Characterization of Carbonic Anhydrases from Riftia pachyptila, a Symbiotic Invertebrate from Deep-Sea Hydrothermal Vents Marie-Ce ´ cile De Cian, 1 * Xavier Bailly, 1 Julia Morales, 2 Jean-Marc Strub, 3 Alain Van Dorsselaer, 3 and Franc ¸ ois H. Lallier 1 1 Equipe Ecophysiologie, CNRS-UPMC UMR 7127 CEOBM, Station Biologique, Roscoff Cedex, France 2 Equipe Cycle Cellulaire et De ´veloppement, CNRS-UPMC UMR 7127 CEOBM, Station Biologique, Roscoff Cedex, France 3 Laboratoire de Spectrome ´trie de Masse Bio-Organique, CNRS-ULP UMR 7509, ECPM, Strasbourg Cedex, France ABSTRACT The symbiotic hydrothermal vent tubeworm Riftia pachyptila needs to supply its internal bacterial symbionts with carbon dioxide, their inorganic carbon source. Our aim in this study was to characterize the carbonic anhydrase (CA) involved in CO 2 transport and conversion at various steps in the plume and the symbiotic tissue, the trophosome. A complete 1209 kb cDNA has been sequenced from the trophosome and identified as a putative -CA based on BLAST analysis and the similarities of total deduced amino-acid sequence with those from the GenBank database. In the plume, the putative CA sequence obtained from cDNA li- brary screening was 90% identical to the tropho- some CA, except in the first 77 nucleotides down- stream from the initiation site identified on trophosome CA. A phylogenetic analysis showed that the annelidan Riftia CA (CARp) emerges clus- tered with invertebrate CAs, the arthropodan Dro- sophila CA and the cnidarian Anthopleura CA. This invertebrate cluster appeared as a sister group of the cluster comprising mitochondrial and cytosolic isoforms in vertebrates: CAV, CAI II and III, and CAVII. However, amino acid sequence alignment showed that Riftia CA was closer to cytosolic CA than to mitochondrial CA. Combined biochemical approaches revealed two cytosolic CAs with differ- ent molecular weights and pI’s in the plume and the trophosome, and the occurrence of a membrane- bound CA isoform in addition to the cytosolic one in the trophosome. The physiologic roles of cytosolic CA in both tissues and supplementary membrane- bound CA isoform in the trophosome in the optimiza- tion of CO 2 transport and conversion are discussed. Proteins 2003;51:327–339. © 2003 Wiley-Liss, Inc. Key words: symbiosis; carbonic anhydrase; cDNA sequence; phylogenetic analysis; MALDI- TOF; cytosolic and membrane-bound isoforms INTRODUCTION Carbonic anhydrases (CAs) are key enzymes for the maintenance of homeostasis in a wide range of organisms. These zinc-containing enzymes catalyze the reversible hydration of CO 2 into bicarbonate and a proton. 1 CAs are presently ordered in three distinct classes of gene families: , , and , corresponding schematically to animal, plant, and bacterial CA. 2 The occurrence of a new type of CA without any significant identity to the three classes, has been recently demonstrated in the marine diatom Thalas- siosira weissflogii by Roberts and coworkers. 3 The -CA group constitutes a multigenic family coding for cytosolic (CA-I, CA-II, CA-III, CA-VII, CA-VIII), mitochondrial (CA- V), membrane-associated (CA-IV, CA-IX), or secreted (CA- VI) isoforms. 4 Depending on their subcellular localization, -CAs are involved in different physiologic processes, including acid– base homeostasis, CO 2 and ion transport, respiration 5 or calcification, 6 reproduction, and neurosen- sitive processes. 7 Historically, the first discovered CA isoforms were -CAs from human red blood cells. 8,9 How- ever, in the last 20 years, a remarkable number of -CAs have been described from nonmammalian organisms, in- cluding zebrafish, 10 flounder, 11 oysters, 12 crabs, 13 sea anemones, 14 and viruses. 15 CA activity was also demon- strated in autotrophic organisms such as plants (- CA) 16,17 or bacteria (-CA), 18 in which CA appears directly involved in carbon supply for the Rubisco (ribulose 1,5- biphosphate carboxylase) enzyme responsible for carbon fixation. Deep-sea hydrothermal vent environments are consid- ered extreme given the high pressure and temperature conditions, the toxicity of surrounding chemicals, and the total lack of phototrophic production for animal nutrition. The ability of hydrothermal vent communities to thrive and survive under such conditions is mainly due to molecu- lar, biochemical, and physiologic adaptations that enable the organisms to maintain vital functions. This study investigates CA in the model organism Riftia pachyptila, a hydrothermal vent annelid. In this worm, adaptation to extreme conditions led to the emergence of Contract grant sponsor: French Research Department; Contract grant sponsor: DORSALE *Correspondence to: Marie-Ce ´cile De Cian, Ecophysiologie, Station Biologique, Place Georges Teissier, BP 74, 29682 Roscoff Cedex, France. E-mail : decian@sb-roscoff.fr Received 1 July 2002; Accepted 5 September 2002 PROTEINS: Structure, Function, and Genetics 51:327–339 (2003) © 2003 WILEY-LISS, INC.