Research paper Analysis of a shortened form of human carbonic anhydrase VII expressed in vitro compared to the full-length enzyme Fatemeh Bootorabi a, b , Janne Jänis c , Elona Smith a, b , Abdul Waheed d , Sampo Kukkurainen a , Vesa Hytönen a , Jarkko Valjakka a , Claudiu T. Supuran e , Daniela Vullo e , William S. Sly d , Seppo Parkkila a, b, f, * a Institute of Medical Technology, University of Tampere and Tampere University Hospital, 33014 Tampere, Finland b School of Medicine, University of Tampere and Tampere University Hospital, 33014 Tampere, Finland c Department of Chemistry, University of Eastern Finland, 80101 Joensuu, Finland d Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA e Università degli studi di Firenze, Laboratorio di Chimica Bioinorganica, I-50019 Sesto Fiorentino (Firenze), Italy f Centre for Laboratory Medicine, Tampere University Hospital, 33521 Tampere, Finland article info Article history: Received 18 February 2010 Accepted 13 May 2010 Available online 21 May 2010 Keywords: Carbonic anhydrase Immunohistochemistry Protein modeling Recombinant protein abstract Carbonic anhydrase (CA) enzymes are expressed in all organs of the mammalian body where they participate in important physiological functions. CA VII is a cytosolic isozyme which may be expressed as two forms according to the recent GenBank data. We designed a present study to express and characterize the human CA VII forms: full-length CA VII and short form (predicted to lack 56 residues from the N-terminus). Reverse transcriptase PCR analysis revealed mRNAs for both CA VII forms in the human brain. These different forms were expressed as recombinant proteins to investigate their biochemical properties. The full-length CA VII was used to raise a polyclonal antiserum in a rabbit, and the antiserum was then employed in western blot analyses and immunohistochemistry of mouse tissues. Data from mass spec- trometry and comparative modeling showed that CA VII protein contains a single intramolecular disulfide bridge (Cys-56 to Cys-180) which is lacking in the short form. The computer model suggested distinctly different folding for the different forms. The more exposed structure and the absence of the disulfide bridge in the short form could make this protein more susceptible to degradation. In fact, this was realized in several protein purification efforts in which the short form readily degraded during the experimental procedures. From these results, we conclude that the full-length CA VII is a predominant active form in human brain and also in other tissues. In addition to the brain, CA VII is expressed in several other organs including the stomach, duodenum, colon, liver, and skeletal muscle. The distribution pattern suggests multiple functions for CA VII in different organs. Ó 2010 Elsevier Masson SAS. All rights reserved. 1. Introduction Carbonic anhydrase (CA) forms five families of enzymes (a-, b-, g-, d-, and z-CAs) that catalyze the reversible conversion of carbon dioxide to bicarbonate and proton: CO 2 þ H 2 O HCO 3  þ H þ [1]. Mammalian isozymes, classified as a-CAs, include 13 active iso- forms with different structural and catalytic properties [2,3]. CAs are produced in several different tissues where they have important roles in biological processes such as pH balance, ion transport, bone resorption, respiration, gluconeogenesis, and ureagenesis [2]. The human CA7 gene is 10 Kb long and contains seven exons and six introns found at positions identical to those determined for the previously described CA1, CA2, and CA3 genes [4]. The human CA7 gene encodes a protein containing 263 residues which are 50, 56, and 49% identical with human CA I, CA II, and CA III, respec- tively [4e9]. The human CA7 gene is located in the chromosome 16q22 instead of 8q21 which is the locus for the other highly homologous, cytosolic CAs (CAI, CA II, CA III, and CA XIII) (http://www.ncbi.nlm.nih.gov/gene/). CA7 was originally identi- fied and characterized from a human genomic library by Mont- gomery et al. [10], and even though the enzyme was discovered almost 20 years ago, it has not been thoroughly characterized to date. Previous studies have shown that it is expressed in the brain [11], where it functionally participates as a molecular switch for GABAergic excitation [12]. * Corresponding author at: Institute of Medical Technology and School of Medicine, Biokatu 6, 33014 University of Tampere, Finland. Tel.: þ358 50 3436364; fax: þ358 3 35517710. E-mail address: seppo.parkkila@uta.fi (S. Parkkila). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.05.008 Biochimie 92 (2010) 1072e1080