1 Section of Molecular Cell and Developmental Biology and Institute for Cellular
and Molecular Biology, University of Texas at Austin, Austin, TX, 78712,
USA
2 Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx,
NY 10461, USA
* Author for correspondence (e-mail: d.stein{at}mail.utexas.edu)
Accepted 27 June 2005
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SUMMARY |
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Key words: Pipe, Windbeutel, Dorsal group, Dorsoventral polarity, Alcian Blue, Mucopolysaccharide, GAG, Proteoglycan, PAPS
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Introduction |
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Eight additional protein isoforms are encoded by the pipe locus,
one of which has also been reported to be expressed in ventral follicle cells
(Sergeev et al., 2001). The
functional consequences of its expression there are unknown. All isoforms
share the same N-terminal 95 amino acids, which are encoded by three common
exons, but the C-terminal region of each isoform is distinct because of the
existence of 10 alternate sets of exons
(Fig. 1A). Like the vertebrate
GAG-modifying enzymes, the Pipe isoforms are predicted to exhibit a typical
type II transmembrane topology, with a short, N-terminal hydrophilic region
followed by a short membrane-spanning hydrophobic region that precedes a
catalytic domain residing in the lumen of the Golgi.
Because DV signaling involves an extracellular serine protease cascade, it
is notable that serine proteolytic activity during blood coagulation is
controlled by a complex formed between heparin and antithrombin
(Furie and Furie, 1988).
Heparan sulfate and heparin are polymers of repeating disaccharides made up of
glucuronic acid and/or iduronic acid residues in ß1,4 linkage to N-acetyl
glucosamine. Heparin is structurally similar to heparan sulfate, with the
distinction that heparin is much more highly sulfated. Of particular interest
is the finding that the high affinity antithrombin/heparin interaction occurs
via a specific pentasaccharide sequence, the distinguishing feature of which
is the 3-0-sulfate group on the internal glucosamine unit
(Petitou et al., 2003
).
The crucial dependence of DV patterning upon the existence of an
extracellular serine proteolytic cascade, and the similarity of Pipe to
vertebrate HS2ST, has led to the hypothesis that Pipe-ST2 modifies a
glycoprotein that is secreted from the ventral follicle cells and localized
ventrally within the perivitelline space
(Sen et al., 1998). It is
proposed that Pipe-dependent modification mediates an interaction between the
glycoprotein and components of the serine protease cascade that lead to
ventral processing of the Spätzle ligand. The hypothesis that embryonic
DV patterning requires sulfotransferase activity in the follicle cell layer is
supported by the finding that females carrying follicle cell clones homozygous
for loss-of-function mutations in the gene slalom produce dorsalized
progeny embryos (Lüders et al.,
2003
). slalom encodes the transporter that mediates
uptake of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the
universal sulfate donor, into the Golgi
(Kamiyama et al., 2003
;
Lüders et al., 2003
).
In the studies reported here, we have used molecular and genetic methods to
investigate whether heparan sulfate is the substrate of Pipe enzymatic
activity. First, we characterized the molecular lesions associated with eleven
pipe mutant alleles. Two of these mutations map to the putative
binding site for PAPS (Kakuta et al.,
1998), the high energy donor molecule in sulfation reactions. We
also demonstrate that in addition to its function in the adult ovary,
pipe expression in the embryonic salivary glands is correlated with
the presence of a material that binds Alcian Blue, a histochemical stain that
interacts with highly sulfated molecules
(Scott et al., 1964
;
Scott and Dorling, 1965
).
To investigate whether the stained material might represent a heparan sulfate-containing molecule, we took advantage of the existence of mutations in several of the genes involved in heparan sulfate biosynthesis and modification in Drosophila. When we examined the salivary glands of embryos that were maternally and zygotically mutant for these genes, the Alcian Blue-stained material was still present. To investigate whether Pipe activity in the ovary is also independent of heparan sulfate synthesis, we generated females carrying follicle cell clones mutant for the heparan sulfate-related genes. In no case did these mutant females produce dorsalized embryos. Together, these observations indicate that proteins encoded by the pipe locus play a crucial role in the generation of sulfated macromolecules in the embryonic salivary gland, and by extension during egg formation. Our results, however, strongly argue against the suggestion that a heparan sulfate GAG is the target of Pipe activity.
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Materials and methods |
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Sequencing of pipe alleles
Genomic DNA was prepared from transheterozygous adult flies carrying each
of the mutant pipe alleles in trans to Df(3L)kto2, which
uncovers the pipe locus. Oligonucleotide primers were generated that
permitted PCR amplification of each of the six exons that constitute the
Pipe-ST2 ovary-specific isoform. For each of the 11 alleles, exon-specific
amplification products were purified and subjected to direct sequence
analysis.
Staining of embryos with Alcian Blue
Stocks were constructed in which chromosomes with mutations of interest
were carried in trans to balancers carrying insertions of
Krüppel-Gal4 and UAS-GFP. Overnight collections of embryos were
dechorionated in 50% bleach, then transferred to a glass plate and covered in
hydrocarbon 27 oil (Sigma). Stage 12-16 embryos were collected and separated
into groups containing fluorescent wild-type or non-fluorescent mutant embryos
using a Leica MZFLIII dissecting microscope equipped for detection of GFP.
Sorted embryos were transferred to a solution of 4% formaldehyde in PEMS
buffer (0.1 M PIPES, 2 mM MgSO4, 1 mM EGTA, pH 6.9):heptane (4.5
ml:5 ml) and fixed for 20 minutes with shaking. Following fixation, the lower
phase containing fixative was aspirated. Methanol (5 ml) was then added and
the embryos shaken vigorously for 1 minute to remove vitelline membranes from
the embryos. The devitellinized embryos were then rinsed several times with
methanol and stored in methanol at 20°C.
Alcian Blue is a cationic histochemical stain that has been used
extensively for the in situ detection of sulfated molecules
(Scott et al., 1964;
Scott and Dorling, 1965
;
Goso and Hotta, 1994
;
Schumacher and Adam, 1994
).
For Alcian Blue staining, fixed embryos were incubated for 30 minutes each in
70% methanol:30% PBT (PBS containing 0.1% Tween-20), 50% methanol:50% PBT, 30%
methanol:70% PBT, and finally PBT. The PBT was then aspirated and the embryos
were resuspended in a solution of 0.00125% Alcian Blue-tetrakis
(Methyl-Pyradinium) chloride in 0.3 MgCl2, 0.1 M sodium acetate
(CH3COONa) (pH 5.8). Following staining overnight, the staining
solution was aspirated and embryos were destained for several hours in a
solution of 0.7 M MgCl2, 0.1 M sodium acetate (pH 5.8).
Generation of P-element transformants expressing PAPS synthetase
A plasmid carrying a full-length cDNA encoding Drosophila PAPS
synthetase (Jullien et al.,
1997) cloned in pBS (SK) was obtained from
Genome Systems (St Louis, MO 63134). The PAPS synthetase
(papss) cDNA was excised and subcloned into phs-CaSPer
(Bang and Posakony, 1992
) at
the unique XbaI site downstream of the hsp70 promoter.
Transgenic lines carrying phs-CaSPeR-papss were generated by
conventional microinjection (Rubin and
Spradling, 1982
) with a P-element transposase-expressing helper
plasmid.
Immunostaining and in situ hybridizations
A peptide of the sequence AFKYRRIPYPKRSVE, corresponding to amino acid
residues 9-23, which are common to all Pipe isoforms, was synthesized by
SynPep Corporation and purified by HPLC. Peptide (5.0 mg) was conjugated to
Keyhole limpet hemocyanin using glutaraldehyde as a crosslinking reagent.
Antibodies directed against the immunogen were generated in a rabbit at
Covance Research Products. Staining of embryos was carried out according to
Macdonald and Struhl (Macdonald and
Struhl, 1986) using antibody preabsorbed against wild-type embryos
at a dilution of 1:1000. The rabbit polyclonal antibody directed against the
Windbeutel protein (Ma et al.,
2003
) was used at a dilution of 1:2000. Primary antibodies were
used in conjunction with a biotinylated goat anti-rabbit secondary antibody
(1:500 diluted, pre-absorbed against wild-type embryos) and visualized with
avidin/HRP complex (Vector Laboratories).
To examine pipe-ST2 RNA expression in pipeC14/pipeC14 embryos, we balanced the pipeC14 mutation over TM3, Sb, Krüppel-Gal4, UAS-GFP. Progeny embryos from this stock were sorted into fluorescent (wild-type) and non-fluorescent (pipeC14/pipeC14) groups.
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Generation of follicle cell and germline clones
To test whether follicle cell expression of genes previously implicated in
the synthesis or modification of GAGs is required maternally for embryonic DV
patterning, we generated follicle cell clones that were homozygous for
mutations in genes of interest by FLP/FRT-mediated site specific recombination
(Golic and Lindquist, 1989).
Clones were generated in females carrying a mutation-bearing FRT chromosome in
trans to an FRT-bearing, but otherwise wild-type chromosome. To identify
embryos derived from follicles containing mutant clones, we used the marking
system of Nilson and Schüpbach
(Nilson and Schüpbach,
1998
).
Embryos lacking both maternal and zygotic expression of sgl, sfl,
frc and papss were generated using the dominant female-sterile
technique of Chou and Perrimon (Chou and
Perrimon, 1996).
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Results |
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Deviations from the wild-type pipe sequence were identified in each of the 11 EMS-derived mutant alleles (Fig. 1B). In 10 of these mutations, the lesions identified were associated with Pipe-ST2 specific exons. The pipe3 allele carries a stop codon in the third exon, which is common to all pipe isoforms. The pipeC14 allele was identified in a screen for P-element mutations in the pipe locus. Although we have not identified the lesion associated with the pipeC14 allele, we believe the mutation is in a 5' regulatory region of the gene that affects all isoforms (see below).
These results suggest that Pipe-ST2 is specifically required in the ovary and that its loss does not affect viability. At least some of the other isoforms, however, are required for viability. In addition to the ovary, pipe is expressed in the embryonic salivary gland. Antibody staining and in situ hybridization demonstrated that in pipeC14 mutants, neither pipe RNA nor protein were detectable in either the ovary or the embryonic salivary gland (Fig. 2B,E,H). This finding is consistent with the idea that the pipeC14 mutation affects the transcription of all of the pipe isoforms. These results suggest that Pipe is required for salivary gland development or function, which may explain the effect of pipeC14 on viability.
Both pipe1 and pipe7 affect valine 123 (Fig. 1B), which is located within a stretch of amino acids extending from residues 120 to 127, PKGVSQTF, that is predicted to be within the binding site for PAPS, the high energy, small molecule donor in sulfation reactions. In the strong pipe1 allele, the nonconservative substitution of an aspartic acid residue for valine results in an apparently nonfunctional protein. Embryos from females carrying this allele are completely dorsalized (Fig. 3G,H). In pipe7, however, the relatively conservative substitution of isoleucine for valine results in a hypomorphic allele. Females carrying pipe7 in trans to a deficiency uncovering pipe produce embryos that are only weakly dorsalized (Fig. 3D) and exhibit residual polarity during gastrulation (Fig. 3C), indicating that the protein retains considerable activity.
The location of the pipe7 mutant lesion within the
putative PAPS-binding site suggested that its weak phenotype might result from
a lowered affinity of the pipe7-encoded protein for PAPS.
To test this hypothesis, we fed
pipe7/Df(3L)pipeA13 flies
yeast containing 1 M sodium chlorate, a compound known to inhibit the activity
of PAPS synthetase (Lansdon et al.,
2004; Baeuerle and Huttner,
1986
; Greve et al.,
1988
). We reasoned that if the pipe7 mutant
protein has reduced affinity for PAPS, then under conditions of decreased PAPS
availability,
pipe7/Df(3L)pipeA13 females
would be expected to produce relatively more dorsalized progeny than untreated
pipe7/Df(3L)pipeA13 females.
Indeed, 94% of the cuticles (n=192) of the embryonic progeny of
treated pipe7/Df(3L)pipeA13
females exhibited a completely dorsalized D0 cuticular phenotype
(Roth et al., 1991
) and apolar
gastrulation movements (Fig.
3E,F; Table 1). By
contrast, the dorsalized D0 phenotype was exhibited by only 1% of the progeny
of untreated
pipe7/Df(3L)pipeA13 females
(Table 1). Wild-type flies fed
sodium chlorate do not produce dorsalized progeny, which implies that the
activity of the wild-type PipeST2 protein is not detectably affected by the
sodium chlorate-induced decrease in PAPS availability, at least as measured by
embryonic DV patterning. Our finding that the pipe7 mutant
protein is sensitive to the concentration of PAPS, however, is consistent with
our designation of Pipe-ST2 as a sulfotransferase.
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Although only one of the ten pipe isoforms, Pipe-ST2, appears to
function in the follicle cell layer and to be required for embryonic DV
patterning, multiple isoforms from the pipe locus are expressed in
the salivary gland (Sergeev et al.,
2001). As noted above, the pipeC14 and
pipe3 mutations affect all of the Pipe isoforms, whereas
the other 10 alleles examined, including the pipe2 allele,
specifically affect the Pipe-ST2 isoform. We examined
pipeC14/pipeC14,
pipe3/pipe3 and
pipe2/pipe2 mutant embryos for Alcian
Blue staining in their salivary glands. We detected no Alcian Blue staining in
the salivary glands of pipeC14/pipeC14
and the pipe3/pipe3 mutant embryos
(Fig. 4D,F). By contrast, the
salivary glands of pipe2/pipe2 mutant
embryos did exhibit Alcian Blue staining
(Fig. 4G). These findings
suggest that pipe activity is required for the presence of a sulfated
molecule in the embryonic salivary glands. Furthermore, although the Pipe-ST2
isoform is required for the maternal function of pipe, our data
suggest that the expression of other Pipe isoforms in the embryonic salivary
gland is sufficient for the production of the Alcian Blue staining
material.
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wind encodes a homologue of the vertebrate endoplasmic reticulum
protein Erp29 (Konsolaki and
Schüpbach, 1998), and we have previously shown that Wind
protein is required for the correct subcellular localization of the Pipe
protein to the Golgi apparatus (Sen et
al., 2000
). As observed for the
pipeC14/pipeC14 and
pipe3/pipe3 mutant alleles, embryos
homozygous for all three of the wind alleles tested lacked Alcian
Blue staining (Fig. 4H-J). The
most straightforward interpretation of these results is that the Pipe isoforms
expressed in the salivary gland function as sulfotransferases that are
directly involved in the formation of the Alcian Blue-staining material. Wind
protein is likely to be required for the Golgi localization, and therefore the
function, of all Pipe isoforms. Embryos homozygous for mutations in the dorsal
group genes nudel (ndl), gastrulation defective, snake,
easter and spätzle exhibited normal Alcian Blue staining,
(data not shown), demonstrating that dorsal group genes other than Pipe and
Wind are not required for the production of this material. These results are
consistent with the idea that the staining material is a direct product of the
catalytic activity of one or more of the Pipe isoforms.
Mutations in genes encoding GAG synthesis proteins do not affect Alcian Blue staining
The similarity between Pipe-ST2 and HS2ST suggested that the Alcian
Blue-staining material in embryonic salivary glands might represent a heparan
sulfate GAG. If so, we would expect that mutations in genes previously shown
to be involved in the synthesis or modification of heparan sulfate would also
affect Alcian Blue staining in the embryonic salivary glands. We therefore
assayed for the presence of Alcian Blue-staining material in the salivary
glands of embryos homozygous for mutations in the following genes:
sugarless (sgl) (Binari
et al., 1997; Häcker et
al., 1997
; Haerry et al.,
1997
) encodes the fly homologue of UDP-glucose-6 dehydrogenase,
which converts UDP-glucose to UDP-glucuronic acid, a required step in the
synthesis of the uronic acid residues present in heparan sulfate;
sulfateless (sfl) encodes a protein with similarity to
vertebrate N-deacetylase/N-sulfotransferases
(Lin and Perrimon, 1999
),
which are known to mediate deacetylation and sulfation of the N-acetyl group
on N-acetylglucosamine (GlcNac) of heparan sulfate; and fringe
connection (frc), which encodes a Golgi transporter that is
required for the Golgi uptake of nucleotide-sugars involved in the synthesis
of heparan sulfate (Goto et al.,
2001
; Selva et al.,
2001
).
In contrast to embryos mutant for papss, Alcian Blue staining was
clearly evident in embryos homozygous for mutations in sgl, sfl and
frc (data not shown), suggesting that this stained substance does not
represent a conventional heparan sulfate GAG. However, the segment polarity
phenotypes that allowed the initial identification of mutations in sgl,
sfl and frc are only observed in zygotically mutant embryos that
are derived from mutant germline clones
(Perrimon et al., 1994). This
raised the possibility that maternal loading of transcripts might provide
sufficient levels of protein expression to enable homozygous mutant embryos to
produce Alcian Blue-stained material. To address this issue, we generated
embryos lacking both maternal and zygotic expression of sgl, sfl and
frc. Although cuticles of these embryos exhibited a typical segment
polarity phenotype (Fig.
6F,I,L), embryos lacking both maternal and zygotic function of
these three genes did exhibit Alcian Blue staining in structures that appeared
to be salivary glands (Fig.
6D,G,J). Staining with an antibody against Wind confirmed that the
Alcian Blue-stained structures corresponded to salivary glands
(Fig. 6E,H,K). In contrast to
sgl, sfl and frc, the salivary glands of embryos lacking
both maternal and zygotic expression of papss failed to stain with
Alcian Blue (Fig. 6A), even
though Windbeutel staining demonstrated the presence of the salivary glands
(Fig. 6B). Based on its central
role in sulfation reactions, embryos derived from germline clones mutant for
papss would also be expected to exhibit a segment polarity phenotype
because of the loss of heparan sulfate, which was observed
(Fig. 6C). Taken together,
these observations indicate that the production of the Pipe-dependent Alcian
Blue-stained material in the embryonic salivary glands does not require the
function of genes known to be involved in heparan sulfate GAG synthesis.
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Discussion |
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Our finding that the pipe7 mutant phenotype is significantly enhanced by sodium chlorate treatment strongly supports the identification of Pipe as a sulfotransferase. This identification is further bolstered by our demonstration that the presence of a Pipe-dependent Alcian Blue-stained material in the embryonic salivary glands requires the function of two other genes essential for the sulfotransferase reaction: slalom, which encodes the Drosophila PAPS Golgi transporter; and papss, the PAPS synthetase gene. The finding that embryos mutant for pipe, slalom or papss all lack Alcian Blue staining in their salivary glands is strong evidence that the stained material represents a sulfated macromolecule.
The original molecular identification of Pipe as a putative sulfotransferase was made on the basis of its similarity to HS2ST. Consequently, it has been assumed that heparan sulfate is the likely substrate of Pipe activity. We reasoned that if Pipe acts as a heparan sulfate sulfotransferase, then the presence of the Alcian Blue-stained material in the embryonic salivary glands would be dependent upon the activity of genes whose products have been demonstrated to participate in heparan sulfate synthesis and modification in Drosophila. In contrast to this expectation, we found Alcian Blue staining to be present in the salivary glands of embryos mutant for sgl, sfl or frc.
We used a similar strategy to investigate the possibility that heparan
sulfate is the target of Pipe activity in the ovary. We anticipated that genes
encoding products involved in the sulfotransferase reaction, or in the
synthesis of the Pipe substrate, would be required in the ventral follicle
cells. Females carrying follicle cell clones mutant for pipe
(Nilson and Schüpbach,
1998) (this work) or slalom
(Lüders et al., 2003
)
produce embryos with a dorsalized phenotype. By contrast, embryos derived from
females carrying ventral clones of follicle cells mutant for sgl, sfl
or frc exhibited normal DV polarity. This suggests that like the
Alcian Blue-stained material in the embryonic salivary glands, the target of
Pipe function in the ovary does not correspond to heparan sulfate.
Surprisingly, females carrying papss mutant follicle cell clones
did not produce dorsalized embryos. Although this result could be interpreted
as an argument against Pipe acting as a sulfotransferase in the ovary, we do
not believe this to be the explanation. Because PAPS, the product of PAPS
synthetase activity, is a small molecule (507 Da), it may be able to
pass through the gap junctions that exist between the oocyte and follicle cell
layer (Giorgi and Postlethwait,
1985; Bohrmann and
Haas-Assenbaum, 1993
;
Waksmonski and Woodruff,
2002
). Gap junctions are known to allow passage of molecules of
approximately 1 kDa in mass (Goldberg et
al., 2004
), which would permit passage of PAPS from a wild-type
oocyte into mutant follicle cells. Another gene whose mutant alleles may
behave nonautonomously for the same reason is sgl, which encodes
UDP-glucose dehydrogenase. The product of Sugarless activity, UDP-glucuronic
acid, is also a small molecule (577 Da) that may be capable of passing
through gap junctions. Although the result for sgl mutant follicle
cell clones may therefore be inconclusive, neither sfl nor
frc mutations would be expected to exhibit nonautonomous behavior.
sfl encodes N-deacetylase/N-sulfotransferase, a Golgi resident enzyme
of Type II transmembrane topology. The product of Sfl activity, sulfated
heparan sulfate, is too large to move between cells through gap junctions. The
product of frc mediates the uptake into the Golgi of nucleotide
sugars required for GAG synthesis and thus could not be rescued
nonautonomously. Therefore, the finding that females carrying ventral follicle
cell clones of sfl or frc did not give rise to dorsalized
embryos provides the strongest evidence that heparan sulfate plays no role in
the function of Pipe in embryonic DV patterning.
Although sgl mutations may behave nonautonomously in the ovary, this explanation cannot be invoked to explain the lack of effect of sgl mutations on the Alcian Blue staining in the embryonic salivary glands. Because these embryos were both maternally and zygotically mutant for sgl, there would be no wild-type cells present to supply UDP-glucuronic acid to the sgl mutant cells. By contrast, even though a role for papss could not be demonstrated in the ovary because of the possibility of nonautonomous rescue, its function was clearly necessary for the formation of the Pipe-dependent Alcian Blue-stained material in the embryonic salivary glands.
In addition to heparan sulfate, the ability of the Alcian Blue-stained material to form in the absence of sgl activity also rules out the possibility that Pipe is involved in the sulfation of dermatan/chondroitin sulfate, at least in that tissue. This is because UDP-glucuronic acid, the product of Sugarless activity, is required not only for the synthesis of heparan sulfate, but also for the synthesis of dermatan/chondroitin sulfate polysaccharide chains. Two other pieces of evidence also argue against a role for Pipe in the sulfation of either heparan sulfate or dermatan/chondroitin sulfate GAGs. First, expression in the follicle cell layer of cDNAs corresponding to hamster HS2ST and the human dermatan/chondroitin sulfate 2-O-sulfotransferase failed to rescue the dorsalized phenotypes of the progeny of pipe/pipe mutant females (Z. Zhang and D.S., unpublished). The Drosophila genome contains another gene, CG10234, that encodes a protein that is much more similar to vertebrate HS2ST than are the Pipe isoforms (http://flybase.bio.indiana.edu/); the product of this gene is likely to represent the bona fide Drosophila heparan sulfate 2-O sulfotransferase. Second, we have not been able to detect heparan sulfate sulfotransferase or dermatan/chondroitin sulfate sulfotransferase activity in vitro using Pipe-ST2 protein expressed in cell culture (A. Amiri and D.S., unpublished). Although our data argue against a role for Pipe in the sulfation of uronic acid residues in heparan sulfate, the similarity of the Pipe isoforms to heparan sulfate 2-O sulfotransferase and dermatan/chondroitin sulfate 2-O-sulfotransferase suggests that Pipe acts on the 2-O position of a monosaccharide component of an as yet unidentified glycoprotein or glycolipid.
The existence of multiple Pipe isoforms is an intriguing feature of the pipe gene in Drosophila melanogaster. Blast analysis of the D. pseudobscura genome (http://flybase.net/blast/) indicates that multiple isoforms of Pipe exist in that species as well. By contrast, only a single Pipe isoform is encoded in the mosquito (http://www.anobase.org/cgibin/blast.pl) and flour beetle (http:bioinformatices.ksu.edu/blast/blast.html) genomes. Similarly, only a single Pipe isoform was detected in a database of silk moth ESTs (http://papilio.ab.a.u-tokyo.ac.jp/silkbase/index.html). In each of these three organisms, the single Pipe isoform exhibits strong sequence similarity to Drosophila Pipe-ST2. It therefore appears likely that only the Pipe-ST2 isoform was present in the common ancestor of true flies, mosquitoes, moths and beetles. This suggests that the ancestral role of the pipe gene was to act during oogenesis to regulate embryonic DV patterning. Multiple Pipe isoforms were probably generated via genomic duplication in Drosophila, where they appear to be required for salivary gland development and/or function. Lack of Pipe activity in the salivary gland may lead to a disruption of the feeding behavior of the larvae, which in turn reduces their growth rate and viability. The generation and expression of multiple protein isoforms may be a mechanism to produce extremely high levels of Pipe protein, if each isoform has a similar enzymatic specificity. Alternatively, each isoform may have a distinct substrate specificity that contributes uniquely to salivary gland development and/or function.
The elucidation of Pipe-ST2 function is crucial to understanding the spatial regulation of the serine protease cascade whose ventrally restricted activity defines embryonic DV polarity. The simplest model of Pipe action posits that Pipe-ST2 functions as a sulfotransferase, and that the target of Pipe must be sulfated in order to exert its function. Although the target of Pipe may be present throughout the follicle cell layer, it would be sulfated only in the ventral follicle cells and following its secretion it would be deposited into the ventral side of the egg. There, it would assemble or activate the dorsal group serine protease cascade, leading to ventrally restricted processing of the Spätzle ligand. Although the specific targets of Pipe action in the follicle cell layer and the salivary gland may not be the same molecule, the general class of glycan on which Pipe acts in the two tissues is likely to be related. Current efforts are directed towards identifying these molecules and defining their roles in DV patterning and salivary gland function.
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ACKNOWLEDGMENTS |
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