Discussion 20 Glycans and Development

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Discussion 20Glycans and Development
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Worms and Flies - Ideal Model Organisms

• Entire genomes are now available
• 19,099 genes in C.elegans, 959 cells, all fate
  mapped
• http//elegans.swmed.edu/
• http//www.sanger.ac.uk
• 13,600 genes in D. melanogaster
• http//www.fruitfly.org/
• Can study differentiation, morphogenesis, and
  behavior

D. melanogaster
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Xenopus laevis
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Few structures, lots of biology

• In comparison to vertebrate systems, few glycan
  structures are currently known in invertebrates
• Homologs for most the genes found in vertebrates
  have been described, but the corresponding
  activity has not been demonstrated in vitro
• Model organisms allow use of reverse genetic
  methods and gene silencing to dissect the
  importance of these genes in development
• In many cases, the developmental biologists have
  stumbled into glycobiology

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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

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Questions

• Compare and contrast what happens to the first
  N-acetylglucosamine residue attached to the
  mannosyl core of an N-glycan in Drosophila, C.
  elegans, and vertebrates

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Vertebrates
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C. elegans
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D. melanogaster
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Questions

• Compare and contrast what happens to the first
  N-acetylglucosamine residue attached to the
  mannosyl core of an N-glycan in Drosophila, C.
  elegans, and vertebrates
• Drosophila, like most invertebrates, have little
  terminal galactose on N-glycans, but instead have
  N-acetylgalactosamine in the form of LacdiNAc.
  What evolutionary change took place to bring
  about this difference?

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Questions

• How does altering the structure of N-glycans
  genetically affect development or physiology?
• If glycans have roles in almost every aspect of
  systemic physiology, why can one sometimes alter
  glycan structure without observing any obvious
  effect?

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C. elegans O-Glycans
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D. melanogaster O-Glycans
If you were an enzymologist, how would you go
about studying the synthesis of these glycans?
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Vertebrate O-GalNAc Glycans
Sialyl Tn antigen
T antigen
Tn antigen
Branched
Keratan Sulfate
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Compare the core structure of GSLs of Drosophila
with those present in C. elegans in vertebrates.
How do the outer chains differ?
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D. melanogaster Glycolipids
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Vertebrate GSLs
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Questions

• Propose some evolutionary forces driving the
  large expansion of some glycosyltransferase
  families in C. elegans (e.g., fucosyltransferases)
  compared to others (e.g. mannosyltransferases)?
• Given the absence of sialic acids in C. elegans,
  what might you predict about the types and
  specificity of glycan-binding proteins in C.
  elegans?
• Compare and contrast chondroitin proteoglycan
  synthesis in C. elegans and in vertebrates.

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Vulval Morphogenesis in C. elegans
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sqv Mutants of C. elegans

• Herman and Horvitz (1999) described a set of C.
  elegans mutants defective in vulval development
  (sqv, squashed vulva)

sqv mutations alter epithelial invagination
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cpg-1 and cpg-2 are Requiredfor Embryonic Events
WT and cpg-2(RNAi)
cpg-1/-2(RNAi)
sqv-5(RNAi)
Jon Audhya
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SQV Proteins Encode the Enzymes Required for
Chondroitin Biosynthesis
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Glycosyltransferase activities were assayed using
cell-free extracts prepared from wild type (/)
and sqv-5(n3611) heterozygotes (-/) and
homozygotes (-/-).
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C. elegans Lacks Mammalian Homologs
Mammalian CSPGs Aggrecan Versican Neurocan Decorin
Biglycan Endocan Epiphycan ?2(IX)
collagen Testicans 1-3 Leprecan Serglycin Syndecan
-1 and -3 NG2 Phosphacan Neuroglycan
C Betaglycan CD44 Thombomodulin Invariant chain
C. elegans CPGs Syndecan
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Biochemical Core Protein Purification
Purification
Crude extract
250 mg
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cpg-1 and cpg-2 are Requiredfor Embryonic Events
WT and cpg-2(RNAi)
cpg-1/-2(RNAi)
sqv-5(RNAi)
Jon Audhya
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Chondroitin Proteoglycan
Xyl
Gal
GlcA
Chondroitin is one type of glycosaminoglycan
(GAG) chain
GalNAc
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sqv
wild-type
One theory of cellular invagination is that the
adjacent epithelial cells may secrete a
chondroitin sulfate proteoglycan in a polarized
fashion Hydration of the matrix might cause
expansion and an inward curvature of the cell
layer.
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Questions

• If you were to discover a new glycan in humans,
  which model organism would you pick for further
  studies?