Molecular and functional evolution of hemoglobin in perissodactyl mammals (equids, tapirs, and rhinos) by Margaret Clapin A thesis submitted to the Faculty of Graduate Studies of the University of Manitoba in partial fulfillment of the requirements for the degree of Master of Science Department of Biological Sciences University of Manitoba Winnipeg, Manitoba Canada © 2019 Abstract: In this thesis, the oxygen binding characteristics of recombinant hemoglobin (Hb) isoforms (HbA [α2β2] and HbA2 [α2δ2]) from the extinct woolly rhinoceros (Coelodonta antiquitatis) are compared with Sumatran rhino (Dicerorhinus sumatrensis) and black rhino (Diceros bicornis) Hb. The major Hb component (HbA) of horses (Equus caballus) was also examined, as its blood O2 affinity has a low thermal sensitivity. This trait is commonly associated with cold-adaptation as it permits O2 to be offloaded at the cool peripheral tissues of regionally endothermic mammals, though the mechanism(s) by which the oxygenation enthalpy is reduced in horse Hb is unknown. It was hypothesized that the woolly rhino Hb isoforms would have similarly low thermal sensitivities to that of horses, either through enhanced effector binding or by altering the energetic transition from the tense to the relaxed state of hemoglobin. To test this hypothesis the hemoglobin coding sequences for each of the above species were determined and their Hb isoforms expressed using E. coli and purified. Oxygen equilibrium curves were then determined in the presence and absence of allosteric effectors at 25 and 37°C. Horse HbA had a low sensitivity to 2,3- diphosphoglycerate (DPG), though its low temperature sensitivity was primarily driven by increased DPG binding at the lower test temperature. By contrast, each of the extant rhino Hb isoforms was shown to be largely insensitive to DPG, and to have a lower sensitivity to Cl- than horse Hb; notably, effector binding wasn’t temperature sensitive. My results further demonstrated that the major HBA woolly rhino isoform possessed nearly identical inherent O2 affinities, allosteric effector sensitivities, and thermal sensitivities to those of the extant rhinoceros species. Thus, the extinct woolly rhino did not evolve cold-adapted hemoglobin which argues against strong regional heterothermy in this species. The woolly rhino evolved a rare residue replacement at a highly conserved position of the δ globin chain (δ104Arg→Ser) which abolishes DPG binding ii - to woolly rhino HbA2 while also dramatically reducing the effects of both pH and Cl on Hb–O2 affinity. It is postulated this seemingly maladaptive trait likely became fixed during a population bottleneck in this species. iii Acknowledgements: I would like to thank my advisor Dr. Kevin Campbell for his support through this entire process including the development of this topic, advise on the methods used and revisions. I would also like to thank my committee, Dr. Jennifer van Wijngaarden and Dr. Kenneth Jeffries, for their helpful suggestions and revisions. Lastly, I would like to thank Anthony Signore, Michael Gaudry, and Diana Hanna for their instruction on the protocols used throughout the research. iv Table of Contents: Abstract: .............................................................................................................................. ii Acknowledgements: ........................................................................................................... iv List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii List of Abbreviations: ......................................................................................................... x Chapter 1: General Introduction ......................................................................................... 1 Chapter 2: Temperature dependent DPG binding underlies thermal adaptation of horse hemoglobin ......................................................................................................................... 8 2.1 Abstract ......................................................................................................................... 8 2.2 Introduction ................................................................................................................... 9 2.3 Materials and Methods ................................................................................................ 11 2.3.1 Horse Hb gene inserts .............................................................................................. 11 2.3.2 Hemoglobin expression ........................................................................................... 12 2.3.3 Hemoglobin purification .......................................................................................... 15 2.3.4 Oxygen binding tests................................................................................................ 15 2.4 Results ......................................................................................................................... 16 2.5 Discussion ................................................................................................................... 23 2.6 Conclusions ................................................................................................................. 26 Chapter 3: Functional characterization of hemoglobin from the rhinoceros clade reveals a lack of temperature adaptation in the extinct woolly rhino. ......................................................... 27 3.1 Abstract ....................................................................................................................... 27 3.2 Introduction ................................................................................................................. 28 3.3 Materials and Methods ................................................................................................ 30 3.3.1 Assembly and annotation ......................................................................................... 31 3.3.2 Hemoglobin expression ........................................................................................... 35 3.3.3 Hemoglobin purification .......................................................................................... 35 3.3.4 Oxygen binding tests................................................................................................ 35 3.4 Results ......................................................................................................................... 36 3.5 Discussion ................................................................................................................... 49 3.6 Conclusions ................................................................................................................. 57 Literature Cited ................................................................................................................. 59 v Appendices ........................................................................................................................ 68 vi List of Tables Table 2.1 PCR protocols used for filling in Hb gene gaps, Sanger sequencing, amplification of gene inserts, and colony screening………………………………………………………………..13 Table 2.2 Heterotropic binding characteristics of horse HbA at 37℃ and 25℃ and at pH 7.2....21 Table 2.3 Effect of temperature on oxygen binding in horse HbA……………………………...22 Table 3.1 Woolly rhino specimen information including lab ID, museum/sample ID, material, 14C date, latitude, and longitude………………………………………………………………………32 Table 3.2 Oxygen affinity and heterotropic effects of woolly, Sumatran, and black rhino major hemoglobin isoforms at 37 and 25℃ at pH 7.2. The oxygen affinity and Bohr effect of each isoform was measured under four conditions; stripped of cofactors, 0.1 M Cl-, 0.5 mM DPG, and 0.1 M Cl- + 0.5 mM DPG………………………………………………………………………...46 Table 3.3 Oxygen affinity and heterotropic effects of woolly and Sumatran minor hemoglobin isoforms at 37 and 25℃ at pH 7.2; data for a woolly rhino 104Ser→Arg mutant are also presented. The oxygen affinity and Bohr effect of each isoform was measured under four conditions; stripped of cofactors, 0.1 M Cl-, 0.5 mM DPG, and 0.1 M Cl- + 0.5 mM DPG……….47 Table 3.4 Enthalpy of oxygen binding of woolly rhinoceros, Sumatran rhinoceros, and black rhinoceros Hb isoforms at pH 7.2. Enthalpies were calculated under four conditions; stripped of cofactors, 0.1 M Cl-, 0.5 mM DPG, and 0.1 M Cl- + 0.5 mM DPG………………………………..48 vii List of Figures Figure 1.1 Phylogenetic tree of Perissodactyla (modified from Orlando et al. 2003, Gaudry 2017)………………………………………………………………………………………………6 Figure 2.1 Oxygen binding characteristics of 0.25 mM horse hemoglobin at 25℃ (closed symbols) and 37℃ (open symbols) as a function of pH. Data is presented for stripped hemoglobin (circles), and hemoglobin in the presence of 0.1 M chloride (squares), 0.5 mM DPG (triangles), and in the combined presence of these effectors (diamonds). Corresponding cooperativity coefficients (n50) are also presented for each experimental condition………………………………………………17 Figure 2.2 Half saturation O2 partial pressures (log P50) of horse HbA plotted over a DPG concentration gradient at 25℃ (closed symbols) and 37℃ (open symbols) and pH 7.2. Concentrations are plotted as a log ratio of DPG/Hb. Arrows denote the log