Transcript Lecture_28.pps

Essentials of Glycobiology
May 16, 2002
Jeff Esko
Lecture 28
Developmental glycobiology in model organisms:
Drosophila and C. elegans
Worms and Flies - Ideal Model Organisms
s Entire genomes are now
available
C. elegans
D. melanogaster
s 19,099 genes in
C.elegans, 959 cells, all
fate mapped
elegans.swmed.edu
www.sanger.ac.uk
s ~13,600 genes in D.
melanogaster,
www.fruitfly.org/
s Can study differentiation,
morphogenesis, and
behavior
Few structures, lots of biology
• In comparison to vertebrate systems, few glycan
structures are currently known in model
organisms
• Homologs for most vertebrate “glyco” genes
have been described, but in only a few cases
have corresponding activities been demonstrated
in vitro
• In many cases, developmental biologists have
stumbled into glycobiology
• Reverse genetic methods (mutations, RNAi) allow
one to dissect the importance of these genes in
development
In Comparison, Vertebrate Systems...
• …have lots of glycans of known structure
• …have hundreds of genes cloned and
biochemically characterized….
• But, fewer genetic tools are available
• Reverse genetics is possible in mice, which
provide models for human disease
Some people say that the best organism in the world to
work on is
the fruitfly, Drosophila melanogaster
Drosophila melanogaster
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•
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Easily studied developmental program
Complex neural system
Behavior
Obviously discernable phenotypes
Virtually all vertebrate pathways of glycosylation
are present, except….
• No sialic acids or sialic acid binding proteins
• No acquired immunity
• Higher order brain functions absent
Let’s look at some examples…….
Example 1: O-Mannosylation
Cuticular phenotypes of rt mutations
(A) Cuticle of a third instar rt 2
hemizygous larvae showing a correct
alignment of cuticle landmarks. (B)
Ventral view of abdominal segments of
an adult fly of the same genotype
showing the staggering of sternites
along the anterior/posterior axis.
• In bilateral animals, the
left and right sides of
the body usually have
asymmetric structures.
• In Drosophila
melanogaster,
mutations in the rotated
abdomen (rt) locus
cause a clockwise
helical rotation of the
body
• rotated abdomen (rt) encodes a putative integral
membrane glycoprotein homologous to yeast
mannosyltransferases (Pmts) that utilize dolicholP-Man for protein O-mannosylation….
• ….but the activity has not yet been demonstrated
in vitro nor has the substrates been identified in
vivo
Hydropathy plot
Example 2: O-Fucose Glycans
- Notch, a cell surface
receptor, is part of a
multicomponent signaling
system that regulates cell
differentiation along tissue
borders
- Notch is expressed on one
cell and notch ligands
(Delta, Serrate and
Scabrous) are presented by
adjacent cells
…and Fringe
- Fringe (fng) and Fringe Connection (frc) also
influence the wing margin. Fringe expression
boundaries coincide with Notch-dependent
patterning centers and with Notch-ligand
expression boundaries.
- Fringe adds GlcNAcb1,3 to O-linked fucose on the
EGF modules of Notch.
- Fringe Connection encodes a multifunctional
UDP-sugar transporter (UDP-GlcA, UDP-GlcNAc)
Moloney et al (2000) Nature 406:369
Bruckner et al (2000) Nature 406:411
Selva et al (2001) Nat Cell Biol 3:809
Example 3: Genome Comparisons
- Compare genes involved in vertebrate fucosylation to
the D. Melanogaster genome
- Fruit fly GDP-fucose formed exclusively by the de
novo pathway from GDP-mannose. No orthologs for
salvage pathway enzymes
- Two novel fucosyltransferases predicted to catalyze
a1,3- and a1,6-specific linkages by sequence
homology
- No genes encoding a1,2-specific fucosyltransferases
- A fucosidase enzyme discovered
- Two novel human genes putatively coding for new
fucosyltransferases identified
Roos et al (2002) J.Biol.Chem. 277:3168
Example 4: Glycome Comparisons
• Drosophila makes
numerous glycolipids
• Notice that the second
sugar is mannose
instead of galactose
as in vertebrate
glycolipids
• Predict new
glycosyltransferases
Seppo & Tiemeyer (2000) 10:751
Example 5: Proteoglycans
- The fly body plan is divided into segments.
Normally, each segment contains an anterior denticle
band, and a more posterior region of naked cuticle.
- In wingless mutants, the naked cuticle is absent,
replaced by a disordered array of denticles
wild-type
wingless
Dally
• Dally mutations delay key cell division events required
for maturation of the eye, antennae, wings, etc.
• Dally mutants also have a modest wg-like patterning
defect
• Dally shows genetic interactions with sgl (UDP-Glc
dehydrogenase) based on the enhanced cuticle
phenotype. UDP-Glc DH makes UDP-GlcA.
dally/dally
-note loss of naked cuticle
dally/+ x sgl/+
- phenotype accentuated
Mutations that enhance wingless phenotype
• dally
Glypican proteoglycan
• sugarless (sgl)
UDP-glucose dehydrogenase
that makes UDP-GlcA
• sulfateless (sfl)
GlcNAc N-deacetylase/Nsulfotransferase
• tout-velu (ttv)
Heparan sulfate copolymerase
sgl, sfl, and ttv define essential steps in heparan
sulfate synthesis, suggesting that wingless
requires heparan sulfate to bind to its receptor
Dally acts as a coreceptor
• Notice the similarity of
this model to one
proposed for FGF-2
signaling in vertebrate
cells
Proteoglycan
Heparan sulfate
FGF FGF
Signaling Event
Mitogenesis
Selective Effects of Mutants
Another Gene?
Chondroitin sulfate might substitute
for heparan sulfate?
Another
Proteoglycan?
Vertebrate Mutants
Example 6: Morphogen gradients
• Loss of tout-velu
function does not
abrogate the ability of
cells to respond to Hh
• Instead it alters the
distribution of Hh,
preventing its
dispersal across
domains of 10-12 cell
diameters
Proteoglycans may facilitate the diffusion of the
morphogen or permit transcytosis
Hedgehog is produced
posteriorly to anteriorposterior (A/P)
boundary in wing
imaginal disc and
diffuses to anterior side
(Blue lines indicate
anterior/posterior
boundary).
Hh induces Patched
expression (green)
anterior to A/P
boundary.
ptc
clone
Patched
expression
V
A
P
D
Notice that Patched
is not induced to the
anterior side of the
ttv clone. However,
when the ttv clone is
located posteriorly,
patched is induced.
ttv clone
anterior
ttv clone
posterior
The ttv clone is
acting like a barrier
to Hh signaling on
the anterior side
V
A
P
D
ptc clone
ttv/ptc
clone
V
A
Hedgehog
Patched
P
D
This time Patched is green, and Hh is stained
red. The lack of ttv expression in the clone
prevents Hh diffusion.
Example 7: C. elegans sqv mutations
• In 1999, Herman and Horvitz described a set of
mutants defective in vulval development (sqv,
squashed vulva). These mutations affect epithelial
invagination (and other developmental events)
• Of 8 complementation groups, 6 genes have been
cloned sqv-2, sqv-3, sqv-5, sqv-6, sqv-7, and sqv-8.
wild-type
sqv
sqv Mutations Affect GAG Biosynthesis
[GalNAc-GlcA]n-GalNAc-GlcA-Gal-Gal-Xyl-O-Ser
wild-type
sqv
One theory of cellular invagination is that the
adjacent epithelial cells may secrete a
polyanion in a polarized fashion
Hydration of the matrix might cause expansion
and an inward curvature of the cell layer
Could the missing link be a chondroitin sulfate
proteoglycan?
Vertebrate Mutants
Zebrafish
— Zebrafish have transparent embryos which
allows one to visualize development
— Saturation mutagenesis has been undertaken
to identify developmental phenotypes
— Excellent model for vertebrate development
— Disadvantage: Reverse genetics, but
morpholinos can be used to inactivate genes
Example 8: Chitin
— DG42, originally identified in Xenopus, is
expressed between midblastula/neurulation
stages
DG42 shows homology to nodC and has chitin
synthase activity ([GlcNAcb1,4GlcNAc]n)
— DG42 also has HA synthase activity, suggesting
the possibility that small chitin oligosaccharides
may act as a primer for HA synthesis
— nodZ is a rhizobial gene that adds fucose a1,3 to
chitin oligosaccharides
— Injection of nodZ, antibodies to DG42, or chitinase
causes defects in trunk and tail development
Zebrafish
Zebrafish express many of the same genes found in
higher vertebrates
- jekyll (UDP-Glc dehydrogenase) mutations
cause cardiac valve malformation
- knypek, glypican homolog. Defects impair
cellular movements during convergent
extension, but not cell fates)
- biglycan is present
- chondroitin sulfate and chondroitinases inhibit
axon growth