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The importance of a single amino acid substitution in reduced red blood cell carbonic anhydrase function of early-diverging fish

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Abstract

In most vertebrates, red blood cell carbonic anhydrase (RBC CA) plays a critical role in carbon dioxide (CO2) transport and excretion across epithelial tissues. Many early-diverging fishes (e.g., hagfish and chondrichthyans) are unique in possessing plasma-accessible membrane-bound CA-IV in the gills, allowing some CO2 excretion to occur without involvement from the RBCs. However, implications of this on RBC CA function are unclear. Through homology cloning techniques, we identified the putative protein sequences for RBC CA from nine early-diverging species. In all cases, these sequences contained a modification of the proton shuttle residue His-64, and activity measurements from three early-diverging fish demonstrated significantly reduced CA activity. Site-directed mutagenesis was used to restore the His-64 proton shuttle, which significantly increased RBC CA activity, clearly illustrating the functional significance of His-64 in fish red blood cell CA activity. Bayesian analyses of 55 vertebrate cytoplasmic CA isozymes suggested that independent evolutionary events led to the modification of His-64 and thus reduced CA activity in hagfish and chondrichthyans. Additionally, in early-diverging fish that possess branchial CA-IV, there is an absence of His-64 in RBC CAs and the absence of the Root effect [where a reduction in pH reduces hemoglobin’s capacity to bind with oxygen (O2)]. Taken together, these data indicate that low-activity RBC CA may be present in all fish with branchial CA-IV, and that the high-activity RBC CA seen in most teleosts may have evolved in conjunction with enhanced hemoglobin pH sensitivity.

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References

  • Alderman SL, Harter TS, Wilson JM, Supuran CT, Farrell AP, Brauner CJ (2016) Evidence for a plasma-accessible carbonic anhydrase in the lumen of salmon heart that may enhance oxygen delivery to the myocardium. J Exp Biol 219:719–724

    PubMed  Google Scholar 

  • Berenbrink M (2006) Evolution of vertebrate haemoglobins: histidine side chains, specific buffer value and Bohr effect. Resp Physiol Neurobiol 154:165–184

    CAS  Google Scholar 

  • Berenbrink M, Bridges C (1994a) Active Na+–, Cl–and HCO3–dependent acid extrusion in Atlantic cod red blood cells in winter activated by hypercapnia. J Exp Biol 192:239–252

    CAS  PubMed  Google Scholar 

  • Berenbrink M, Bridges C (1994b) Catecholamine-activated sodium/proton exchange in the red blood cells of the marine teleost Gadus morhua. J Exp Biol 192:253–267

    CAS  PubMed  Google Scholar 

  • Berenbrink M, Koldkjær P, Kepp O, Cossins AR (2005) Evolution of oxygen secretion in fishes and the emergence of a complex physiological system. Science 307:1752–1757

    CAS  PubMed  Google Scholar 

  • Bohr C, Hasselbalch K, Krogh A (1904) Über einen in biologischer Beziehung wichtigen Einfluss, den die Kohlensäurespannung des Blutes auf dessen Sauerstoffbindung übt. Acta Physiol 16:402–412

    Google Scholar 

  • Brauner C, Berenbrink M (2007) Gas transport and exchange. Fish physiology, vol 26. Academic Press, Cambridge, pp 213–282

    Google Scholar 

  • Brauner C, Randall D (1998) The linkage between oxygen and carbon dioxide transport. Fish physiology, vol 17. Elsevier, Amsterdam, pp 283–319

    Google Scholar 

  • Brittain T (2005) Root effect hemoglobins. J Inorg Biochem 99:120–129

    CAS  PubMed  Google Scholar 

  • Chegwidden WR, Carter ND (2000) Introduction to the carbonic anhydrases. The carbonic anhydrases. Birkhäuser, Basel, pp 13–28

    Google Scholar 

  • Christianson DW, Fierke CA (1996) Carbonic anhydrase: evolution of the zinc binding site by nature and by design. Accounts Chem Res 29:331–339

    CAS  Google Scholar 

  • Clifford AM, Weinrauch AM, Edwards SL, Wilkie MP, Goss GG (2017) Flexible ammonia handling strategies using both cutaneous and branchial epithelia in the highly ammonia-tolerant Pacific hagfish. Am J Physiol Reg I 313:R78–R90

    Google Scholar 

  • Ellory J, Wolowyk M, Young J (1987) Hagfish (Eptatretus stouti) erythrocytes show minimal chloride transport activity. J Exp Biol 129:377–383

    CAS  PubMed  Google Scholar 

  • Esbaugh A, Tufts B (2006a) The structure and function of carbonic anhydrase isozymes in the respiratory system of vertebrates. Resp Physiol Neurobiol 154:185–198

    CAS  Google Scholar 

  • Esbaugh AJ, Tufts BL (2006b) Tribute to R. G. Boutilier: evidence of a high activity carbonic anhydrase isozyme in the red blood cells of an ancient vertebrate, the sea lamprey Petromyzon marinus. J Exp Biol 209:1169–1178

    CAS  PubMed  Google Scholar 

  • Esbaugh AJ, Lund SG, Tufts BL (2004) Comparative physiology and molecular analysis of carbonic anhydrase from the red blood cells of teleost fish. J Comp Physiol B 174:429–438

    CAS  PubMed  Google Scholar 

  • Esbaugh AJ, Gilmour K, Perry S (2009) Membrane-associated carbonic anhydrase in the respiratory system of the Pacific hagfish (Eptatretus stouti). Resp Physiol Neurobiol 166:107–116

    CAS  Google Scholar 

  • Esbaugh AJ, Secor SM, Grosell M (2015) Characterization of carbonic anhydrase XIII in the erythrocytes of the Burmese python, Python molurus bivittatus. Comp Biochem Phys B 187:71–77

    CAS  Google Scholar 

  • Ferguson R, Sehdev N, Bagatto B, Tufts B (1992) In vitro interactions between oxygen and carbon dioxide transport in the blood of the sea lamprey (Petromyzon marinus). J Exp Biol 173:25–41

    CAS  Google Scholar 

  • Ferreira-Martins D, McCormick S, Campos A, Lopes-Marques M, Osório H, Castro LFC, Wilson JM (2016) A cytosolic carbonic anhydrase molecular switch occurs in the gills of metamorphic sea lamprey. Sci Rep 6:339–354

    Google Scholar 

  • Forster R, Steen J (1968) Rate limiting processes in the Bohr shift in human red cells. J Physiol 196:541–562

    CAS  PubMed  PubMed Central  Google Scholar 

  • Geers C, Gros G (2000) Carbon dioxide transport and carbonic anhydrase in blood and muscle. Physiol Rev 80:681–715

    CAS  PubMed  Google Scholar 

  • Giardina B, Mosca D, De Rosa M (2004) The Bohr effect of haemoglobin in vertebrates: an example of molecular adaptation to different physiological requirements. Acta Physiol 182:229–244

    CAS  Google Scholar 

  • Gilmour K (2010) Perspectives on carbonic anhydrase. Comp Biochem Phys A 157:193–197

    CAS  Google Scholar 

  • Gilmour K, Perry S (2009) Carbonic anhydrase and acid–base regulation in fish. J Exp Biol 212:1647–1661

    CAS  PubMed  Google Scholar 

  • Gilmour KM, Henry RP, Wood CM, Perry SF (1997) Extracellular carbonic anhydrase and an acid-base disequilibrium in the blood of the dogfish, Squalus acanthias. J Exp Biol 200:173–183

    CAS  PubMed  Google Scholar 

  • Gilmour K, Perry S, Bernier N, Henry R, Wood C (2001) Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion. Physiol Biochem Zool 74:477–492

    CAS  PubMed  Google Scholar 

  • Gilmour K, Bayaa M, Kenney L, Mcneill B, Perry S (2007) Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias). Am J Physiol-Reg I 292:R556–R567

    CAS  Google Scholar 

  • Gilmour KM, Perry SF (2004) Branchial membrane-associated carbonic anhydrase activity maintains CO2 excretion in severely anemic dogfish. Am J Physiol-Reg I 286:R1138–R1148

    CAS  Google Scholar 

  • Gilmour KM, Shah B, Szebedinszky C (2002) An investigation of carbonic anhydrase activity in the gills and blood plasma of brown bullhead (Ameiurus nebulosus), longnose skate (Raja rhina), and spotted raffish (Hydrolagus colliei). J Comp Physiol B 172:77–86

    CAS  PubMed  Google Scholar 

  • Harter TS, Brauner CJ (2017) The O2 and CO2 transport system in teleosts and the specialized mechanisms that enhance Hb–O2 unloading to tissues. Fish physiolpgy, vol 36. Academic Press, Cambridge, pp 1–106

    Google Scholar 

  • Harter TS, Zanuzzo FS, Supuran CT, Gamperl AK, Brauner CJ (2019) Functional support for a novel mechanism that enhances tissue oxygen extraction in a teleost fish. In: Proceedings of the Royal Society B, p 286

  • Henry RP (1991) Techniques for measuring carbonic anhydrase activity in vitro: the electrometric delta pH and pH statmethods. The carbonic anhydrases. Springer, New York, pp 119–125

    Google Scholar 

  • Henry RP, Swenson ER (2000) The distribution and physiological significance of carbonic anhydrase in vertebrate gas exchange organs. Resp Physiol 121:1–12

    CAS  Google Scholar 

  • Henry RP, Tufts BL, Boutilier RG (1993) The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate exchange. J Comp Physiol B 163:380–388

    CAS  Google Scholar 

  • Henry RP, Gilmour KM, Wood CM, Perry SF (1997) Extracellular carbonic anhydrase activity and carbonic anhydrase inhibitors in the circulatory system of fish. Physiol Zool 70:650–659

    CAS  PubMed  Google Scholar 

  • Jensen FB (1999) Haemoglobin H+ equilibria in lamprey (Lampetra fluviatilis) and hagfish (Myxine glutinosa). J Exp Biol 202:1963–1968

    CAS  PubMed  Google Scholar 

  • Jensen FB (2004) Red blood cell pH, the Bohr effect, and other oxygenation-linked phenomena in blood O2 and CO2 transport. Acta Physiologica 182:215–227

    CAS  Google Scholar 

  • Lindskog S, Silverman DN (2000) The catalytic mechanism of mammalian carbonic anhydrases. The carbonic anhydrases. Birkhäuser, Basel, pp 175–195

    Google Scholar 

  • Maren TH, Swenson ER (1980) A comparative study of the kinetics of the Bohr effect in vertebrates. J Physiol 303:535–547

    CAS  PubMed  PubMed Central  Google Scholar 

  • Maren TH, Friedlandl BR, Rittmaster RS (1980) Kinetic properties of primitive vertebrate carbonic anhydrases. Comp Biochem Physiol B 67:69–74

    Google Scholar 

  • McMillan OJL, Dichiera AM, Harter TS, Wilson JM, Esbaugh AJ, Brauner CJ (2019) Blood and gill carbonic anhydrase in the context of a chondrichthyan model of CO2 excretion. Phys Biochem Zool 92(6):554–566

    Google Scholar 

  • Morrison PR, Gilmour KM, Brauner CJ (2015) Oxygen and carbon dioxide transport in elasmobranchs. Fish physiology, vol 34. Academic Press, Cambridge, pp 127–219

    Google Scholar 

  • Nikinmaa M (1986) Red cell pH of lamprey (Lampetra fluviatilis) is actively regulated. J Comp Physiol B 156:747–750

    CAS  Google Scholar 

  • Nikinmaa M (1993) Haemoglobin function in intact Lampetra fluviatilis erythrocytes. Resp Physiol 91:283–293

    CAS  Google Scholar 

  • Nikinmaa M, Huestis WH (1984) Adrenergic swelling of nucleated erythrocytes: cellular mechanisms in a bird, domestic goose, and two teleosts, striped bass and rainbow trout. J Exp Biol 113:215–224

    CAS  Google Scholar 

  • Nikinmaa M, Airaksinen S, Virkki LV (1995) Haemoglobin function in intact lamprey erythrocytes: interactions with membrane function in the regulation of gas transport and acid-base balance. J Exp Biol 198:2423–2430

    CAS  PubMed  Google Scholar 

  • Nikinmaa M, Berenbrink M, Brauner CJ (2019) Regulation of erythrocyte function: multiple evolutionary solutions for respiratory gas transport and its regulation in fish. Acta Physiol 227(2):e13299

    Google Scholar 

  • Pelster B, Weber R (1991) The Physiology of the Root Effect. Advances in comparative and environmental physiology. Springer, New York, pp 51–77

    Google Scholar 

  • Randall D, Rummer J, Wilson J, Wang S, Brauner C (2014) A unique mode of tissue oxygenation and the adaptive radiation of teleost fishes. J Exp Biol 217:1205–1214

    CAS  PubMed  Google Scholar 

  • Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542

    PubMed  PubMed Central  Google Scholar 

  • Root R (1931) The respiratory function of the blood of marine fishes. Biol Bull 61:427–456

    CAS  Google Scholar 

  • Rummer JL, Brauner CJ (2011) Plasma-accessible carbonic anhydrase at the tissue of a teleost fish may greatly enhance oxygen delivery: in vitro evidence in rainbow trout, Oncorhynchus mykiss. J Exp Biol 214:2319–2328

    CAS  PubMed  Google Scholar 

  • Rummer JL, Brauner CJ (2015) Root effect haemoglobins in fish may greatly enhance general oxygen delivery relative to other vertebrates. PloS One 10:e0139477

    PubMed  PubMed Central  Google Scholar 

  • Rummer JL, Mckenzie DJ, Innocenti A, Supuran CT, Brauner CJ (2013) Root effect hemoglobin may have evolved to enhance general tissue oxygen delivery. Science 340:1327–1329

    CAS  PubMed  Google Scholar 

  • Scholander PF, Van Dam L (1954) Secretion of gases against high pressures in the swimbladder of deep sea fishes, I. Oxygen dissociation in blood. Biol Bull 107:247–259

    Google Scholar 

  • Silverman DN, Lindskog S (1988) The catalytic mechanism of carbonic anhydrase: implications of a rate-limiting protolysis of water. Accounts Chem Res 21:30–36

    CAS  Google Scholar 

  • Stams T, Christianson DW (2000) X-ray crystallographic studies of mammalian carbonic anhydrase isozymes. The carbonic anhydrases. Birkhäuser, Basel, pp 159–174

    Google Scholar 

  • Swenson ER (2000) Respiratory and renal roles of carbonic anhydrase in gas exchange and acid-base regulation. The carbonic anhydrases. Birkhäuser, Basel, pp 281–341

    Google Scholar 

  • Takezaki N, Figueroa F, Zaleska-Rutczynska Z, Klein J (2003) Molecular phylogeny of early vertebrates: monophyly of the agnathans as revealed by sequences of 35 genes. Mol Biol Evol 20:287–292

    CAS  PubMed  Google Scholar 

  • Tashian RE (1992) Genetics of the mammalian carbonic anhydrases. Advances in genetics, vol 30. Academic Press, Cambridge, pp 321–356

    Google Scholar 

  • Tu C, Silverman DN, Forsman C, Jonsson BH, Lindskog S (1989) Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant. Biochem 28:7913–7918

    CAS  Google Scholar 

  • Tufts B, Perry SF (1998) Carbon dioxide transport and excretion. Fish physiology, vol 17. Academic Press, Cambridge, pp 229–281

    Google Scholar 

  • Wang Q, Arighi CN, King BL, Polson SW, Vincent J, Chen C, Huang H, Kingham BF, Page ST, Farnum RM, Thomas WK, Udwary DW, Wu CH, The North East Bioinformatics Collaborative Curation, T (2012) Community annotation and bioinformatics workforce development in concert—Little Skate Genome Annotation Workshops and Jamborees. Database, 2012

  • Wells R, Forster M, Davison W, Taylor H, Davie P, Satchell G (1986) Blood oxygen transport in the free-swimming hagfish, Eptatretus cirrhatus. J Exp Biol 123:43–53

    CAS  PubMed  Google Scholar 

  • Wilson JM, Randall DJ, Vogl AW, Harris J, Sly WS, Iwama GK (2000) Branchial carbonic anhydrase is present in the dogfish Squalus acanthias. Fish Physiol Biochem 22:329–336

    CAS  Google Scholar 

  • Wyffels J, King BL, Vincent J, Chen C, Wu CH, Polson SW (2014) SkateBase, an elasmobranch genome project and collection of molecular resources for chondrichthyan fishes. F1000es 3:191

    Google Scholar 

Download references

Acknowledgements

This study was funded by the National Science Foundation Grant to A.J.E. (EF 1315290). The authors would like to thank everyone who kindly donated or helped obtain blood samples: Dr. Jim Gelsleichter, The University of North Florida; Dr. Till Harter, Ellen Jung, Dr. Phill Morrison, and Dr. Chris M. Wood, University of British Columbia; Dr. C. Sepulveda, Pflüger Institute for Environmental Research; Dr. Diego Bernal, UMass Amherst; Dr. Martin Haulena and the veterinary team, Vancouver Aquarium; Dr. Adalberto Val, National Institute for Research of the Amazon; Dr. Gary Anderson, University of Manitoba; the research and foreshore staff at Bamfield Marine Sciences Centre, Bamfield, BC. Additionally, the authors gratefully acknowledge Dr. Kiley Seitz and Dr. Nina Dombrowski for their expertise and guidance in Bayesian phylogenetics. No competing interests declared.

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Authors and Affiliations

  1. Marine Science Institute, The University of Texas at Austin, 750 Channel View Drive, Port Aransas, TX, 78373-5015, USA

    Angelina M. Dichiera & Andrew J. Esbaugh

  2. Zoology Department, The University of British Columbia, 6270 University Blvd., Vancouver, BC, V6T 1Z4, Canada

    Olivia J. L. McMillan & Colin J. Brauner

  3. Scripps Institute of Oceanography, The University of California, San Diego, 9500 Gilman Drive #0202, La Jolla, CA, 92093-0202, USA

    Alexander M. Clifford

  4. Department of Biological Sciences, The University of Alberta, 116 St. and 85 Ave., Edmonton, AB, T6G 2R3, Canada

    Greg G. Goss

  5. Bamfield Marine Sciences Centre, 100 Pachena Road, Bamfield, BC, V0R 1B0, Canada

    Greg G. Goss

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  1. Angelina M. Dichiera
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  2. Olivia J. L. McMillan
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Correspondence to Angelina M. Dichiera.

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Dichiera, A.M., McMillan, O.J.L., Clifford, A.M. et al. The importance of a single amino acid substitution in reduced red blood cell carbonic anhydrase function of early-diverging fish. J Comp Physiol B 190, 287–296 (2020). https://doi.org/10.1007/s00360-020-01270-9

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