From the Howard Hughes Medical Institute, Department
of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139 and the § Department of Cellular and
Molecular Medicine, Glycobiology Research and Training Center,
University of California, San Diego, La Jolla, California
92093-0687
Received for publication, September 11, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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In mutants defective in any of eight
Caenorhabditis elegans sqv (squashed
vulva) genes, the vulval extracellular space fails to
expand during vulval morphogenesis. Strong sqv mutations
result in maternal-effect lethality, caused in part by the failure of the progeny of homozygous mutants to initiate cytokinesis and associated with the failure to form an extracellular space between the
egg and the eggshell. Recent studies have implicated glycosaminoglycans in these processes. Here we report the cloning and characterization of
sqv-2 and sqv-6. sqv-6 encodes a
protein similar to human xylosyltransferases. Transfection
of sqv-6 restored xylosyltransferase activity to and
rescued the glycosaminoglycan biosynthesis defect of a
xylosyltransferase mutant hamster cell line. sqv-2 encodes
a protein similar to human galactosyltransferase II. A recombinant
SQV-2 fusion protein had galactosyltransferase II activity with
substrate specificity similar to that of human galactosyltransferase
II. We conclude that C. elegans SQV-6 and SQV-2 likely act
in concert with other SQV proteins to catalyze the stepwise formation
of the proteoglycan core protein linkage tetrasaccharide
GlcA Glycosaminoglycans
(GAGs)1 are important in
animal development, and defects in GAGs are responsible for certain
human disorders. For example, mutations in the Drosophila
melanogastger genes tout-velu (1) and
sulfateless (2), which encode homologs of heparan sulfate
co-polymerase and heparan sulfate
N-deacetylase/N-sulfotransferase, respectively,
cause zygotic lethality and defects in segmentation. Mutations in the
mouse tout-velu homolog EXT1 disrupt gastrulation and the
generation of mesoderm (3), while mutations in human EXT1 and EXT2 have
been associated with hereditary multiple exostoses (reviewed in Ref.
4). Mutations in the human galactosyltransferase I have been associated
with a progeroid variant of the connective-tissue disorder
Ehlers-Danlos syndrome (EDS) (5-7). EDS is a group of heritable
disorders characterized by hyperelasticity of the skin and hypermobile
joints. Tout-velu, EXT-1, EXT-2, and Sulfateless affect the
biosynthesis of heparan sulfate specifically, while galactosyltransferase I deficiency affects the biosynthesis of both
chondroitin and heparan sulfate.
The backbones of chondroitin and heparan sulfate consist of repeating
disaccharide units: GlcA Eight sqv (squashed vulva) genes
were genetically identified in a screen for Caenorhabditis
elegans mutants defective in vulval morphogenesis (9). All
sqv mutants fail to form a large fluid-filled vulval
extracellular space and have a reduced separation of the anterior and
posterior halves of the vulva from the early to middle phases of L4
larval development. Strong mutant alleles of all eight sqv
genes also cause maternal-effect lethality. Most progeny of mothers
homozygous for a strong sqv mutant allele arrest at the
one-cell stage (9). The nuclei of the arrested progeny divide normally,
but the extrusion of the polar bodies and the initiation of cytokinesis
are impaired (10). These mutant eggs fail to form the normal
fluid-filled extracellular space between the membrane of the egg and
the eggshell. We have postulated that the sqv genes control
the biosynthesis of GAGs that are secreted and become hydrated to form
fluid-filled extracellular spaces (10, 11).
The molecular identification of five sqv genes has led to a
model implicating the biosynthesis of chondroitin and/or heparan sulfate in C. elegans development. sqv-1,
-3, -4, -7, and -8 encode UDP-GlcA decarboxylase (10), galactosyltransferase I (12), UDP-glucose
dehydrogenase (13), UDP-GlcA/UDP-Gal/UDP-GalNAc transporter (14), and
glucuronosyltransferase I (12), respectively. sqv-3 was used
to identify the human galactosyltransferase I, which has been
implicated in the progeroid variant of EDS (7, 15). In this paper, we
show that sqv-6 encodes the xylosyltransferase that adds Xyl
to the protein core, thus initiating GAG biosynthesis. sqv-2
encodes a galactosyltransferase that adds the second Gal residue to the
linkage tetrasaccharide.
C. elegans Maintenance--
Strains were cultured as described
(16) and were grown at 20-22 °C unless indicated otherwise.
Molecular Biology--
Standard molecular biological techniques
were used (17). The sequences of all PCR-amplified DNAs used for
cloning were confirmed to exclude unintended mutations. Oligonucleotide
sequences used for amplification or mutagenesis of DNA are shown in
Supplementary Materials and Methods.
Rescue of C. elegans sqv-2 and sqv-6 Mutants--
For germ line
rescue, we injected cosmids carrying genomic DNA into
sqv-2(n2821) and sqv-6(n2845)
unc-60(e677)/unc-34(s138) animals with the dominant roller marker
pRF4, as described by Mello et al. (18). Rol lines were
established, and Rol animals and Unc-60 Rol animals were examined for
rescue of the sqv-2 and the sqv-6 mutant
phenotype, respectively. We injected sqv-2(n2821) hermaphrodites with plasmids containing the sqv-2 open
reading frame (ORF) under the control of the C. elegans
heat-shock promoters (19) and pRF4 as the coinjection marker. We
injected sqv-6(n2845)/nT1(n754) hermaphrodites with plasmids
containing the sqv-6 ORF under the control of the C. elegans heat-shock promoters (19) and pRF4. Rol lines were
established, and Rol (non-Unc) animals were examined for rescue of the
sqv-2 and sqv-6 mutant phenotype following
induction of sqv-2 and sqv-6 expression by 30 min
of heat-shock treatment at 33 °C.
SQV-2 Galactosyltransferase II Assay--
A sequence encoding
amino acids 25-330 of SQV-2, thus lacking the presumptive
transmembrane domain at the amino terminus, was cloned into
pDEST-CMV-protA. This plasmid was designed to express a secreted fusion
protein containing protein A and SQV-2 amino acids 25-330. COS7 cells
were transiently transfected with pDEST-CMV-protA-sqv-2
using LipofectAMINE (Invitrogen) according to the
manufacturer's instructions. After 72 h of incubation, the fusion
protein was recovered from the cell culture supernatant by affinity
chromatography using IgG-agarose (20). Galactosyltransferase II
activity was assayed as described by Bai et al. (21).
Rescue of the Xylosyltransferase Defect in Chinese Hamster Ovary
(CHO) pgsA-745 Cells by sqv-6--
The xylosyltransferase-deficient
CHO pgsA-745 cells (22) were transfected with sqv-6 ORF,
which was cloned into pcDNA3.1. Stable transfectants were selected
with 400 µg/ml geneticin (Invitrogen). Several drug-resistant
colonies were isolated and screened by flow cytometry for
sqv-6 expression based on binding of biotinylated FGF-2 as
described (23). Incorporation of 35SO4 into GAG
chains of wild-type CHO or pgsA-745 cells with or without
sqv-6 was assayed essentially as described by Bame and Esko
(24), labeling cells overnight at 30 °C with 50 µCi/ml 35SO4 (PerkinElmer Life Sciences).
SQV-6 Xylosyltransferase Assay--
Cell extracts of wild-type
CHO, pgsA-745, and sqv-6 or empty vector stable
transfectants of pgsA-745 were prepared as described previously (22).
Xylosyltransferase activity was assayed essentially as described (22)
by incubating 25 µg crude cell extract with 50 µg of soluble silk
acceptor and 6 × 105 cpm
UDP- [1-3H]xylose (PerkinElmer Life Sciences, 8.9 Ci/mmol) at 26 °C for 5 h. Product formation was dependent on
the addition of silk. The concentration of substrate was saturating.
Molecular Identification of sqv-2--
sqv-2 was
previously mapped to the left of lin-31 on LGII (25). We
further mapped sqv-2 to an interval between sup-9
and lin-31 (see Supplementary Materials and Methods). We
assayed 27 cosmids in this interval for the ability to rescue the
sqv-2 mutant phenotype, but none showed rescuing activity
(Fig. 1A).
We examined the DNA sequence corresponding to the gaps between the
cosmids in this interval and found a predicted gene, Y110A2AL.14, that
is weakly similar to galactosyltransferases. Because all previously
cloned sqv genes are implicated in the biosynthesis of
chondroitin and/or heparan sulfate, we suspected that sqv-2 also encodes a protein involved in GAG biosynthesis.
Specifically, it seemed plausible that Y110A2AL.14 encodes the
galactosyltransferase II involved in the formation of the protein core
linkage tetrasaccharide and that had not been identified molecularly in
any organism at the time.
We identified three molecular lesions corresponding to
three of the four identified alleles of sqv-2 in the ORF of
Y110A2AL.14 (Fig. 1B). The two stronger alleles of
sqv-2, n3037 and n3038, cause a
maternal-effect lethal phenotype and are an opal nonsense mutation at
arginine 225 and a methionine-to-isoleucine missense mutation of the
predicted start codon, respectively. A weak allele, n2826,
that results in live progeny is a missense mutation causing a
glycine-to-arginine substitution at amino acid position 99. The
molecular lesion of the weakest allele, n2840, has not been identified yet.
We determined the sequences of two cDNA clones, yk94e4 and yk292g2
(see Supplementary Materials and Methods), that correspond to
Y110A2AL.14. The yk292g2 clone contains 990 bases of ORF, 17 bases of
5'-untranslated region (UTR), and 121 bases of 3'-UTR. The 5' end
contains three bases that correspond to the sequence of 5' SL1
trans-spliced leader, which is found at the 5' end of many
C. elegans transcripts (26). The 3' end contains a poly(A) sequence. The longest ORF in this cDNA is identical to Y110A2AL.14 and is predicted to encode a protein of 330 amino acids. The yk94e4 clone lacks the 5' end of Y110A2AL.14. Expression of the longest ORF in
yk292g2 under the control of the C. elegans heat-shock promoters (19) rescued the defect in sqv-2 vulval
morphogenesis in all five isolated lines.
sqv-2 Encodes a Protein Similar to Galactosyltransferase
II--
The predicted SQV-2 protein contains a putative transmembrane
domain near the amino terminus, suggesting it may be a type II
transmembrane protein (Fig. 1B). Most known
glycosyltransferases that act in the lumen of the ER and the Golgi
apparatus are type II transmembrane proteins. Of 330 amino acids of
SQV-2, 93 (28%) and 133 (40%) are identical to the
Drosophila and human homologs, respectively (Fig.
1B). Recently, the human homolog of SQV-2 was identified as
galactosyltransferase II (21).
SQV-2 Has Galactosyltransferase II Activity--
We assayed
recombinant protein A-SQV-2 fusion protein expressed in COS7 cells for
galactosyltransferase II activity (see "Experimental Procedures").
The SQV-2 fusion protein specifically catalyzed the addition of
galactose to a disaccharide acceptor, Gal Molecular Identification of sqv-6--
sqv-6 was
previously mapped to the left of the polymorphism stP3 on
LGV (9). We further mapped sqv-6 to the left of the cosmid
W07B8 and within about 0.2 map units of unc-34 (see
Supplementary Materials and Methods). We assayed 11 cosmids to the
right of unc-34 for the ability to rescue the
sqv-6 mutant phenotype, but none showed rescuing
activity (Fig. 2A).
We examined the DNA sequences in the gaps in the cosmid coverage near
the cosmid W07B8 and unc-34 and found a gene, Y50D4C.d, that
is similar to two human xylosyltransferases (27). Using DNA from the
only allele of sqv-6, n2845, we identified in the ORF of Y50D4C.d an amber nonsense mutation causing the elimination of
the last 42 amino acids of the predicted protein product (Fig. 2B).
We determined the sequence of PCR-amplified cDNA and 5'-rapid
amplification of cDNA ends products corresponding to
Y50D4C.d (see Supplementary Materials and Methods). We found that this cDNA contains a 5' SL1 trans-spliced leader, 23 bases of
5'-UTR, and 2418 bases of ORF, including two additional 5' exons not in Y50D4C.d. The longest ORF in this cDNA including the additional exons is predicted to encode a protein of 806 amino acids. Expression of this ORF under the control of the C. elegans heat-shock
promoters (19) prior to the start of vulval morphogenesis rescued the sqv-6 vulval morphogenesis defect in all animals
(n = 13) and the maternal-effect lethality of the
progeny of sqv-6 homozygotes generated by +/sqv-6
heterozygous parents for three of 13 sqv-6 homozygotes studied.
sqv-6 Encodes a Protein Similar to Xylosyltransferases--
Of the
806 amino acids of the SQV-6 protein, 182 (23%) and 193 (24%) are
identical to human xylosyltransferases I and II, respectively (Fig.
2B). Both the predicted SQV-6 protein and the human
xylosyltransferase II contain a putative transmembrane domain near the
amino terminus and are likely to be type II transmembrane proteins.
Neither the start codon nor a presumptive transmembrane domain has been
defined for human xylosyltransferase I (27).
sqv-6 Can Correct a Xylosyltransferase Defect in CHO Cells--
We
tested the ability of sqv-6 to act as a xylosyltransferase
by testing its ability to complement GAG-deficient CHO mutant cells
lacking this enzymatic activity (22). Mutant pgsA-745 cells were
transiently transfected with a plasmid containing sqv-6 under the control of a cytomegalovirus (CMV) promoter. These cells showed partial rescue of the defect, as assayed by the restored abilities to incorporate 35SO4 into GAGs
(16-27% that of the wild type) and to bind biotinylated FGF-2, which
binds cell surface heparan sulfate as assayed by flow cytometry (data
not shown). From these transiently transfected cells, we obtained a
clonal cell line stably expressing sqv-6. This cell line
showed full restoration of FGF-2 binding to heparan sulfate on the cell
surface (Fig. 3A). Stable
expression of sqv-6 in pgsA-745 cells enhanced the
incorporation of 35SO4 into GAGs to ~50% of
wild-type levels, compared with 1% for the untreated mutant or mutant
transfected with empty vector (Fig. 3B). The
35SO4 incorporation into GAGs was similar in
wild-type CHO cells and pgsA-745 cells transfected with
sqv-6: 30-40% was released by treatment with
chondroitinase ABC and 55-65% by a heparin lyase mixture in both
cells, indicating that the composition of chondroitin and heparan
sulfate was comparable. Expression of sqv-6 also resulted in
restoration of xylosyltransferase activity, as measured by the transfer
of xylose from UDP-xylose to a soluble silk acceptor (22), whereas
pgsA-745 cells transfected with empty vector had virtually no
activity (Fig. 3C).
The sqv-2 and sqv-6 Genes Act in the C. elegans Chondroitin and
Heparan Sulfate Biosynthesis Pathway--
Our findings indicate that
sqv-2 and sqv-6 encode galactosyltransferase II
and xylosyltransferase, respectively. With the previously identified
sqv-3 galactosyltransferase I and sqv-8 glucuronosyltransferase I, all four C. elegans genes
responsible for the biosynthesis of the proteoglycan core protein
linkage tetrasaccharide of chondroitin and heparan sulfate have now
been defined (Fig. 4). Three previously
identified genes, sqv-4 UDP-glucose dehydrogenase,
sqv-1 UDP-GlcA decarboxylase and sqv-7
UDP-GlcA/UDP-Gal/UDP-GalNAc transporter, act in earlier steps of GAG
biosynthesis. All sqv genes identified to date affect the
biosynthesis of both chondroitin and heparan sulfate. Based upon these
observations, we conclude that in C. elegans early embryonic
cytokinesis and epithelial invagination during vulval development
depend on the expression of GAGs.
1,3Gal
1, 3Gal
1,4Xyl
-O-(Ser), which is common to the
two major types of glycosaminoglycans in vertebrates, chondroitin and
heparan sulfate. Our results strongly support a model in which C. elegans vulval morphogenesis and zygotic cytokinesis depend on
the expression of glycosaminoglycans.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1,3GalNAc
1,4 for chondroitin and
GlcA
1,4GlcNAc
1,4 for heparan sulfate (reviewed in Ref. 8). Their
polymerization occurs on a tetrasaccharide primer
(GlcA
1,3Gal
1,3Gal
1,4Xyl
-) that is linked to the protein
core of a proteoglycan. The addition of these four sugars is catalyzed
stepwise in the lumen of the Golgi apparatus and requires three
nucleotide sugars, UDP-Xyl, UDP-Gal, and UDP-GlcA, and four glycosyltransferases.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (62K):
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Fig. 1.
SQV-2 is similar to galactosyltransferase
II. A, genetic and physical maps showing
sqv-2. The dashed horizontal lines depicting
ccDf11 and ccDf1 indicate the possible extents of
the left end points of these deletions (C. Spike and R. Herman
(University of Minnesota), personal communication; E. Davison and H. R. Horvitz, unpublished observations). Short solid lines
represent cosmid clones that were assayed in germ line transformation
experiments. Below is the structure of the sqv-2 gene as
deduced from genomic and cDNA sequences. Solid boxes
indicate exons, and open boxes indicate untranslated
sequences. The trans-spliced leader SL1 is indicated, and
the arrow indicates the poly(A) tail. B,
alignment of SQV-2, the Drosophila homolog, and human
galactosyltransferase II. Identities between at least two proteins are
shaded in black. The predicted transmembrane
domains are underlined. The three sqv-2 mutant
alleles are indicated. The numbers on the right
indicate amino acid positions.
1,4Xyl
-O-benzyl that had
been used to demonstrate the acceptor substrate specificity of the
human galactosyltransferase II (21) (Table
I).
The SQV-2 fusion protein has acceptor specificity consistent with its
being galactosyltransferase II
1,4Xyl
-O-Bn was 141,000-142,000 cpm.
Bn, benzyl; NM, naphthalenemethanol; C10, O-decenyl
(CH2)8CH
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Fig. 2.
SQV-6 is similar to xylosyltransferases.
A, genetic and physical maps showing sqv-6.
Short solid lines represent cosmid clones that were assayed
in germline transformation experiments. Below is the structure of the
sqv-6 gene as deduced from genomic and cDNA sequences.
Solid boxes indicate exons. The trans-spliced
leader SL1 and the start codon (ATG) are indicated. B,
alignment of SQV-6 and two human xylosyltransferases. Identities
between at least two proteins are shaded in
black. The predicted transmembrane domains are underlined.
The single sqv-6 nonsense allele is indicated. The
numbers on the right indicate amino acid
positions.
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Fig. 3.
sqv-6 rescues a
xylosyltransferase-deficient CHO cell line. A, FGF-2
binding to cell-surface heparan sulfate as assayed by flow cytometry
(23). Light gray shading, wild-type CHO-K1. Dark gray
shading, mutant pgsA-745. Dashed line, pgsA-745 with
empty vector. Solid line, pgsA-745 with sqv-6.
B, 35SO4 incorporation into GAGs
(see "Experimental Procedures"). Black bars,
[35S]heparan sulfate (HS). White
bars, [35S]chondroitin sulfate (CS). The
average values ± S.D. (n = 3) are shown.
C, xylosyltransferase activity in crude cell extracts (see
"Experimental Procedures"). Average incorporation of
[3H]xylose from UDP-[1-3H]xylose into
soluble silk acceptor ± S.D. (n = 3) are
shown.
View larger version (27K):
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Fig. 4.
Model for the role of seven sqv
genes in glycosaminoglycan biosynthesis. SQV-4 converts
UDP-glucose to UDP-GlcA (13). SQV-7 transports UDP-GlcA, UDP-Gal, and
UDP-GalNAc from the cytoplasm to lumen of the Golgi apparatus (14).
SQV-1 converts UDP-GlcA to UDP-Xyl in the lumen of the Golgi apparatus
(10). SQV-6 is xylosyltransferase (this study). SQV-3 is
galactosyltransferase I (12). SQV-2 is galactosyltransferase II (this
study). SQV-8 is glucuronosyltransferase I (12). In other organisms,
two additional sets of glycosyltransferases act in later steps of the
biosynthesis of chondroitin and heparan sulfate (8).
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ACKNOWLEDGEMENTS |
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We thank Beth Castor for help with DNA sequence determination, Ewa Davison and Ignacio Perez de la Cruz for communicating the locations of the lin-8 and sup-9 loci, respectively, and Mark Alkema and Melissa Harrison for helpful suggestions concerning this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health (NIH) Grant GM24663 (to H. R. H.) and NIH Grant GM33063 (to J. D. E.).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 GenBankTM/EBI Data Bank with the accession number(s) AY241927 and AY241928.
The on-line version of this article (available at
http://www.jbc.org) contains a Supplementary Materials and Methods and
Refs. 1-4.
¶ Investigator of the Howard Hughes Medical Institute. To whom correspondence and reprint requests should be addressed: Howard Hughes Medical Inst., Dept. of Biology, Massachusetts Inst. of Technology, Rm. 68-425, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-253-4671; Fax: 617-253-8126; E-mail: horvitz@mit.edu.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.C200518200
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ABBREVIATIONS |
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The abbreviations used are: GAG, glycosaminoglycan; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; Xyl, xylose; EDS, Ehlers-Danlos syndrome; ORF, open reading frame; CHO, Chinese hamster ovary; UTR, untranslated region; CMV, cytomegalovirus.
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