning of gly-12, gly-13, and gly-14 cDNAs--
The three
C. elegans GnT I cDNA sequences are not shown in this
paper and have been submitted to the GenBankTM data base.
We have determined the 3'-end of gly-13 but not of gly-12 and gly-14. The gly-13 cDNA
has a 342-nt 3'-untranslated region and an AATAAA polyadenylation
initiation sequence 23 nt upstream of a long poly(A) sequence. Over
70% of C. elegans mRNAs carry a 22-nt
trans-spliced 5' leader sequence known as SL1 (66); we
showed the presence of SL1-bearing transcripts at the 5'-ends of all
three C. elegans GnT I cDNAs but have not determined
whether the mRNAs of the three genes are exclusively
trans-spliced to SL1. As expected, there are consensus 3'
intron splice acceptor sites at the 5'-end of the first exon of all
three GnT I genes (data not shown).
Comparison of the gly-12, gly-13, and
gly-14 genomic DNA sequences in the C. elegans
data base with the cDNA sequences indicate that the three genes
contain 14, 12, and 12 exons, respectively, with conservation of all
5'-intron splice donor and 3'-intron splice acceptor sequences. This is
in marked contrast to the mammalian GnT I gene, in which the entire
coding region is on a single exon (55). The exon-intron junctions
predicted by computer analysis (Genefinder) of the C. elegans genomic DNA sequences were correct for the
gly-14 gene. Errors for the gly-13 gene were
relatively minor. Computer analysis of cosmid F48E3
predicted a gene F48E3.1 consisting of 17 exons.
However, we found that the predicted gene F48E3.1 consisted
of two separate genes. The most 3' 11 predicted exons, together with
three additional exons not predicted by the Genefinder program,
constitute an open reading frame that can encode a protein similar to
mammalian GnT I. We have designated this gene as gly-12. The
six predicted exons upstream of gly-12 form a separate gene,
designated F48E3.1a, that can encode a protein of unknown
function with no similarity to mammalian GnT I. A series of PCR
reactions using adaptor-ligated C. elegans cDNA as
template were carried out to establish the presence of these two
distinct genes (data not shown). PCR products of the expected sizes
were obtained with the following primer pairs (Table
I): CEFF8-CEFR11 (from the upstream gene)
and CEFF5-CEFR3 (from the downstream gly-12 gene). No PCR
products were obtained with primer pairs CEFF9-CEFR4 and CEFF8-CEFR10;
CEFF9 and CEFF8 were derived from the upstream gene sequence, whereas
CEFR4 and CEFR10 were derived from the downstream gly-12
gene.
The gly-12, gly-13, and gly-14
cDNA sequences contain open reading frames of 1401, 1347, and 1311 nt encoding putative proteins of 467, 449, and 437 amino acid residues,
respectively (Fig. 1). Hydropathy plots
(67) of all three protein sequences (not shown) predict a domain
structure typical of all previously cloned Golgi-type glycosyltransferases, namely a short N-terminal cytoplasmic domain, a
hydrophobic noncleavable signal-anchor transmembrane domain, a stem
region, and a long C-terminal catalytic domain. Whereas the mammalian
GnT I proteins do not contain any putative N-glycosylation sites (NX(S/T) sequons), all three C. elegans
proteins contain such sequences (GLY-12 at Asn111,
Asn128, Asn337, and Asn402; GLY-13
at Asn159; GLY-14 at Asn129,
Asn188, and Asn242). Comparison of the aligned
full-length protein sequences (Fig. 1) shows 44, 44, and 60%
identities for the GLY-12/GLY-13, GLY-12/GLY-14, and GLY-13/GLY-14
pairs, respectively, and comparison of the 350-394 amino acid
C-terminal catalytic domains with the corresponding mammalian GnT I
sequences shows 36, 47, and 48% identities for the GLY-12, GLY-13, and
GLY-14 sequences, respectively. The three C. elegans GnT I
protein sequences show no similarity to the mammalian GnT I proteins in
the cytoplasmic, transmembrane, and stem regions (Fig. 1). The
gly-13 and gly-14 genes share 9 of 11 intron
positions, whereas four introns occur at the same positions in all
three genes (Fig. 1). Genes gly-12 and gly-13 are
on C. elegans chromosome X, and gly-14 is on
chromosome III. The data indicate that the mammalian GnT I genes and
three C. elegans GnT I genes are derived from a common
ancestor.
Expression of C. elegans GnT I cDNAs in the
Baculovirus/Sf9 System--
Sf9 cell lysates have been
reported to contain GnT I activity (68), but endogenous GnT I activity
was low in both cell lysates and supernatants under our assay
conditions (0.03-3.4 nmol/105 cells/h at 48-120 h after
infection). The culture medium of Sf9 cells infected with
recombinant baculovirus encoding truncated GLY-14 contained levels of
enzyme activity equivalent to those previously obtained with mammalian
GnT I expression (data not shown). Expression of GLY-12 either as a
truncated or full-length protein yielded enzyme activities above
background levels, but this activity was appreciably less than the
intracellular activity of truncated GLY-14 (data not shown). Western
blot analysis using mouse monoclonal antibody raised against the
enterokinase cleavage site (Anti-Xpress antibody kit, Invitrogen) (38)
showed recombinant protein bands at the expected molecular weights for
GLY-12 (weak) and GLY-14 (strong). Attempts to express recombinant
baculovirus encoding truncated GLY-13 were not successful; we could not
detect a protein band by Western analysis, nor could we detect any
enzyme activity either in cell lysates or supernatants (data not
shown). Kinetic analysis of GLY-14 in cell supernatant and GLY-12 in
Sf9 cell extracts gave linear 1/v versus
1/S plots (where v is the initial velocity and
S is the substrate concentration) consistent with an ordered
sequential Bi Bi mechanism if one assumes steady state rather than
rapid equilibrium kinetics (53). Kinetic constants (data not shown)
indicate no major differences between C. elegans and
previously published data on rabbit GnT I (54); GLY-12 was assayed only
with M3-octyl, whereas GLY-14 and the rabbit enzyme were
assayed with both M3-octyl and M5-glycopeptide.
Rabbit GnT I had a higher temperature optimum (37 °C) than GLY-12
and GLY-14 (20-30 °C, data not shown), and the rabbit enzyme
remained active at pH 5.0-5.5, whereas GLY-14 did not (data not
shown). The rabbit and GLY-14 enzymes showed very similar metal
requirements (data not shown); there was an absolute requirement for
Mn2+ and little (<20% of maximum activity) or no activity
with Mg2+, Ni2+, Ba2+,
Ca2+, Cd2+, Fe2+, or
Cu2+.
The products of GLY-12 and GLY-14 with M3-octyl were
analyzed by thin layer chromatography and shown to co-migrate with
standard Man Expression of GnT I mRNA at Various Stages in C. elegans
Development--
As a first step toward understanding the role of
N-glycans in development, we studied the expression of GnT I
mRNA in six developmental stages of C. elegans, an
embryo fraction containing a mixture of embryo stages, the four larval
stages L1-L4, and adult worms (70). Northern analysis detected
messages for both gly-12 (a major band at ~2.1 kb, Fig.
2A) and gly-13 (a
major band at ~1.9 kb, Fig. 2B) in all six developmental
stages. Assuming that the 3'-untranslated regions of gly-12
and gly-14 are not excessively long, the mRNA sizes are
consistent with the cDNA lengths determined by sequencing (>1982,
1719, and >1322 nt for gly-12, gly-13, and
gly-14, respectively). We used quantitative RT-PCR (Figs.
3, A-C) to establish the
relative abundance of gly-12 and gly-14 mRNA.
There were no major variations between different stages of development
except for a higher level of gly-12 message at the embryo
stage (Fig. 3, A and C). The gly-12
mRNA levels are 6-38 times higher than the gly-14
mRNA (Fig. 3C).
Expression of GnT I Promoters at Various Stages in C. elegans
Development--
Expression of the
The F1 progeny of worms injected with the gly-13 promoter
construct p11B/prom-ORF expressed
Transgenic animals carrying the gly-14 promoter construct
p57M/prom2.6, containing a 2.6-kb promoter fragment and the 3'-UTR of
the unc-54 gene, expressed the reporter gene only in the gut (Fig. 4G and data not shown). The same expression pattern
was observed in animals ranging from L1 to adulthood. The most anterior gut cells (int 1) and those on the posterior third of the
body expressed the reporter gene most strongly. The remainder of the gut often failed to express the reporter. We did not detect reporter expression in embryos, despite finding that embryos contain
approximately the same amount of gly-14 mRNA as other
stages (Fig. 3, B and C). As is typical of
reporter transgenes in C. elegans (71), none of our reporter
constructs directed expression in the germ line. Therefore, we cannot
exclude the possibility that gly-14 mRNA is maternally
contributed to the embryo. Injection of p11M/prom-ORF (lacking the
3'-UTR of the unc-54 gene) and the truncated constructs p57M/prom1.3 and p57M/prom0.6 did not result in reporter gene expression.
Heat Shock-induced Overexpression of GnT I--
Transgenic worms
that overexpress gly-12, gly-13, or
gly-14 under the control of the hsp-16 heat shock
promoters show no obvious phenotypic defects. To test whether
functional GnT I was produced in these transgenic worms, microsomal
fractions were prepared to determine enzyme activity. Heat shock
induction of gly-13 resulted in little or no increase in GnT
I activity compared with wild type N2 worms (wild type GnT I
activity = <0.1 nmol/h/mg) (Table II). Dramatic increases in enzyme
activity were observed on heat shock induction of both
gly-12 (27-157-fold) and gly-14 (39-182-fold) (Table II). Lysates of all three transgenic lines overexpressing GnT I
showed protein bands of the expected size by Western blotting (Fig.
5). This finding suggests that the low
GLY-13 activity is not due to poor expression but rather to a low
specific activity, at least with the acceptor substrate used for assay;
GLY-13 may have a high activity with an as yet unknown physiological
acceptor. Immunolocalization experiments showed that all three GnT I
gene products stained as focal areas in the perinuclear region of the cytoplasm, suggesting a Golgi complex location (Fig.
6 and data not shown).
Our studies on "knockout" mice with a null mutation in the GnT
I gene (3) and on humans with carbohydrate-deficient glycoprotein syndrome type II (13) have indicated that interference with complex
N-glycan synthesis is associated with severe defects in embryonic development particularly of the nervous system. We have therefore initiated studies on the expression of GnT I in C. elegans in the hope that this relatively simple and thoroughly
characterized organism will provide important information on the role
of complex N-glycans in development.
A search of the genomic DNA data base using the BLASTP algorithm (39)
indicated the presence of three C. elegans genes that show
significant similarity to mammalian GnT I. These genes have been
designated gly-12, gly-13, and gly-14.
Only a single functional copy of the GnT I gene has been reported in
mammals. We have cloned the C. elegans cDNAs and have
demonstrated that two of them (gly-12 and gly-14)
encode an active GnT I in both a heterologous (Sf9 insect cells)
and homologous (transgenic worms) host. However, no protein was
detected by Western analysis when we attempted to express
gly-13 in Sf9 insect cells. Expression of
gly-13 in transgenic worms yielded a protein of the expected
size on Western blots, but this protein showed no GnT I enzyme
activity. The data suggest that gly-13 may encode a
glycosyltransferase with a specificity different from GnT I. Similarly,
only 5 of the 11 C. elegans UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase cDNA homologues cloned
by Hagen and Nehrke (41) were shown to possess enzyme activity. Bakker et al. (72) attempted to clone a snail
UDP-GalNAc:GlcNAc In contrast to the mammalian GnT I genes in which the entire open
reading frame is on a single exon, the gly-12,
gly-13, and gly-14 genes have multiple exons (14, 12, and 12, respectively). However, although the identity between the
C. elegans and mammalian GnT I amino acid sequences is less
than 50%, the GLY-12 and GLY-14 enzymes show kinetic parameters very
similar to the rabbit enzyme. The major differences detected were the
pH profiles (rabbit GnT I maintains its activity to a significantly
lower pH than GLY-14) and the lower temperature optimum for GLY-12 and
GLY-14 relative to rabbit GnT I. C. elegans
GalNAc-transferase also has a lower temperature optimum than the
mammalian enzyme (41).
We have previously shown that removal of 106 amino acids from the N
terminus of rabbit GnT I does not inactivate the enzyme (38). This
region contains the cytoplasmic, transmembrane, and stem domains and
shows marked differences in amino acid sequence between the mammalian
enzymes and each of the three C. elegans enzymes (Fig. 1).
The catalytic domain of GnT I contains 341 amino acids for the
mammalian enzymes and 350-394 amino acids for the C. elegans enzymes. Comparison of mammalian and C. elegans
sequences indicates five highly conserved regions that are probably
essential for catalytic activity (118-159, 200-211, 221-265,
277-330, and 431-461 in Fig. 1). Of the five cysteine residues in the
consensus sequence (128, 137, 159, 256, and 322 in Fig. 1), only two
are conserved for all mammalian and C. elegans sequences
(256 and 322). Site-directed mutagenesis of invariant amino acids is
being carried out to determine whether these residues are indeed
essential for enzyme activity.
All three gene messages (gly-12, gly-13, and
gly-14) are expressed in all six stages of worm development.
Except for a relative increase of gly-12 expression in the
embryo, there is no significant difference in expression between the
various developmental stages. Analysis of reporter gene expression in
transgenic animals confirms the expression of gly-12 and
gly-13 at all stages of development and shows that the
gly-12 and gly-13 promoters are expressed in most
cell types. Expression of the gly-14 promoter was detected only postembryonically and only in gut cells, suggesting a
tissue-specific expression of enzyme activity. Confirmation of its
gut-specific expression will require detection of endogenous
gly-14 gene products.
Overexpression of the three C. elegans GnT I genes in
transgenic worms under the control of worm heat shock promoter caused no obvious phenotypic changes despite marked increases in the enzyme
activities of GLY-12 and GLY-14 in worm lysates. This is perhaps not
surprising, since mammalian GnT I is probably a housekeeping gene (55)
and is expressed in non-rate-limiting amounts. However, mice with a
null mutation in the GnT I gene do not survive beyond 10 days of
embryonic life, and it is therefore of great interest to study the
effects of null mutations in the three C. elegans genes.
Attempts to create mutant worms lacking GnT I by injection of
single-stranded and double-stranded RNA have to date been unsuccessful in demonstrating any obvious phenotypes. Attempts to create mutant worms by other methods are under way.
The C. elegans genomic DNA and expressed sequence tag data
bases contain sequences that show significant homologies to at least 17 enzymes involved in the synthesis of N- and
O-glycans, glycosyl-phosphatidylinositol anchors, and
proteoglycans, e.g. UDP-GalNAc:polypeptide
N-acetylgalactosaminyltransferase (41), UDP-GlcNAc:polypeptide N-acetylglucosaminyltransferase (73), GnT I (this study), UDP-Gal:GlcNAc-R -3-D-mannoside
-1,2-N-acetylglucosaminyltransferase I (GnT I) is
a key enzyme in the synthesis of Asn-linked complex and hybrid glycans.
Studies on mice with a null mutation in the GnT I gene have indicated
that N-glycans play critical roles in mammalian
morphogenesis. This paper presents studies on N-glycans
during the development of the nematode Caenorhabditis elegans. We have cloned cDNAs for three predicted C. elegans genes homologous to mammalian GnT I (designated
gly-12, gly-13, and gly-14). All
three cDNAs encode proteins (467, 449, and 437 amino acids,
respectively) with the domain structure typical of previously cloned
Golgi-type glycosyltransferases. Expression in both insect cells and
transgenic worms showed that gly-12 and gly-14,
but not gly-13, encode active GnT I. All three genes were
expressed throughout worm development (embryo, larval stages L1-L4,
and adult worms). The gly-12 and gly-13
promoters were expressed from embryogenesis to adulthood in many
tissues. The gly-14 promoter was expressed only in gut
cells from L1 to adult developmental stages. Transgenic worms that
overexpress any one of the three genes show no obvious phenotypic
defects. The data indicate that C. elegans is a suitable
model for further study of the role of complex N-glycans in development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-3-D-mannoside
-1,2-N-acetylglucosaminyltransferase I (GnT
I)1 is a key enzyme in the
synthesis of Asn-linked complex and hybrid glycans because branching
cannot occur until GnT I has acted (1, 2). Recent work on mice and
humans in which the synthesis of complex N-glycans is
defective has provided excellent evidence that these structures play
essential roles in development. Although somatic Chinese hamster ovary
cell mutants lacking the GnT I gene show essentially normal growth,
mouse embryos with a null mutation in this gene do not survive beyond
10.5 days postfertilization and show severe developmental abnormalities
particularly of the brain (3, 4). Mice with a homozygous null mutation
in the gene encoding
UDP-N-acetylglucosamine:
-6-D-mannoside
-1,2-N-acetylglucosaminyltransferase II (GnT II) survive
to term but are born stunted with various congenital abnormalities and
die shortly after birth (5). Carbohydrate-deficient glycoprotein
syndrome (CDGS) is a group of congenital diseases in which there is a
defect in protein N-glycosylation (6). Children with CDGS
types 1 and 2 show severe psychomotor retardation and other
multisystemic abnormalities. About 80% of CDGS type 1 children have a
defect in the phosphomannomutase gene (7-9). Another variant of
CDGS type 1 has been described recently with a defect in the
phosphomannose isomerase gene (10). Two children with CDGS type 2 have
inactivating point mutations in the GnT II gene (11-13). Several other
congenital diseases are associated with defective complex
N-glycan synthesis (14).
MATERIALS AND METHODS
-32P]dCTP
(Amersham Pharmacia Biotech; 3000 Ci/mmol) using the Amersham Pharmacia
Biotech "Ready To Go" DNA labeling kit. The labeled probes were
purified using Sephadex G50 DNA grade nick columns (Amersham Pharmacia
Biotech). An oligo(dT)-primed C. elegans cDNA library in
gt10 (provided by Drs. S. Kim and H. R. Horvitz, MIT) was
screened with a mixture of probes A, B, and C. Positive plaques were
rescreened with each probe individually. Only one plaque hybridized to
all three probes, and it was purified for further analysis.
gt10 plaque DNA with EcoRI, subcloned into pGEM7Zf(+)
(Promega), and sequenced. A partial cDNA was also produced by the
RT-PCR/3'-RACE procedure (49) using a preparation of C. elegans total RNA as template. An oligo(dT) primer was used for
reverse transcription. A gene-specific forward primer (CEBF5, Table I)
and a reverse adaptor primer (AP2) were used for 3'-RACE (Marathon
cDNA amplification kit, CLONTECH).
1-6(Man
1-3)Man
-octyl (M3-octyl, kindly provided by Dr. Hans Paulsen, University
of Hamburg) (50, 52) and as donor substrate 1 mM
UDP-[3H]GlcNAc (NEN Life Science Products) diluted with
nonradioactive UDP-GlcNAc (Sigma) to a specific activity of 2500 dpm/nmol for routine enzyme assays, 15,000 dpm/nmol for worm lysates,
and 30,000-140,000 dpm/nmol for kinetic studies. The assay mixture
also contained 2.5 mM AMP, 50 mM GlcNAc, 20 mM MnCl2, 1 mg ml
1 bovine serum
albumin, 0.1% Triton X-100, 0.1 M MES buffer, pH 6.1, and
0.010 ml of enzyme in a 0.050-ml total volume (50). Time of incubation
was 0.5-1 h at 37 °C for all baculovirus/Sf9-expressed enzymes and at 26 °C for enzymes expressed in C. elegans.
Kinetic parameters were determined on the recombinant rabbit (50) and C. elegans enzymes by a series of reciprocal
velocity-substrate plots at four concentrations of both substrates
(0.5, 1.0, 1.5, and 2.0 mM M3-octyl and 0.02, 0.04, 0.06 and 0.08 mM UDP-GlcNAc) (50, 53). Kinetic
analyses were also carried out using
(Man
1-6(Man
1-3)Man
1-6)(Man
1-3)Man
1-4GlcNAc
1-4GlcNAc-Asn (M5-glycopeptide) (54) as acceptor substrate (0.22, 0.44, 0.66, and 0.88 mM). Control GnT I assays were carried out
with uninfected Sf9 cells and cells infected with wild type
baculovirus. Metal requirements (20 mM divalent cation) and
pH and temperature optima were determined. Protein content was measured
by the BCA assay (Pierce).
phage DNA (5 µg) with EcoRI and HindIII; DNA fragments were
end-labeled with [
-32P]dATP (3000 Ci/mmol, NEN Life
Science Products) and Klenow DNA polymerase (New England Biolabs).
Probes for gly-12 (126-1504 nt relative to the ATG start
codon at +1) and gly-13 (114-1350 nt relative to the ATG
start codon at +1) were made by excision of the truncated
gly-12 and gly-13 inserts from the respective recombinant baculovirus transfer vectors (see above). Probes were randomly labeled with [
-32P]dATP (3000 Ci/mmol, NEN
Life Science Products). After hybridization of Northern blots with
these probes (1 × 107 cpm/ml for gly-12
and 3.2 × 106 cpm/ml for gly-13), the
blots were stripped with 0.1% SDS at 100 °C and reprobed with a
32P-labeled probe for the fem-1 gene (1-2 × 106 cpm/ml) as a sample loading control (56).
-32P]dCTP (800 Ci/mmol, Amersham Pharmacia Biotech)
using the Advantage RT-for-PCR kit (CLONTECH).
Competitor cDNA was made by PCR with primer pairs (Table I)
CEFF-DEL/CEFR3 (gly-12, 660-nt product, internal deletion of
233 nt) and CEMF7/CEMR-DEL (gly-14, 726 nt product, internal
deletion of 213 nt) using the respective gly-12 and
gly-14 cDNAs as templates. PCR was then carried out with
gene-specific primer pairs (Table I) CEFF5/CEFR3 (gly-12,
893-nt product) and CEMF7/CEMR4 (gly-14, 939-nt product)
using as template a mixture of [
-32P]dCTP-labeled
total cDNA (at a constant concentration) and purified competitor
cDNA (at variable concentrations). The PCR products were resolved
in a 1.5% agarose gel stained with ethidium bromide. Since the amount
of added competitor cDNA is known, and on the assumption that the
molar ratio of wild type cDNA to competitor cDNA remains
approximately constant throughout the amplification, estimates can be
made of the amount of gly-12 and gly-14 cDNA present at the start of the PCR reaction by scanning of the agarose gels. The expression level of message at each worm developmental stage
was normalized with the amount of [
-32P]dCTP-labeled
total cDNA added to each PCR reaction.
) (Stratagene). In some experiments,
the F1 progeny of injected animals were analyzed directly for reporter
expression; in these experiments, the concentration of GnT
I::lac Z constructs was 100 ng/µl. LacZ
expression was examined in a smg-1 (e1228) background. The
smg-1 mutation stabilizes aberrant transcripts with long
3'-UTRs (59). Transgenic lines or F1 progeny were cultured at 25 °C.
For F1 lac Z assays, 15-20 adult hermaphrodites were
injected on each of three consecutive days.
-galactosidase staining
of late larvae and adults was carried out as described by Xie et
al. (60). Staining of embryos and young larvae was carried out by
the method of Fire (61). All animals were co-stained with 1 µg/ml of
4,6-diamidino-2-phenylindole to visualize the cell nuclei. In the
figures, anterior is to the left and dorsal is
up.
) (30 ng/µl)); (ii) p78B-Myc/p83B-Myc mixture (p78B-Myc (25 ng/µl), p83B-Myc (25 ng/µl), pRF4 (50 ng/µl)); (iii) p78M/p83M
mixture (p78M (10 ng/µl), p83M (10 ng/µl), pRF4 (50 ng/µl),
pBlueScript II(
) (30 ng/µl)); (iv) p78M-Myc/p83M-Myc mixture
(p78M-Myc (10 ng/µl), p83M-Myc (10 ng/µl), pRF4 (50 ng/µl), pBlueScript II(
) (30 ng/µl)); (v) p78F-Myc/p83F-Myc mixture
(p78F-Myc (10 ng/µl), p83F-Myc (10 ng/µl), pRF4 (50 ng/µl),
pBlueScript II(
) (30 ng/µl)). Transgenic lines carrying the
injected DNA on extrachromosomal arrays were established from F2 Rol
progeny of injected N2 animals.
70 °C.
Worms were lysed by sonication, five times with 5-s pulses at 30-s
intervals. The sonicate was centrifuged at 3500 rpm in a Beckman JA17
rotor for 10 min, and the supernatant was centrifuged at 55,000 rpm for
1 h (Beckman 100.3 rotor). The microsomal pellet was resuspended
in lysis buffer (25 mM MES, pH 6.1, 1% Triton X-100 and
protease inhibitor mixture).
RESULTS
Oligonucleotide primers
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Fig. 1.
Alignment of GnT I amino acid
sequences from mammals (mouse, rat, Chinese hamster, rabbit, and human)
and C. elegans (gly-12, gly-13, and
gly-14) using the GCG PileUp program (78) (Genetics
Computer Group Inc., Madison, WI). Dashed
line, same as consensus sequence; dotted
line, gap; vertical bar, exon-exon
boundaries; asterisk, STOP codon.
1-6(GlcNAc
1-2Man
1-3)Man
-O-octyl
(69) in the following solvent systems: (i) acetonitrile/water (5:1) and
(ii) dichloromethane/methanol/water (55:35:6) (data not shown). The
product of GLY-14 was purified and shown to have an NMR spectrum at 500 MHz identical to the Man
1-6(GlcNAc
1-2Man
1-3)Man
-O-octyl standard
(69) (data not shown).
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Fig. 2.
Northern blot analyses of GnT I mRNA
expression during C. elegans development. Total RNA
(~20 µg/lane) and poly(A)+ RNA (~2.5 µg/lane) were
probed with gly-12 cDNA (A) and
gly-13 cDNA (B). The blots were stripped with
boiling 0.1% SDS and reprobed with fem-1 cDNA as a
sample loading control. RNA was obtained from embryos (E),
the four larval stages (L1-L4), adult worms
(Ad), and a mixture of all stages (Mix). The
~2.1-kb gly-12 mRNA was expressed in all developmental
stages, with the highest level in embryos and the lowest in adults. The
~1.9-kb gly-13 mRNA was expressed in all developmental
stages without significant variations.
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Fig. 3.
Quantitation of GnT I mRNA expression by
competitive RT-PCR. The procedure is described under "Materials
and Methods." A and B show electrophoretograms
of PCR products in 1.5% agarose gels stained with ethidium bromide.
The triangles above the gels represent
the amount of competitor DNA template added to the samples during the
PCR, as follows. A, gly-12 cDNA (2, 0.2, and
0.02 amol, respectively); B, gly-14 cDNA
(2 × 10 2, 2 × 10
3, and 2 × 10
4 amol, respectively). C shows a
semilogarithmic plot of the relative abundance of gly-12
(filled diamonds) and gly-14
(open squares) mRNAs obtained by scanning the
above gels; values were normalized by the amount of radioactive
reverse-transcribed total cDNA added to the PCR reaction.
E, embryo; L1-L4, larval stages; Ad,
adult.
-galactosidase reporter gene in
the F1 progeny of worms injected with the gly-12 promoter
construct p57F/prom was observed throughout all developmental stages
and in many tissues (intestine, muscle, hypodermis, and other
epithelial cells and in ganglia in the head and tail region) (Fig.
4, A-C, and data not
shown).
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Fig. 4.
Light microscopic images of transgenic
C. elegans carrying gly-12::lacZ,
gly-13::lacZ, and gly-14::lacZ
reporter constructs. A-C, expression of
gly-12 reporter in late embryo (A), L2 larva
(B), and L3 larva (C). D-F,
expression of gly-13 reporter in L1 larva (D), L4
larva (E), and adult hermaphrodite (F).
G shows the expression of gly-14 reporter in an
L4 hermaphrodite. hyp, hypodermal cell; mus,
muscle cell; int, intestinal cell; gan, ganglia;
vul, vulval cell; vnc, ventral nerve cord. Worms
are oriented with anterior to the left and dorsal
up except for the worms in C, which is a ventral
view, and G, which is a dorsal view. Animals in
D-F are displayed at 0.5 times the magnification shown in
the other panels.
-galactosidase from late
embryogenesis to adulthood. Expression in L1 larva was confined to the
gut cells (Fig. 4D). From L2 to adulthood,
-galactosidase
was expressed in many different tissues, including gut, muscle,
hypodermis, and other epithelial cells and the nervous system (ganglia
in the head and tail region and the ventral nerve cord) (Fig. 4, E and F, and data not shown).
GnT I activities in normal and transgenic C. elegans
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Fig. 5.
Western blot analysis of Myc-tagged C. elegans GnT I overexpressed in transgenic worms under the control
of heat shock promoter. Left, gly-14
(Ex[HS-GLY-14]) and gly-13 (Ex[HS-GLY-13]) transgenic
worms and wild-type worms (N2) were analyzed before ( ) and after (+)
heat shock induction. About 260 µg of total protein was loaded in
each lane. This corresponds to 0.96 nmol/h of GLY-14 activity after
heat shock. The GLY-13 protein does not show GnT I enzyme activity
(Table II). Right, gly-12 (Ex[HS-GLY-12] lines
2b and 2c) transgenic worms were analyzed before (
) and after (+)
heat shock induction. Sample loading was as follows: ~420 µg of
uninduced Myc-GLY-12 lysate, ~280 µg of heat shock-induced
Myc-GLY-12 lysate (worm line 2b) corresponding to 0.73 nmol/h GnT I
activity, and ~140 µg of heat shock-induced Myc-GLY-12 lysate (worm
line 2c) corresponding to 2.2 nmol/h enzyme activity. The calculated
molecular weights of Myc-tagged GLY-12, GLY-13, and GLY-14 are 58.0, 55.7, and 54.9 kDa, respectively. Protein bands of approximately the
expected sizes are seen for all three genes after heat shock induction
but not in wild type or uninduced transgenic worms.
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Fig. 6.
Immunolocalization of C. elegans
heat shock-induced Myc-GLY-12. The transgenic worm was
stained with antibody 9E10, which recognizes the Myc epitope tag
(right panel) and with
4,6-diamidino-2-phenylindole to visualize nuclei (left
panel). A portion of the intestine has extruded, thereby
permitting a clear view of three gut cells. It is seen that the Myc
epitope is localized to punctate perinuclear areas, suggestive of
localization in the Golgi complex.
DISCUSSION
-R
4-GalNAc-transferase by screening a snail
cDNA library with a UDP-Gal:GlcNAc
-R
4-Gal-transferase probe
but instead cloned a novel UDP-GlcNAc:GlcNAc
-R
4-GlcNAc-transferase. Studies are under way on the large scale
expression of gly-13 so that a search can be made for other
enzyme activities.
1,4-galactosyltransferase,
1,6-N-acetylglucosaminyltransferase V, and
1,3-fucosyltransferase. The only enzymatically active C. elegans glycosyltransferases published to date are the
UDP-GalNAc:polypeptide
N-acetylgalactosaminyltransferases (41) and GnT I (this
study). Lectins such as wheat germ agglutinin (74, 75) and concanavalin
A (76) have been shown to bind to C. elegans tissues,
suggesting the presence of glycoproteins in these organisms. Although
no detailed glycan structures have as yet been reported for C. elegans, both N- (21-23) and O-glycan (77)
fine structures have been determined for several parasitic nematodes
using mass spectrometric analysis. Some of these nematode N-glycan structures contain the
GlcNAc
1,2-Man
1,3-Man
-R moiety indicative of a functional GnT I
enzyme. The data available to date therefore show that C. elegans is highly active in the synthesis of glycoproteins and is
an excellent organism in which to study the role of protein
glycosylation in the development of a multicellular organism.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Asher D. Schachter (The Children's Hospital, Harvard Medical School, Boston, MA) for the quantitative RT-PCR protocol and Jeb Gaudet for advice on many aspects of this work.
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Note Added in Proof |
---|
Catalytic activity has been reported on
expression of C. Elegans genes encoding an
1,3-fucosyltransferase (De Bose-Boyd, R. A., Kwame Nayame, A., and
Cummings, R. D., (1998) Glycobiology 8, 905-917) and
1,6-N-acetylglucosaminyltransferase V (Warren, C. E.,
Roy, P. J., Krizus, A., Culotti, J. G., and Dennis, J. W. (1998)
Glycobiology 8, Abstr. 16, Third Annual Conference of
the Society for Glycobiology, Nov. 11-14, 1998, Baltimore, MD).
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FOOTNOTES |
---|
* This work was supported by grants from the Medical Research Council of Canada (to H. S. and A. M. S.) and by a grant from the Mizutani Foundation for Glycoscience Research (to H. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF082011 (gly-12), AF082010 (gly-13) and AF082012 (gly-14). The accession numbers of the corresponding genomic DNA sequences are U28735 (gly-12), U23516 (gly-13) and Z46381 (gly-14).
To whom correspondence should be addressed: Dept. of
Structural Biology and Biochemistry, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.:
416-813-5915; Fax: 416-813-5022; E-mail: harry{at}sickkids.on.ca.
2 A. Fire, S. Xu, J. Ahnn, and G. Seydoux, personal communication.
3 J. Gaudet and A. Spence, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
GnT I, UDP-N-acetylglucosamine:-3-D-mannoside
-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101);
CDGS, carbohydrate-deficient glycoprotein syndrome;
GnT II, UDP-N-acetylglucosamine:
-6-D-mannoside
-1,2-N-acetylglucosaminyltransferase II (EC 2.4.1.143);
nt, nucleotide(s);
PCR, polymerase chain reaction;
RT-PCR, reverse
transcription-PCR;
RACE, rapid amplification of cDNA ends;
MES, 2-(N-morpholino)ethanesulfonic acid;
M3-octyl, Man
1-6(Man
1-3)Man
-octyl;
M5-glycopeptide, (Man
1-6(Man
1-3)Man
1-6)(Man
1-3)Man
1-4GlcNAc
1-4GlcNAc-Asn;
UTR, untranslated region;
kb, kilobase pair(s)..
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REFERENCES |
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