Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers

Abstract

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30-42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

Figure 1

Amino acid sequences of CA XII aligned below with sequences of other catalytic human CAs. Numbers above the aligned sequences correspond to numbering of amino acids in CA I. The conserved residues are boxed. Arrows above His-94, His-96, and His-119 indicate Zn-binding, active site residues. Stars below NLS and NKS indicate asparagine glycosylation sites. The N-terminal extensions represent the signal sequences of membrane CAs (IV, IX, and XII) and mitochondrial CA V. Cysteines at positions 23 and 203 (CA I numbering) are conserved between CA VI and CA XII. The C-terminal extension of CA XII (amino acid residues 291–354) includes the 26-amino acid hydrophobic domain (underlined) and potential sites for phosphorylation by casein kinase II, protein kinase C, and cAMP-dependent kinase (double underline). The underlined hydrophobic C-terminal extension of CA IV is cleaved off in glycosyl-phosphatidylinositol anchoring and is not found in the mature, glycosyl-phosphatidylinositol-anchored enzyme.

Figure 2

Western blot of proteins in extracts of COS cells transfected with pCXN vector expressing CA XII that were incubated with or without endoglycosidase PNGase F. A 43- to 44-kDa doublet was identified with polyclonal antibody to CA XII that was reduced to ≈39 kDa by treatment with 100 milliunits of endoglycosidase PNGase F.

Figure 3

(Upper) Lanes: Northern blots of mRNAs from normal human tissues and from pairs of specimens derived from RCC (T) and adjacent normal renal tissue (N). Normal tissues were lanes 1, testis; 2, stomach; 3, lung; 4, muscle; 5, liver; 6, spleen; 7, bladder; 8, prostate; 9, breast; 10, lung; 11, colon; 12, peripheral blood lymphocytes; 13, liver; and 14, kidney. Significant signals were seen only in mRNA from kidney and colon. (Lower) Lanes: normal kidney (N) adjacent to mRNA from kidney tumor (RCC) (T).

Figure 4

Chromosome localization of CA XII. Fluorescence in situ hybridization with full-length CA XII showed signal on chromosome 15q22.