Human mitochondrial carbonic anhydrase VB - cDNA cloning, mRNA expression,subcellular localization, and mapping to chromosome X

A cDNA clone for a novel carbonic anhydrase (CA) isozyme was isolated from human pancreas and salivary glands. The cDNA sequence of 1182 base pairs en coded a 317-amino acid protein with a predicted mass of 36.4 kDa, The highe st similarity of this cDNA and the deduced amino acid sequence is to human CAV (mitochondrial GA), hereafter referred to as GA Vk Recombinant protein expressed in COS-7 cells transfected with this cDNA clone was enriched in a mitochondrial fraction. Confocal fluorescence microscopy showed cytoplasmi c granular signals in COS-7 cells expressing a fusion protein of the novel CA and green fluorescent protein. Several lines of evidence suggest that th e cDNA clone presented herein encodes a novel human mitochondrial CA isozym e, designated CA VB. CA VB has a hydrophobic N-terminal mitochondrial signa l sequence (33 amino acid residues). Western blot analysis showed a 36-kDa protein precursor and a 32-kDa mature protein for CA VB. Similar to CAVA, C AVE is a "low activity" enzyme with a sensitivity to acetazolamide. The CA VB gene is located on Xp22.1. Northern blot analysis in normal human tissue s demonstrated expression of a 1.3-kilobase transcript in heart and skeleta l muscle, and reverse transcription-polymerase chain reaction analysis show ed expression of CA VB in pancreas, kidney, salivary glands, and spinal cor d but not in liver, CA VA mRNA expression was observed only in liver. These findings indicate these are two genetically distinct isoforms of human CA V, designated CA VA and CA VB, which have different patterns of tissue-spec ific distribution, suggest different physiological roles for the two mitoch ondrial isozymes.