Received for publication, November 5, 2002, and in revised form, November 25, 2002
The neurogenic Drosophila genes
brainiac and egghead are essential for
epithelial development in the embryo and in oogenesis. Analysis of
egghead and brainiac mutants has led to the
suggestion that the two genes function in a common signaling pathway.
Recently, brainiac was shown to encode a
UDP-N-acetylglucosamine:
Man
1,3-N-acetylglucosaminyltransferase (
3GlcNAc-transferase) tentatively assigned a key role in
biosynthesis of arthroseries glycosphingolipids and forming the
trihexosylceramide, GlcNAc
1-3Man
1-4Glc
1-1Cer. In the
present study we demonstrate that egghead encodes a
Golgi-located GDP-mannose:
Glc
1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac,
Man
1-4Glc
1-1Cer. Egghead is unique among
eukaryotic gly- cosyltransferase genes in that homologous genes are
limited to invertebrates, which correlates with the exclusive existence
of arthroseries glycolipids in invertebrates. We propose that brainiac
and egghead function in a common biosynthetic pathway and that
inactivating mutations in either lead to sufficiently early termination
of glycolipid biosynthesis to inactivate essential functions mediated
by glycosphingolipids.
 |
INTRODUCTION |
The Drosophila genes brainiac and
egghead play essential roles in epithelial development in
the embryo and during oogenesis (1, 2). Brainiac and
egghead encode proteins that are required in the germline to
allow for normal interaction between germ line and somatic cells in the
developing ovary (2). In the absence of brainiac or
egghead in the germ line defects are observed in the
overlying follicular epithelium, which is of somatic origin (1, 2). On
one hand, these follicular epithelial defects resemble defects in
epidermal growth factor receptor signaling between germ line and
follicle cell layers. On the other hand, they resemble a subset of the
follicular defects associated with Notch mutants (1-3).
Defects in female fertility have also been described (4). The diversity
of defects caused by brainiac and egghead mutants
suggests that they may be involved in communication between cells at a
fundamental level and that they can affect multiple signaling pathways.
Brainiac and egghead mutants exhibit similar and
non-additive phenotypes, leading to the proposal that they function in
a common signaling pathway. Based on sequence analysis, Yuan et al. (5) originally proposed that brainiac together with
the distant homologous gene fringe encoded
glycosyltransferases. This hypothesis has subsequently proved correct
and both represent glycosyltransferases with functionally conserved
mammalian homologs (6-9). Brainiac encodes a
UDP-N-acetylglucosamine:
Man
1,3-N-acetylglucosaminyltransferase (
3GlcNAc-transferase)1
with a predicted function in biosynthesis of arthroseries
glycosphingolipids in the Drosophila (8, 9). Brainiac was
shown to catalyze addition of the third monosaccharide residue to form
the trihexosylceramide glycolipid,
GlcNAc
1-3Man
1-4Glc
1-1Cer. Arthroseries glycolipids have
only been found in invertebrates and differ fundamentally from
mammalian glycolipids by having a core disaccharide structure based on
Man
1-4Glc
1-Cer (MacCer) rather than Gal
1-4Glc
1-Cer (LacCer) (10). Interestingly, brainiac was found to transfer
1-3
linked GlcNAc to both MacCer and LacCer, while mammalian homologs only
transfer to LacCer (8, 9, 11). Sequence analysis of egghead
indicates that it could encode a type II transmembrane glycosyltransferase. Homologous genes appear limited to invertebrates, and no similar genes are found in the mammalian databases. In the
present study we tested the hypothesis that egghead encodes a unique invertebrate glycosyltransferase activity in the same biosynthetic pathway as brainiac and present evidence that
egghead indeed encodes a
1,4-mannosyltransferase
predicted to form the MacCer precursor glycolipid substrate for brainiac.
 |
EXPERIMENTAL PROCEDURES |
Expression of egghead in Insect--
An expression construct of
the full coding region of egghead was prepared by reverse
transcriptase-PCR using Drosophila melanogaster mRNA and the sense primer Egh001
(5'-AGCAGATCTCAAGATGAACTCCACCACAAAG-3') with a BglII
restriction site and the antisense primer Egh002 (5'-AATAGTCTAGACAGTCTCCAGTACGCG-3') with a XbaI restriction
site. The resulting 1.37-kb fragment was cloned into the
BglII/XbaI sites of pVL1393 (PharMingen) and
pVL1393-MYC. Baculovirus expression constructs,
pVL-egghead-full and pVL-egghead-Myc-full, were
co-transfected with Baculo-GoldTM DNA (PharMingen) in
Sf9 cells as described (12). Control constructs included
pVL-GalNAc-T4-full (13) and pVL-brainiac-full (8). Standard enzyme assays were performed in 50-µl reaction mixtures containing 25 mM HEPES-KOH (pH 7.4), 10 mM
MgCl2, 0.1% n-octylgalactoside (Sigma), and 100 µM GDP-[14C]Man (2,000 cpm/nmol) (Amersham
Biosciences), and varying concentration of acceptor substrates
(purchased from Fluka, Merck, Sigma, and Toronto Research Chemicals
Inc.; see Table I for structures). Assays with brainiac were
carried out in the same reaction mixture except for addition of
UDP-[14C]GlcNAc (3,000 cpm/nmol) (Amersham Biosciences)
and MnCl2. Enzyme sources were microsomal fractions of
baculovirus-infected Sf9 and High FiveTM cells
prepared essentially as described (14). Briefly, cells were lysed in
lysis buffer (25 mM Tris-HCl (pH 7.4), 250 mM
sucrose); after incubation 30 min on ice cells were homogenized and
lysate centrifuged at 1,000 × g. Glycerol was added to
20%, and membrane pellets were obtained by 100,000 × g. Pellets were used at 10 mg/ml (protein concentration
determined by BCA, Pierce). Reaction products of soluble acceptors were
quantified by chromatography on Dowex AG1-X8 (Sigma). Assays with
glycosphingolipids included 5 mM
2-acetamido-2-deoxy-D-glucono-1,5-lactone (inhibitor of
hexosaminidase activity), and products were purified on
octadecyl-silica cartridges (Supelco) and analyzed by high performance
thin-layer chromatography followed by autoradiography.
Expression of egghead in CHO Cells--
The 1.37-kb fragment
used for baculo constructs was cloned into the
BamHI/XbaI sites of pcDNA3(+). CHO-K1 cells were
stably transfected with the pcDNA3-egghead-Myc-full as
described previously and clones selected with anti-Myc antibodies (13).
Cells were grown to subconfluence and fixed with 3% paraformaldehyde
and immunostained with anti-Myc monoclonal antibody (Invitrogen). Transferase assays were performed in standard reaction mixtures with
cell lysates.
Isolation and Analysis of a Product Formed by egghead--
The
product formed with n-octyl glucoside (1 mg) was
purified on octadecyl-silica cartridges (Bakerbond, J. T. Baker),
followed by stepwise elution with increasing concentrations of methanol in water. The purified glycolipid was deuterium-exchanged by repeated addition of CDCl3-CD3OD 2:1, sonication, and
evaporation under nitrogen, then dissolved in 0.5 ml of
Me2SO-d6, 2% D2O
(0.03% tetramethylsilane) for NMR analysis. One-dimensional
1H, two-dimensional 1H-1H gCOSY,
TOCSY, and ROESY NMR spectra were acquired on a Varian Inova 500 MHz
spectrometer at 35 °C.
 |
RESULTS |
tBLASTn searches performed with D. melanogaster
egghead coding region (GenBankTM accession
number NM_080313) of the National Center for Biotechnology Information
data base and the whole genome data base GadFly released by the
Berkeley Drosophila Genome Project revealed genes with significant
similarity in flies (diptera) and nematodes, including Caenorhabditis elegans. Low sequence similarity was found to
the putative cellulose synthetase CelA (GenBankTM accession
number AAC41435) from Agrobacterium tumefaciens as well as
other bacterial genes predicted to be glycosyltransferases (GenBankTM accession numbers NP_348317 (Clostridium
acetobutylicum) and NP_531181 (A. tumefaciens str.
C58)). No significant similarity was found with mammalian genes.
egghead is predicted to encode a protein of 457 amino acids
with a putative N-terminal signal sequence and a putative hydrophobic
transmembrane retention signal (3), which is typical for Golgi located
glycosyltransferases. SDS-PAGE Western blot analysis with anti-Myc
antibodies of lysates of baculovirus-infected High FiveTM
cells or a stable CHO egghead transfectant revealed a single protein migrating with an apparent molecular weight of 52 kDa (not
shown). Subcellular localization of egghead was analyzed by
immunofluorescense staining of a stable CHO egghead
transfectant, where immunoreactivity was limited to a supranuclear
pattern characteristic for Golgi localization (not shown). A similar
staining pattern was found for a stable CHO transfectant with human
3GnT2 (not shown), as well as transfectants with other human
glycosyltransferases (13). The GadFly data base predicts that egghead
contains a sugar nucleotide donor substrate binding site with potential
DXD/E binding motifs (15).
egghead Encodes a
GDP-Man:
Glc
1,4-Mannosyltransferase--
Initial assays of
activity included a screen with high concentrations of monosaccharide
substrates and different donor substrates as described previously (6,
8). Microsomal fractions of infected High FiveTM cells
expressing the full coding region of egghead exhibited a
marked increase in GDP-Man transferase activity with
D-glucose (Fig. 1). Egghead
exhibited strict donor substrate specificity for GDP-mannose and did
not utilize other donor sugar nucleotides tested (UDP-Gal, UDP-GalNAc,
UDP-GlcNAc). Analysis of a panel of mono- and disaccharide
derivatives showed that egghead exhibits strong preference for
substrates containing terminal
-linked glucose (
-Glc) (Table
I). Interestingly, some
Man
monosaccharide derivatives also served as efficient substrates;
however, no activity was found with the disaccharides Man
1-4GlcNAc
and Man
1-4Glc
1-n-Oct. Analysis of apparent
Km for the most active substrates identified showed
that n-octyl-
-Glc was the preferred acceptor substrate
(apparent Km 0.67 ± 0.08 mM) with
Glc
1-pNph (apparent Km 1.10 ± 0.3 mM) being comparable and Man
1-pNph (apparent Km 2.30 ± 0.5 mM) less
preferred. The apparent Km for GDP-Man with
n-octyl-
-Glc acceptor substrate was 58.0 ± 6.2 µM.

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Fig. 1.
egghead exhibits
GDP-Man: Glc mannosyltransferase activity with
monosaccharides. Microsomes of transfected High FiveTM
cells were used as enzyme sources. Donor sugar nucleotides included
GDP-Man, UDP-Glc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP-Xyl.
Designations are as follows: , egghead with GDP-Man and
D-glucose; , control with GDP-Man and
D-glucose; , egghead with GDP-Man and
L-mannose; , control with GDP-Man and
L-mannose; , egghead with GDP-Man and
D-galactose; , control with GDP-Man and
D-galactose; +, egghead with
D-GlcNAc; *, control with D-GlcNAc. Control
background values represented activity with microsomal fractions
expressing human polypeptide GalNAc-T4.
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Optimization of the enzyme assay using microsomal membranes
demonstrated that Triton X-100, Triton CF-54, and Nonidet P-40 inhibited egghead activity at 0.1%, while
n-octylgalactoside at 3.4 mM (0.1%) and to a
lesser extent CHAPS activated the enzyme. The pH optimum of egghead
activity was pH 7-8. Addition of 5-10 mM
MgCl2 and MnCl2 activated enzyme activity
(Mg2+ being better than Mn2+), and
CaCl2 had no effect, while addition of 10 mM
EDTA destroyed the activity.
Analysis of egghead activity in the established CHO transfectant cells
showed the same properties as when egghead is expressed in insect cells
(not shown). Attempts to visualize in vivo formed products
by lectin staining with Vicia Faba (Sigma) was unsuccessful, and
further characterization of the products formed await large scale
production of cells for chemical analysis of glycolipids.
Egghead Functions in Glycosphingolipid
Biosynthesis--
Glycosphingolipids of the fruit fly are based on the
arthroseries GlcNAc
1-3Man
1-4Glc
1-1Cer core (10). The
finding that egghead exhibits
-mannosyltransferase
activity with
Glc acceptor substrates strongly suggested that
egghead transfers Man to Glc
1-1Cer to form MacCer. As shown in Fig.
2 egghead utilizes Glc
1-1Cer as an
acceptor substrate, whereas LacCer does not serve as substrate. In
addition, Gal
1-1Cer was found not to serve as a substrate (not
shown). Based on this result it was predicted that egghead functions as
the MacCer synthase. Evidence in support hereof was provided by showing
that brainiac utilizes the product formed by egghead (Fig.
3). This assay was carried out with
n-octyl-
-Glc as initial acceptor substrate because it
served as a better substrate than GlcCer under the assay conditions
used.

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Fig. 2.
Egghead transfer Man to
Glc 1-1Cer. Microsomal fraction of
egghead and GalNAc-T4 were incubated with Glc 1-1Cer, LacCer, or no
glycolipid and GDP-Man as described under "Experimental
Procedures." Autoradiography of thin-layer chromatography of reaction
products (4 h) is shown. Plate was run in chloroform-methanol-water
(60/38/10, v/v/v). Migration of standard glycolipids is indicated with
arrows.
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Fig. 3.
The product formed by egghead with
n-octyl- Glc serves as a
substrate for brainiac. High performance
thin-layer chromatography analysis of product developments (2 h) with
combinations of microsomal fractions of egghead
(Egh), polypeptide GalNAc-T4 (GT4), and
brainiac (Brn) expressing High FiveTM
cells and combinations of sugar nucleotides GDP-Man and UDP-GlcNAc are
shown. The upper panel is stained with orcinol, and the
lower panel represents an autoradiography. Plates were run
in chloroform-methanol-water (60/30/8, v/v/v), and the migration of
n-octyl- Glc (NOG) and the disaccharide and
trisaccharide products hereof are indicated in the margins. Man-Glc-Oct
is formed only in the presence of egghead and GDP-Man, and
GlcNAc-Man-Glc-Oct is formed only in the presence both of egghead and
brainiac as well as GDP-Man and UDP-GlcNAc. In lane 7, the
asterisks indicate that the autoradiography assay was
carried out with non-labeled GDP-Man to confirm that the initial added
sugar was Man.
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Structural Characterization of Product Formed by egghead--
A
one-dimensional 1H NMR spectrum of the diglycosyl product
formed with n-octyl-
-glucoside exhibited resonances
consistent with ~55% conversion to
Man
1-4Glc
1-1-n-octyl, i.e. anomeric signals at 4.477 and 4.143 ppm
(3J1,2 = ~1 and 7.9 Hz,
respectively), corresponding to H-1 of Man
1-4 and Glc
1-1
residues of this glycolipid. H-1 of unreacted Glc
1-1 is observed at
4.080 ppm (3J1,2 = 7.6 Hz) (Fig.
4). Following complete assignment of
1H resonances from all three monosaccharide spin systems
present (see Table II) by two-dimensional
1H-1H gCOSY and TOCSY experiments (not shown),
the connectivity between the
-Man and the more abundant
-Glc
(spin system originating from the H-1 at 4.143 ppm) was established as
a 1
4 linkage by a two-dimensional ROESY experiment, which showed a
dipolar cross-relaxation correlation between
-Man H-1 and
-Glc
H-4. This is consistent with the substantial downfield shift of H-4
compared with that observed for unreacted n-octyl
-glucoside (3.350 versus 3.016). Although other
-Glc
resonances are affected by the glycosylation, H-4 is shifted downfield
by the largest increment (
H-4 = 0.334 ppm;

H-3 = 0.244 ppm; 
H-5 = 0.151 ppm).

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Fig. 4.
Downfield region of 500-MHz 1H
NMR spectrum (Me2SO-d6, 2%
D2O, 35 °C) of the
Man 1-4Glc 1-1-n-octyl
product of egghead. Arabic numerals
refer to ring protons of residues designated by standard three-letter
monosaccharide nomenclature in the corresponding structure;
P = product; S = substrate.
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Table II
1H chemical shifts (ppm) and 3J1,2 coupling
constants (Hz, in parentheses) for Glc 1-n-octyl substrate and
biosynthetic Man 4-Glc 1-n-octyl product.
Data were obtained in Me2SO-d6, 2%
D2O at 35 °C. Chemical shifts are referenced to internal
tetramethylsilane (set to 0.000 ppm).
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 |
DISCUSSION |
The original prediction that the neurogenic genes
brainiac and egghead encoded proteins serving
functions in a common pathway has been verified by demonstrating that
both genes encode glycosyltransferases and that egghead can synthesize
the immediate precursor glycolipid substrate for brainiac. The two
enzymes function very early in glycosphingolipid biosynthesis at the
second and third steps in build-up of the glycan chain, and it is
likely that this reflects the severe phenotypes associated with
inactivation of these genes. Glycosphingolipids of
Drosophila have been reported to be based on the
arthroseries and exist as extended oligosaccharide structures such as
Gal
1-3GalNAc
1-4GalNAc
1-4GlcNAc
1-3Man
1-4Glc
1-1Cer, which can be terminated by glucuronic acids and modified with phosphoethanolamine to give charged and zwitterionic glycolipids (10, 16). Specific biological functions of distinct glycolipid structures have not been elucidated in Drosophila, but it is
conceivable that termination of glycolipid biosynthesis at GlcCer and
at MacCer could block biological activity of glycolipids to similar effect.
Genetic approaches to studying glycosphingolipid functions in mammals
have so far provided some insight into defined biological activities.
In contrast to invertebrate glycolipids that appear to be based on one
class, mammalian glycolipids are based on multiple classes. Mice
deficient in ganglioseries glycolipids built on GalNAc
1-4Gal
1-4Glc
1-Cer have yielded significant information (17-21). Globoseries glycolipids built on
Gal
1-4Gal
1-4Glc
1-Cer are dispensable in man as evidenced
from the rare Pk and p blood groups (22, 23). While the
biosynthesis of ganglioseries and globoseries glycolipids are carried
out by unique single copy genes, each step in the biosynthesis of
lacto- and neolactoseries glycolipids based on the fact that
GlcNAc
1-3Gal
1-4Glc
1-Cer is carried out by multiple
isoenzymes, many of which serve functions in the synthesis of
glycoproteins as well (24). Drosophila and C. elegans may in this respect constitute simpler systems for studies
of functions of glycolipids. Recently, the
4GalNAc-transferase acting in sequence after brainiac to form
GalNAc
1-4GlcNAc
1-3Man
1-4Glc
1-1Cer was characterized
(25).
An increasing number of genes involved in biosynthesis of
glycoconjugates have been identified as essential or important for normal development of flies and nematodes. A number of genes involved in biosynthesis of the proteoglycan core region were identified through
an elegant screen for defects in vulval invagination of C. elegans (26), and these include glycosyltransferases functioning in precursor-product relationships and relevant sugar nucleotide transporters (27), fringe, a distant homolog of
brainiac, was found to encode a key enzyme controlling
elongation of O-linked fucose directly on Notch (6,
7), and precursor-product relationships with glycosyltransferases
functioning after fringe have also been implicated (28). To our
knowledge, egghead and brainiac are currently the
only available examples of essential genes in Drosophila with functions in the biosynthesis of glycolipids. Glycolipids are
known to serve important biological functions in mammals including modulation of receptor functions (29). Modulation may be mediated through direct lectin-carbohydrate interactions between the receptor and glycolipids (30) or through organization of lipid rafts, which are
known to be enriched in MacCer in Drosophila (31). Egghead and brainiac offers new tools to decipher
mechanisms of receptor modulation through glycolipids.
Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.C200619200
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