Analysis of a shortened form of human carbonic anhydrase VII expressed in vitro compared to the full-length enzyme.
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 spectrometry 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.
Bootorabi
Fatemeh
F
Institute of Medical Technology, University of Tampere and Tampere University Hospital, 33014 Tampere, Finland.
Jänis
Janne
J
Smith
Elona
E
Waheed
Abdul
A
Kukkurainen
Sampo
S
Hytönen
Vesa
V
Valjakka
Jarkko
J
Supuran
Claudiu T
CT
Vullo
Daniela
D
Sly
William S
WS
Parkkila
Seppo
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
D017931:DNA Primers; D011994:Recombinant Proteins; D002256:Carbonic Anhydrases