Essentials of Glycobiology Lecture 16

Genomics and Evolution Chapters 7 and 19

May 19, 2008

Pascal Gagneux

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?

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?

16s rRNA based phylogenyOlson & Woese 1993

The universal tree of cellular life

You are here

Viruses

105 107106 1010109108 1011

Replicator (Genome) Sizes:C-values, bases in haploid genome complement

MOLLUSKS

BONY FISH

REPTILES

BIRDS

BACTERIA

mycoplasma E.coli

FUNGI

yeast

AMPHIBIANS

newtfrog

PLANTS

bean lily

MAMMALS

human

CARTILAGINOUS FISHshark

INSECTSDrosophila

Viruses

Genetic vocabulary:“genome, gene, allele, haplotype”

• Genome X

• Gene, Locus

• Allele

• Haplotype Exon 1

Intron 1

Exon 2 Exon 3

Intron 2

Primary transcript

Protein

Locus 1 Locus2 Locus 3 Locus 4

Chromosome(ADN)

mRNA

Chromosome 1’

Locus 1Allele 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 1Allele 1*01’

Locus 2Allele 2*01’

Locus 3Allele 1*01’

Haplotype 2

Glycoprotein

GLYCOSYLATION

Glycosyl Transferase

100 million years of:Translocations, duplications, rearrangements

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.

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.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.

http://www.cazy.org/

http://www.cazy.org/

Journal of Molecular BiologyVolume 328, Issue 2, 25 April 2003, Pages 307-317 An Evolving Hierarchical Family Classification for GlycosyltransferasesPedro 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

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.

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

Genomic Evolution of Hox Gene Clusters

Derek Lemons and William McGinnis

Science 29 September 2006

Angata, Takashi 2006, MolecularDiversity10:555–566

Comparisons of the Siglec gene cluster in human, dog, and mouse

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.

Journal of Molecular BiologyVolume 328, Issue 2, 25 April 2003, Pages 307-317 An Evolving Hierarchical Family Classification for GlycosyltransferasesPedro 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:

Carbohydrate active Enzymes and total gene numberin the three kingdoms

Bishop & Gagneux, Glycobiology, 2007

Distribution of various glycan types in nature

Lineage effects in the three domains

Specific: GPI anchors

Glycans and recognition phenomena

endogenous

exogenous

Phylogenetic distribution of Sialic acids

Fungi

Eukarya

Archaea

Common ancestor of cellular life

EuryarchaeotaCrenarchaeota

Protozoa

Proto-stomes

Deutero-stomes

Spirochetes

Chlamydia

Thermus/Deinococcus

Cyanobacteria

Aquifex

Gram-positive

High G+C

Gram-positiveLow G+C

Gram-negative

Bacteria

Angata &Varki 2002 Chem. Rev.presentpossibly present

PlantsHOSTS

PATHOGENS

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.

Convergent Evolution or Mimicry?

• Gangliosides in octopus and squid?

• Ganglioside-like structures in vertebrate pathogens?

Bishop & Gagneux, Glycobiology, 2007

Distribution of various glycan types in nature

Bishop & Gagneux, Glycobiology, 2007

Distribution of various glycan types in nature

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?

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.

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?

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?

Eukaryotic N-Glycan trends?

Ramakrishnan & Qasba

J Mol Biol. 2007 365(3): 570–576.

Glycan phylogenetic “watershed”?e.g. ßGalNAc-T1

Gagneux & Varki 1999 Glycobiology 9:747-755

Herd immunity through glycan polymorphisms?

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!)

Non-nucleated RBC in most mammalsViral Traps – Smoke Screens – Decoys?

NO NUCLEUSNO GENOMENO TRANSCRIPTIONNO TRANSLATION

POLYMORPHIC GLYCANS

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.

Gagneux & Varki 1999 Glycobiology 9:747-755

The search for the essential glycan…Are there selectively neutral glycans?

Roles of endogenous lectin - glycan recognition throughout life

Exogenous Recognition:InfectionVaccinesAllergy

MicrobiomeCancer

Xenotransplantation

Any change selected for under one process is likely to affect many unrelated processes!

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?

Current State of Glycomics:

Ursus Wehrli