Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

Abstract

Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CA VI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension [Fernley, R. T., Wright, R. D., & Coghlan, J. P. (1988b) Biochemistry 27, 2815]. Overall the human CA VI protein has a sequence identity of 35% with human CA II, while residues involved in the active site of the enzymes have been conserved. The human sheep secreted carbonic anhydrases have a sequence identity of 72%. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with neurons and neural cell-adhesion molecules, is an extracellular variant of a receptor-type protein tyrosine phosphatase.

“…The proteoglycan contains an N-terminal domain homologous to carbonic anhydrases. At least seven mammalian carbonic anhydrase isozymes are known, and the highest level of identity (32%) ofphosphacan is with carbonic anhydrase VI, a protein secreted by salivary glands (11). However, because a catalytically obligatory zinc ion is bound to three essential histidine residues in carbonic anhydrases, whereas in both rat phosphacan and human RPTPC/f two of these have been changed to threonine and glutamine (Fig.…”

Carbonic anhydrase IV expression in rat and human gastrointestinal tract regional, cellular, and subcellular localization.

“…While homologous in amino acid sequence, the isozymes differ in catalytic activities and sensitivities to inhibitors. They also differ in their tissue distributions and subcellular localizations; CA I, II, III, and VII are cytoplasmic, CA VI is secretory, CA V mitochondrial, and CA IV membrane-associated (1)(2)(3)(4)(5)(6).…”

Determination of Full-Length cDNA Nucleotide Sequence of Equine Carbonic Anhydrase VI and Its Expression in Various Tissues

“…Interestingly, there were some differences of expressional pattern in alimentary tracts between species [10,15]. Until now, several mammalian CA6 cDNAs have been cloned successfully [1,8,14,16], though that of equine remains to be determined. Here in this study, we investigated and determined the nucleotide sequence of equine CA6 cDNA and examined its expression in various organs as the first step of exploring the physiological roles of CA-VI in equine.…”

“…Total RNA was isolated from the parotid gland of a male 6-year-old thoroughbred using an RNA extraction solution (Isogen, Nippon Gene, Tokyo). Degenerate primers used for the amplification of a partial equine CA6 cDNA was prepared as described previously [1,9]. RT-PCR amplification was performed with the total RNA and these degenerate primers employing a SuperScript first cDNA system kit (Invitrogen Japan, Tokyo) using AmpliTaq DNA polymerase (Applied Biosystems Japan, Tokyo).…”