Homo sapiens sapiens , a recent addition to life on earth

1. Genetic DifferencesBetweenHumans and Great ApesAjit VarkiProfessor of Medicine and Cellular & Molecular Medicine Co-Director, Glycobiology Research and Training Center University of California, San DiegoLa Jolla, CA, USA

2. Modern Humans Ape/Human Common ancestor Cambrian Origin Origin Expansion of Universe of Life Origin of Primates 10 11 10 10 Homo sapiens sapiens, a recent addition to life on earth 4 5 6 7 8 9 10 10 10 10 10 10 YEARS BEFORE PRESENT

3. “Nothing in biology makes sense, except in the light of evolution”. Theodosius Dobzhansky

4. TheProbableEvolution of Life Forms on Earth

5. New World Monkeys Great Apes Old World Monkeys Rodents Lesser Apes Humans Prosimians Evolutionary Relationships of Primates and Rodents 0 10 20 Millions of Years Before Present * 30 40 50 *Precise Timing Uncertain

6. <1.0% “Great Apes” Pongo pygmaeus Gorilla gorilla Homo sapiens Pan paniscus Pan troglodytes Orangutan Gorilla Chimpanzee Human Bonobo MEAN Amino Acid Difference Evolutionary Relationships amongst Humans and the Great Apes 0 5 Millions of Years Before Present* 10 *Precise Timing Uncertain

7. Red Blood Cell White Blood Cell Platelet White Blood Cell Blood Plasma Wright-stained Normal Human Blood Smear

8. Two-Dimensional Separation of proteins The critical first step in Proteomic Analysis Total Plasma Proteins Isoelectric focusing Protein

9. Proteomic Comparison of Human and Great Ape Blood Plasma Reveals Conserved Glycosylation and a Small Numbers of Differences Gagneux et al. American Journal of Physical Anthropology 115: 99-109, 2001 MW PI

10. Human-specific Blood Plasma Protein Differences from Great Apes 1 1 2 2 • Human 1 • Chimpanzee • Gorilla 8 7 • Human 2 • Bonobo • Orangutan 1 = Haptoglobin 2 = Transthyretin Gagneux et al. American Journal of Physical Anthropology 115: 99-109, 2001

11. SOME FEATURES OF HUMANS THAT SEEM DIFFERENT FROM THE GREAT APES Brain size relative to body Relative size of neocerebellum % of brain growth complete at birth Age of closure of major fontanelles/sutures Size of cerebral venous sinuses Age of final pelvic bone fusion Rotation of the neck of the astralagus bone Bicondylar angle of the femur Presence of chin Inner ear canal orientation Foramen magnum relative to basal skull axis Adductive thumb Absence of Pharyngeal Air Sacs Descended larynx Age of first rolling over Maximum protected life span Skeletal muscle strength Bone cortex thickness Penis size relative to body length Penile baculum(penis bone) Earliest age of onset of menarche Menstrual blood loss (amount per cycle) Concealed ovulation Period of sexual receptivity (% of total cycle) Average Breast tissue mass in virgin adult female Increase in breast mass during lactation Modal Chromosome Number Neonatal Cephalhematoma/caput at birth Menopause Presence of Ear Lobes Presence of canine tooth diastema Thickness of tooth enamel Age of first molar eruption Presence of retromolar gap Frequency of Third Molar Impaction Body hair Eccrine/Apocrine sweat gland ratio Dermal fat thickness (relative to total skin) Relative size of frontal air sinuses Postnatal % body water change

12. Are we really a “Third Chimpanzee?” Analysis of 20 structural and functional features of hominoids (C.Wills & A.Varki) presented IN “Children of Prometheus” By Chris Wills

13. A-Z Examples of Human Uniqueness Exercise: Take a dictionary and check entries under each letter from the top. Stop when you reach the first one you think is unique to humans Abbreviating Bag-making Calculus Darts Ear-piercing Face-lifting Gambling Hacking(computers) Illustrating Jet-sking Karate Lacrosse Machining Nailing(wood) Operating (Surgery) Panning for gold Quilting Racing (organized) Sacrificing (others) Tagging (systematic marking) Umpiring Vacationing Wage-earning Xeroxing Yachting Zeroing

14. Examples of Human Uniqueness starting with the letter S Exercise: Take a dictionary and scan all entries under the letter S. Record all the ones that you think are unique to humans. Sacrificing Sack-making Saddling Sailing Salt-making Saluting Sand-castle building Sandwich-making Sawing (wood) Saxaphone playing Schedule making Schooling Science Scoring (points) Scuba-diving Sculling Sculpting Sealing (wax) Selling Semen banking Serum collecting Sewing Shampooing Shaving Ship-building Shooting Shopping Signatures Silver (trading etc.) Singing (e.g., opera) Skating Sketching Skiing Skinning Skipping Sky-diving Slang words Slavery Sledding Sleighing Slimming Smeltering Snowball fighting Snuff-taking Soaping Sobbing Soccer Soliciting Sowing (seeds) Soup making Spaying Spear-throwing Spelunking Spice collection Spending _____________ _____________________ _____________________ _____________________ _____________________ Steel production Stitching Story-telling Sun-tanning Surfing

15. ? ? DNA ORGANISM DNA RNA PROTEIN CELL ORGANISM

16. DNA ORGANISM PHYSICAL ENVIRONMENT

17. DNA DNA BIOLOGICAL ENVIRONMENT Microbes ORGANISM PHYSICAL ENVIRONMENT

18. DNA DNA DNA BIOLOGICAL ENVIRONMENT CULTURAL ENVIRONMENT ORGANISM PHYSICAL ENVIRONMENT

19. DNA DNA DNA DNA BIOLOGICAL ENVIRONMENT CULTURAL ENVIRONMENT ORGANISM HUMANS HUMANS PHYSICAL ENVIRONMENT

20. ENZYMES SUGARS TRANSCRIPTION FACTORS LIPIDS Glycobiology: the study of the structure, biosynthesis and biology of saccharides (sugar chains or “glycans”) in nature. DIET SIGNALLING MOLECULES GLYCOPROTEINS PROTEOGLYCANS GLYCOLIPIDS MICROBES PARASITES CELL MATRIX PHYSICAL ENVIRONMENT TISSUES & ORGANS DNA RNA PROTEINS DNA ORGANISM DNA

21. Red Blood Cell Wright-stained Normal Human Blood Smear

22. Red Blood Cell Membrane Scanning Electron Micrograph of Human Red Blood Cells

23. Scale Model of 1/100,000 of Human Red Blood Cell Surface Sugar chains (Oligosaccharides) (Glycans) Cell Membrane (Lipids) Sialic Acid Neutral Sugars Protein Modified from Viitala & Järnefelt

24. Influenza Malaria Cholera Helicobacter Mycoplasma Rotavirus Polyoma virus Coronavirus Pertussis Tetanus etc. Siglecs Factor H Selectins Uterine Agglutinin Laminins Endogenous Recognition “Self” Exogenous Recognition “Non-self” Molecular Mimicry EXOGENOUS RECEPTOR ENDOGENOUS RECEPTOR E.Coli Gonococcus Meningococcus Campylobacter Trypanosoma Streptococcus Etc. M SELF M = Micro-organism or Toxin Biological Roles of Sialic Acids Structural/Physical Roles SELF =SIALIC ACID CARRYING SUGAR CHAIN

25. (1984) UCSD Medical CenterSarah Anna Varki HEMATOLOGY CONSULT Case: A 22-year old woman with weakness and bleeding. Diagnosis: Aplastic Anemia (bone marrow failure) Treatment: Trial of Equine Anti-Thymocyte Globulin (Horse Serum) Complication: “Serum Sickness Reaction” to Horse Serum Immune Reaction partly against Sialic Acids in Horse Serum!

26. Two major kinds of Sialic Acids in Mammals: Neu5Ac and Neu5Gc Neu5Ac Neu5Gc Great Ape or Other Mammal Human Human Cells are Unusual: Excess of Neu5Ac and Loss of Neu5Gc

27. Sialic acids on Red Cells and Plasma Proteins from Humans and Great Apes Muchmore, E.A., Diaz, S. & Varki, A.: Amer.J.Physical Anthropology 107:187-198,1998 (Great Ape samples from Yerkes Primate Center, Atlanta, GA) Neu5Ac Neu5GcHumans ++++ -Great Apes ++ ++ The Human Loss of Neu5Gc Expression • HOW did it happen? • WHY did it happen? • WHEN did it happen? • WHAT are the consequences for risk of infections in humans? • WHAT are the consequences for human sialic acid receptors? • HOW does Neu5Gc re-expression in tumors and fetuses occur? • WHERE does the small amount of Neu5Gc in humans come from? • WHAT are the consequences of human ingestion of Neu5Gc in food? • WHAT are the consequences for biotechnology products? • WHAT are the consequences for attempts at xenotransplantation? • WHAT are the consequences for the human brain?

28. <1.0% “Great Apes” Neu5Ac Neu5Gc ~0.5% Pongo pygmaeus Gorilla gorilla Homo sapiens Pan paniscus Pan troglodytes Orangutan Gorilla Chimpanzee Human Bonobo Genetic Mutation Causing loss Of Neu5Gc MEAN Amino Acid Difference Evolutionary Relationships amongst Humans and the Great Apes 0 5 Millions of Years Before Present* 10 *Precise Timing Uncertain

29. Neu5Ac Neu5GcHumans ++++ -Great Apes ++ ++ The Human Loss of Neu5Gc Expression • HOW did it happen? • WHY did it happen? • WHEN did it happen? • WHAT are the consequences for risk of infections in humans? • WHAT are the consequences for human sialic acid receptors? • HOW does Neu5Gc re-expression in tumors and fetuses occur? • WHERE does the small amount of Neu5Gc in humans come from? • WHAT are the consequences of human ingestion of Neu5Gc in food? • WHAT are the consequences for biotechnology products? • WHAT are the consequences for attempts at xenotransplantation? • WHAT are the consequences for the human brain?

30. “Intelligent Design” “Optimal Design” Biologists EVOLUTIONISTS MANY BIOLOGISTS ASSUME THAT EVOLUTION USUALLY RESULTS IN OPTIMAL DESIGN CREATIONISTS

31. Human-Specific Loss of Neu5Gc (CMAH mutation) • Random mutation that drifted to fixation due to small effective population size? • Selection due to Neu5Gc-recognizing pathogen(s)? • Signature of other past “selective sweep” affecting hominid ancestors? Regardless of the cause, what were the consequences for human evolution?

32. Neu5Ac Neu5GcHumans ++++ -Great Apes ++ ++ The Human Loss of Neu5Gc Expression • HOW did it happen? • WHY did it happen? • WHEN did it happen? • WHAT are the consequences for risk of infections in humans? • WHAT are the consequences for human sialic acid receptors? • HOW does Neu5Gc re-expression in tumors and fetuses occur? • WHERE does the small amount of Neu5Gc in humans come from? • WHAT are the consequences of human ingestion of Neu5Gc in food? • WHAT are the consequences for biotechnology products? • WHAT are the consequences for attempts at xenotransplantation? • WHAT are the consequences for the human brain? CMAH Mutation found in Homozygous State in all Human Populations studied

33. Modern Humans - a very recent “success story”

34. Modern Humans - a very recent “success story”

36. Neandertals Us Emergence of Moderns Brain size Brain size Stone tools Hunting Meat Eating Loss of Neu5Gc Bipedal Posture Collaboration with Meave Leakey, Svante Paabo, Yuki Takahata Modified from: Bernard Wood, Nature, 418:133-5, 2002 Java Man “Lucy”

37. Neu5Ac Neu5GcHumans ++++ -Great Apes ++ ++ The Human Loss of Neu5Gc Expression • HOW did it happen? • WHY did it happen? • WHEN did it happen? • WHAT are the consequences for risk of infections in humans? • WHAT are the consequences for human sialic acid receptors? • HOW does Neu5Gc re-expression in tumors and fetuses occur? • WHERE does the small amount of Neu5Gc in humans come from? • WHAT are the consequences of human ingestion of Neu5Gc in food? • WHAT are the consequences for biotechnology products? • WHAT are the consequences for attempts at xenotransplantation? • WHAT are the consequences for the human brain?

38. Influenza Malaria Cholera Helicobacter Mycoplasma Rotavirus Polyoma virus Coronavirus Pertussis Tetanus etc. Siglecs Factor H Selectins Uterine Agglutinin Laminins Endogenous Recognition “Self” Exogenous Recognition “Non-self” Molecular Mimicry EXOGENOUS RECEPTOR ENDOGENOUS RECEPTOR E.Coli Gonococcus Meningococcus Campylobacter Trypanosoma Streptococcus Etc. M SELF M = Micro-organism or Toxin Biological Roles of Sialic Acids Structural/Physical Roles SELF SIALYLATED OLIGOSACCHARIDE =

39. Neu5Ac Neu5GcHumans ++++ -Great Apes ++ ++ The Human Loss of Neu5Gc Expression • HOW did it happen? • WHY did it happen? • WHEN did it happen? • WHAT are the consequences for risk of infections in humans? • WHAT are the consequences for human sialic acid receptors? • HOW does Neu5Gc re-expression in tumors and fetuses occur? • WHERE does the small amount of Neu5Gc in humans come from? • WHAT are the consequences of human ingestion of Neu5Gc in food? • WHAT are the consequences for biotechnology products? • WHAT are the consequences for attempts at xenotransplantation? • WHAT are the consequences for the human brain?

40. Influenza Malaria Cholera Helicobacter Mycoplasma Rotavirus Polyoma virus Coronavirus Pertussis Tetanus etc. Siglecs Factor H Selectins Uterine Agglutinin Laminins Endogenous Recognition “Self” Exogenous Recognition “Non-self” Molecular Mimicry EXOGENOUS RECEPTOR ENDOGENOUS RECEPTOR E.Coli Gonococcus Meningococcus Campylobacter Trypanosoma Streptococcus Etc. M SELF M = Micro-organism or Toxin Biological Roles of Sialic Acids Structural/Physical Roles SELF SIALYLATED OLIGOSACCHARIDE =

41. Neu5Ac Neu5GcHumans ++++ -Great Apes ++ ++ The Human Loss of Neu5Gc Expression • HOW did it happen? • WHY did it happen? • WHEN did it happen? • WHAT are the consequences for risk of infections in humans? • WHAT are the consequences for human sialic acid receptors? • HOW does Neu5Gc re-expression in tumors and fetuses occur? • WHERE does the small amount of Neu5Gc in humans come from? • WHAT are the consequences of human ingestion of Neu5Gc in food? • WHAT are the consequences for biotechnology products? • WHAT are the consequences for attempts at xenotransplantation? • WHAT are the consequences for the human brain?

42. Neu5Ac Neu5GcHumans ++++ traceGreat Apes ++ ++ The Human Loss of Neu5Gc Expression • HOW did it happen? • WHY did it happen? • WHEN did it happen? • WHAT are the consequences for risk of infections in humans? • WHAT are the consequences for human sialic acid receptors? • HOW does Neu5Gc re-expression in tumors and fetuses occur? • WHERE does the small amount of Neu5Gc in humans come from? • WHAT are the consequences of human ingestion of Neu5Gc in food? • WHAT are the consequences for biotechnology products? • WHAT are the consequences for attempts at xenotransplantation? • WHAT are the consequences for the human brain?

43. Uptake and Excretion of Neu5Gc in Normal Humans Intensity Detection by DMB-HPLC and Mass Spectrometry Elaine Muchmore Sandra Diaz Pascal Gagneux Tangvoranuntakul et al. Proc.Nat'l.Acad.Sci.U.S.A. 100:12045-12050, 2003

44. Fruits, vegetables and grains - none? (no sialic acids) Chicken and Turkey - traces Fish - variable, but very low Bovine milk and milk products - low Lamb, Pork and Beef - high Distribution of Neu5Gc in Common Foods Sandra Diaz Pascal Gagneux Tangvoranuntakul et al. Proc.Nat'l.Acad.Sci.U.S.A. 100:12045-12050, 2003

45. Detection of Anti-Neu5Gc antibodies in Normal Human Sera Pam Tang Pascal Gagneux A492 Frequency much higher than previously reportedLikely due to improved assay with lower background and better negative controlsHigher values are in range for anti-alpha-Gal antibodies Tangvoranuntakul et al. Proc.Nat'l.Acad.Sci.U.S.A. 100:12045-12050, 2003

46. Neu5Ac Neu5GcHumans ++++ -Great Apes ++ ++ The Human Loss of Neu5Gc Expression • HOW did it happen? • WHY did it happen? • WHEN did it happen? • WHAT are the consequences for risk of infections in humans? • WHAT are the consequences for human sialic acid receptors? • HOW does Neu5Gc re-expression in tumors and fetuses occur? • WHERE does the small amount of Neu5Gc in humans come from? • WHAT are the consequences of human ingestion of Neu5Gc in food? • WHAT are the consequences for biotechnology products? • WHAT are the consequences for attempts at xenotransplantation? • WHAT are the consequences for the human brain?

47. Neu5Ac Neu5GcHumans ++++ tracesGreat Apes ++ ++ The Human Loss of Neu5Gc Expression • HOW did it happen? • WHY did it happen? • WHEN did it happen? • WHAT are the consequences for risk of infections in humans? • WHAT are the consequences for human sialic acid receptors? • HOW does Neu5Gc re-expression in tumors and fetuses occur? • WHERE does the small amount of Neu5Gc in humans come from? • WHAT are the consequences of human ingestion of Neu5Gc in food? • WHAT are the consequences for biotechnology products? • WHAT are the consequences for attempts at xenotransplantation? • WHAT are the consequences for the human brain?

48. Neu5GC expression No Neu5GC expression Even Mammals With High Levels of Neu5Gc in Non-neural Tissues, it is Difficult to Detect Neu5Gc in the Brain Pascal Gagneux CMAH gene expression is down-regulated in mammalian brains

49. Multiple Human-Specific Changes in Sialic Acid Biology - What do they mean? • Random mutations that have drifted to fixation due to small effective population size? Unlikely, too many changes. • Selection due to Neu5Gc-recognizing pathogen(s)? • Signature of a past selective sweep affecting hominid ancestors? Likely - cause or consequence? • What are consequences for humans today? • Innate immune system? • Susceptibility/resistance to pathogens? • Brain development? • Dietary ingestion of Neu5Gc? • Biotechnology and Xenotransplantation?

50. Susceptibility to Neu5Ac-binding pathogens? Increased Expression of Neu5Ac Increased Ligands for Siglec-1 (Sialoadhesin) Loss of Neu5Gc Expression Resistance to Neu5Gc-requiring pathogens? Altered Distribution of Siglec-1-positive Tissue Macrophages No Neu5Gc in Brain Loss of Sialic Acid Recognition by Siglec-L1 (Neu5Gc preferred by chimp) ? Dietary Neu5Gc as a foreign sugar Sudden Unmasking of CD33-related Siglecs Universal Expression of Siglec-1 in Tissue Macrophages Traces of Neu5Gc in human tissues Epithelial excretion? Multiple mutations in Sialic Acid-binding domains of CD33-related Siglecs Natural Anti- Neu5Gc Antibodies Human-specific Expression of Siglec-6 in Placenta (promoter mutation?) CD33-related Siglec binding specificity relaxed to include Neu5Ac Effects on timing of labor and birth? Genomic mutation in CMP-Neu5Ac Hydroxylase (CMAH) Difference: Probable Possible Definite Multiple Differences in Sialic Acid Biology Between Humans & Great Apes Homozygosity of CMAH mutation Point mutation Eliminating critical arginine residue in Siglec-L1 Altered regulation of ST6Gal-I gene expression (promoter mutation?) Human expression of a2-6-linked Sias on bronchial epithelium and certain other cell types and loss of expression on goblet cells Susceptibility to Human Influenza Strains