Human mitochondrial carbonic anhydrase VB. cDNA cloning, mRNA expression, subcellular localization, and mapping to...

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 encoded a 317-amino acid protein with a predicted mass of 36.4 kDa. The highest similarity of this cDNA and the deduced amino acid sequence is to human CA V (mitochondrial CA), hereafter referred to as CA VA. Recombinant protein expressed in COS-7 cells transfected with this cDNA clone was enriched in a mitochondrial fraction. Confocal fluorescence microscopy showed cytoplasmic granular signals in COS-7 cells expressing a fusion protein of the novel CA and green fluorescent protein. Several lines of evidence suggest that the cDNA clone presented herein encodes a novel human mitochondrial CA isozyme, designated CA VB. CA VB has a hydrophobic N-terminal mitochondrial signal 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 CA VA, CA VB 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 tissues demonstrated expression of a 1.3-kilobase transcript in heart and skeletal muscle, and reverse transcription-polymerase chain reaction analysis showed expression of CA VB in pancreas, kidney, salivary glands, and spinal cord 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-specific distribution, suggest different physiological roles for the two mitochondrial isozymes.