1. Essentials of Glycobiology Lecture 16 Genomics and Evolution Chapters 7 and 19 May 19, 2008 Pascal Gagneux

2. Questions for Lecture 16 Genomics and Evolution, Monday, May 19. 2008 1.Explain what is a sequence-based classification of glycosyltransferases. 2. Describe the ways that gene sequence predicts or fails to predict functionality in transferases, hydrolases, and glycan- binding proteins. 3. Give examples of bifunctional enzymes involved in glycosylation. Suggest the driving force for the evolution of bifunctional transferases? 4. What can you learn about the way of life of an organism (“ecology”) based on the relative number of glycosyl hydrolases and glycosyltransferase 5. How could an organism effectively augment the number of glycosyl hydrolasesand or glycosyltransferases at its disposal. 6. Do viruses entirely rely on their host cells for glycosylation?

3. Questions for Lecture 16 Genomics and Evolution, Monday, May 19. 2008 7. Discuss the concept of "glycan genes.” 8. What processes could be responsible for maintaining glycan polymorphisms (i.e., structural heterogeneity) within populations? 9. What changes in sialic acid biology occurred during human evolution? 10. Can you think of evolutionary trends in glycosylation? 11. What are the problems in using “comparative glycobiology” for determining evolutionary relationships (phylogeny)? 12. How could glycans on mammalian red blood cells protect against viral infection?

4. 16s rRNA based phylogeny Olson & Woese 1993 The universal tree of cellular life You are here Viruses

5. 10 5 10 7 10 610 10 9 10 8 10 11 Replicator (Genome) Sizes : C-values, bases in haploid genome complement MOLLUSKS BONY FISH REPTILES BIRDS BACTERIA mycoplasmaE.coli FUNGI yeast AMPHIBIANS newtfrog PLANTS beanlily MAMMALS human CARTILAGINOUS FISH shark INSECTS Drosophila Viruses

6. Genetic vocabulary: “genome, gene, allele, haplotype” Genome X Gene, Locus Allele Haplotype Exon 1 Intron 1 Exon 2Exon 3 Intron 2 Primary transcript Protein Locus 1Locus2Locus 3Locus 4 Chromosome (ADN) mRNA Chromosome 1’ Locus 1 Allele 1*01’ Allele 1*02’ Locus 1 Chromosome 1 Haplotype 1 Allele 1*02’ Locus 1 Allele2*02’ Locus 2 Allele 3*02’ Locus 3 Locus 1 Allele 1*01’ Locus 2 Allele 2*01’ Locus 3 Allele 1*01’ Haplotype 2 Glycoprotein GLYCOSYLATION Glycosyl Transferase

7. 100 million years of: Translocations, duplications, rearrangements

8. Genomics 500 genomes fully sequenced Ranging in sizes: – 450 Kb archea, 3 Gb primate, some plants and amphibians 100 Gb Number of genes: –a few hundred (Mycoplasma) to ~20 500 (H.sap). Making up ~ 1.5% of total genome. Comparative genomics: –5% of mammalian genome under evolutionary constraint. ENCODE Project Consortium for comparative mammalian genomics: –Many novel non-protein coding transscripts –Many novel transcription start sites –Regulatory regions symmetrically distributed upstream and down stream from start sites. –Many functional elements are surprisingly unconstrained. Large pool of neutral elements with biological activity –“warehouse for natural selection”? –Source of lineage-specific elements and functionally conserved but non-orthologous elements between species.

9. Genomics of Glycosylation Glycosyl transferases GT, Glycosyl hydrolases GH (glycosidases) and glycan binding proteins GBP (lectins). Prediction of function based on sequence similarity often limited. Carbohydrate Active enZymes, www.cazy.orgwww.cazy.org Listing candidate enzymes based on genomic sequence and predicted folding pattern of proteins. 5% of the vertebrate genome encoding genes involved in glycan synthesis – degradation – recognition In H. sapiens: ~250 GTs, ~250 GHs, and 100-200 GBP. Jointly comparable to the number of Kinase genes. Reduction in symbionts and parasites. But, B. thetaiotaomicron has 2.3 times more GH than humans! Increase of GT’s in plants (450in A. thaliana, 560 in rice, and 800 in poplar. Increase of GHs in fungi Some large viruses e.g. mimivirus: 12 putative GTs., Bacteriophage T4 glycosylates its DNA with Glucose.

10. http://www.cazy.org/

11. Journal of Molecular Biology Volume 328, Issue 2, 25 April 2003, Pages 307-317 An Evolving Hierarchical Family Classification for Glycosyltransferases Pedro M. Coutinho1, Emeline Deleury1, Gideon J. Davies2 and Bernard Henrissat1, Corresponding Author Contact Information B. subtilis SpsA phage T4  -glucosylT TAXONOMY OF GT ENZYMES: Two basic different topologies

12. Rates of Evolution Extremely conserved: think signalling in development Fucosyl Transferase: –Fucosylates the cell signal molecule Notch and modifies its interactions with ligands serrate and delta conserved between insects and Primates. Much less conserved: think blood groups in primates Fucosyl Transferase IV –Secretor only in primates Under strong adaptive selection: Xylosyl Transferase 1 in humans….. –Initiates GAG synthesis on proteoglycan core peptides.

13. Ortholog or Paralog ? Xie et al. Genome Biology 2003 4:R14 Speciation Duplication Or Both? Hayakawa et al. Science 2005 Partial gene conversion in Human Siglec 11

14. Genomic Evolution of Hox Gene Clusters Derek Lemons and William McGinnis Science 29 September 2006

15. Angata, Takashi 2006, MolecularDiversity10:555–566 Comparisons of the Siglec gene cluster in human, dog, and mouse

16. Genomics of Glycosylation Evolving by expanding and modifying glycan modifying tool kits: Gene numbers, gene families: Analogies from non glycan related genes: –G-coupled proteins, such as OR, >1000 loci in many mammals. –Kinases, 500 functional genes, plus many pseudogenes, many of these possibly functional. GH gene copy number variation as mechanism for dosage and functional adaptation: –Salivary amylase genes in humans: agriculture vs foraging. Polyploidy, i.e. gain of additional gene copies – (plants, fish e.g. salmon). Symbionts and contribution of their combined glycan modifying genomes.

17. Journal of Molecular Biology Volume 328, Issue 2, 25 April 2003, Pages 307-317 An Evolving Hierarchical Family Classification for Glycosyltransferases Pedro M. Coutinho1, Emeline Deleury1, Gideon J. Davies2 and Bernard Henrissat1, Corresponding Author Contact Information monocatalytic appended with non-catalytic module (A), tandem GTs on same polypeptide (B), GT with appended trans glycosidase module (C). Modularity of GTs:

20. Carbohydrate active Enzymes and total gene number in the three kingdoms

21. Bishop & Gagneux, Glycobiology, 2007 Distribution of various glycan types in nature Lineage effects in the three domains Specific: GPI anchors

23. Glycans and recognition phenomena endogenous exogenous

24. Phylogenetic distribution of Sialic acids Fungi Eukarya Archaea Common ancestor of cellular life Euryarchaeota Crenarchaeota Protozoa Proto- stomes Deutero- stomes Spirochetes Chlamydia Thermus/ Deinococcus Cyanobacteria Aquifex Gram-positive High G+C Gram-positive Low G+C Gram-negative Bacteria Angata &Varki 2002 Chem. Rev. present possibly present Plants HOSTS PATHOGENS

25. Mimicry Hyaluronan in pathogenic bacteria (Pasteurella multicoda) Polysialic acid in Neisseria meningitidis and E. coli K1. Disialylated gangliosides on LOS of H. influenzae Sialylated Siglec ligands by Group B Streptococcus Gullain Barré Syndrome: associated with central nervous system glycan bearing pathogens and resulting anti-GM1, Gd1a, GT1a, GQ1a autoantibodies: Campylobacter jejuni, cytomegalovirus, Epstein-Barr virus, Mycoplasma pneumoniae, Brucella melitensis. All these pathogens carry ganglioside-like glycans. Fucosylation of Bacteroides fragilis capsular glycans and induction of hosts gut epithelial fucose expression. freshwater snail Biomphalaria glabrata host N-glycans mimicked by helminth parasite Schistosoma mansoni. Questions: –Parasites with multiple hosts belonging to very different animal lineages face spectacular challenges in adapting to glycans in each of their hosts and vectors e.g. Plasmodium in insect and vertebrate host, Schistosomes in mollusk and mammalian hosts! –Anisakis simplex (herring worm) nematodes in marine mammals, curstaceans and herring/cod.

26. Convergent Evolution or Mimicry? Gangliosides in octopus and squid? Ganglioside-like structures in vertebrate pathogens?

27. Bishop & Gagneux, Glycobiology, 2007 Distribution of various glycan types in nature

28. Bishop & Gagneux, Glycobiology, 2007 Distribution of various glycan types in nature

29. Discrete Domains of Life? Pick your ploysaccharide: –Plants: cellulose and pectins, –Vertebrates hyaluronan, GAGs and polysialic acids, –insects chitin, –fungi chitin, –bacteria peptidoglycans and LOS Polysaccharides were likely among the first cell constituents for structural roles and biochemical properties? How did different lineages get stuck with different types?

30. Nature of constraints Internal Constraints Once a lineage has elaborated upon a set of glycan types, change may become more difficult. No radical re-design possible for living organisms! Both because of: –the integration of the glycan in important features –Irreversible loss of enzymatic machinery. (“Use it or lose it”). External Constraints The use of non-self glycan types for innate and adaptive immunity, tends to rule out the use of the same glycan types in the future.

31. Functions for Discrete Domains of Life? Viruses are classified by the type of organism they infect: –Plant viruses almost never infect animals –Bacterial viruses (phages) do not infect animals or plants –Fungal viruses semm highly specialized on fungi. Unlike bacteriophages and animal viruses, plant viruses do not seem to exploit host cell membrane surface glycans, rather plant viruses carry characteristic movement protein, which interact with plasmodesmata of plants and allow entry. Most plant viruses are non-enveloped, most animal viruses are enveloped (I.e. the latter inherit cell membrane characteristics including certain glycans from their animal host cells). Discrete glycan types as “firewalls” for horizontal infection?

32. Evolutionary trends? Galactosylceramide and its derivatives in deuterostome animals versus glucocerebrosides in protostomes. Loose to structured myelin? Increase in sialic acid content of gangliosides between reptiles, fish and mammals. Cold blooded animals express many polysialylated gangliosides in the brain. N-glycans Trends: core relatively conserved but trimming and extension is key feature of vetrebrate and plant N-glycans. GPI-anchors as eukaryotic invention?

33. Eukaryotic N-Glycan trends?

35. Ramakrishnan & Qasba J Mol Biol. 2007 365(3): 570–576. Glycan phylogenetic “watershed”? e.g. ßGalNAc-T1

36. Gagneux & Varki 1999 Glycobiology 9:747-755 Herd immunity through glycan polymorphisms?

37. Sialic Acid Sugar chains =“ Glycan ” Cell Membrane (Lipids) Protein Modified from Viitala & Järnefelt, 1985 RBC’s as viral traps? 350 X 350 Å of the Human Red Blood Cell Surface Glycophorin (Missing in some healthy humans!)

38. Non-nucleated RBC in most mammals Viral Traps – Smoke Screens – Decoys? NO NUCLEUS NO GENOME NO TRANSCRIPTION NO TRANSLATION POLYMORPHIC GLYCANS

39. Contingency and the primate hand With very few exceptions (colobus and spider monkeys), all primates have five digits on all four limbs. Analogy: Once you use sialic acid as a common terminal monosaccharide, it may be virtually impossible to abolish it. The SO4-GalNAcß1- 4GlcNAcß1- terminal unit on pituitary glycproteins, conserved throughout vertebrate evolution.

40. Gagneux & Varki 1999 Glycobiology 9:747-755 The search for the essential glycan… Are there selectively neutral glycans?

41. Roles of endogenous lectin - glycan recognition throughout life Exogenous Recognition: Infection Vaccines Allergy Microbiome Cancer Xenotransplantation Any change selected for under one process is likely to affect many unrelated processes!

42. Agenda for Research How much neutral glycan variationis there? How rapid is glycan evolution and how much time is needed for targeting innate immunity to novel non-self glycans? What is the scope of intrinsic constraints on glycan-mediated escape options from pathogens? What is the cost of a successful escape? –E.g. loss of Neu5Gc in humans? –Both in terms of functional consequences and future evolution. What are the constraints on pathogens? –A master of all trades is a master of none? Why are there not more pathogens pretending to be symbionts?

43. Current State of Glycomics: Ursus Wehrli