Essentials of Glycobiology

May 16, 2002Jeff Esko

Lecture 28

Developmental glycobiology in model organisms: Drosophila and C. elegans

Worms and Flies - Ideal Model Organisms

Entire genomes are now available

19,099 genes in C.elegans, 959 cells, all fate mapped elegans.swmed.edu www.sanger.ac.uk

~13,600 genes in D. melanogaster, www.fruitfly.org/

Can study differentiation, morphogenesis, and behavior

C. elegans

D. melanogaster

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

• 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

• 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

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.

• rotated abdomen (rt) encodes a putative integral membrane glycoprotein homologous to yeast mannosyltransferases (Pmts) that utilize dolichol-P-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 GlcNAc1,3 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 406:369Bruckner et al (2000) Nature 406:411Selva 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 1,3- and 1,6-specific linkages by sequence homology

- No genes encoding 1,2-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

wild-type wingless

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

dally/dally dally/+ x sgl/+

-note loss of naked cuticle

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.

- phenotype accentuated

sgl, sfl, and ttv define essential steps in heparan sulfate synthesis, suggesting that wingless requires heparan sulfate to bind to its receptor

Mutations that enhance wingless phenotype

• dally Glypican proteoglycan

• sugarless (sgl) UDP-glucose dehydrogenase that makes UDP-GlcA

• sulfateless (sfl) GlcNAc N-deacetylase/N-sulfotransferase

• tout-velu (ttv) Heparan sulfate copolymerase

Dally acts as a coreceptor

• Notice the similarity of this model to one proposed for FGF-2 signaling in vertebrate cells

Signaling EventMitogenesis

FGF

Heparan sulfate

Proteoglycan

FGF

Selective Effects of Mutants

Another Gene?

Chondroitin sulfate might substitute for heparan sulfate?

AnotherProteoglycan?

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 anterior-posterior (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.

Patchedexpression

V

D

PA

ptcclone

ttv clone anterior

ttv cloneposterior

V

D

PA

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.

The ttv clone is acting like a barrier to Hh signaling on the anterior side

This time Patched is green, and Hh is stained red. The lack of ttv expression in the clone prevents Hh diffusion.

ptc clone ttv/ptcclone

HedgehogPatched

V

D

PA

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 stagesDG42 shows homology to nodC and has chitin synthase activity ([GlcNAc1,4GlcNAc]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 1,3 to chitin oligosaccharides

— Injection of nodZ, antibodies to DG42, or chitinase causes defects in trunk and tail development

Zebrafish express many of the same genes found in higher vertebrates

Zebrafish

- 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