Production and Characterization of Carbonic Anhydrase VII. Acetaldehyde-derived modifications and comparison to the other cytosolic isozymes

Abstract

Väitöskirjatutkimuksessa tuotettiin bakteerisoluissa ihmisen hiilihappoanhydraasi VII isoentsyymi (CA VII), jota karakterisoitiin tutkimuksen aikana. Paikantamistutkimuksissa todettiin, että CA VII isoentsyymiä esiintyy runsaasti erityisesti maksassa ja aivokasvaimissa.

Tulosten perusteella CA VII on lupaava merkkiproteiini aivokasvainten diagnostiikassa. Tutkimuksen erityisenä tavoitteena oli selvittää muutoksia, joita alkoholin aineenvaihduntatuote, asetaldehydi, aiheuttaa hiilihappoanhydraasien proteiinirakenteessa.

Kohteeksi valittiin kaikki solulimassa ilmentyvät hiilihappoanhydraasit, CA I, CA II, CA III, CA VII ja CA XIII. Tulosten perusteella asetaldehydi sitoutui voimakkaimmin CA II isoentsyymiin. Vaikka yhteen entsyymimolekyyliin sitoutui jopa 19 asetaldehydimolekyyliä, tämä rakennemuutos laski odotettua vähemmän hiilihappoanhydraasin entsymaattista aktiivisuutta.

Väitöskirjatutkimuksesta saadut tiedot auttavat ymmärtämään paremmin hiilihappoanhydraasien toimintaa sekä asetaldehydin sitoutumista ja vaikutuksia entsyymeissä.

Carbonic anhydrase (CA) enzymes are ubiquitous metalloenzymes which are able to maintain acid-base homeostasis by catalyzing the reversible conversion of carbon dioxide to bicarbonate ions and protons. This family of enzymes plays an important role for the survival of the cell and whole organism by contributing to various physiological functions. The ?-CA family consists of fifteen isozymes of which thirteen are expressed and have catalytically active forms in mammals. The CA isozymes have shown unique expression patterns, kinetic and inhibitory properties, as well as several different subcellular localizations: five isozymes are cytosolic, five are membrane-associated, two are mitochondrial, and one is a secretory form.

One major aim of our study was to produce and characterize human CA VII, a cytosolic isozyme. Two potential variants of CA VII were discovered based on GeneBank data. The investigations on the CA VII expression in the human cerebrum and hippocampus showed positive mRNA signals for both variants. The shorter variant turned out to be unstable and was not detectable in any murine tissues at the protein level. The full-length CA VII was expressed in the liver, colon, and muscle. In addition to certain normal tissues, CA VII was detected in gliomas. Our results indicated that CA VII expression became higher in high-grade tumors, suggesting an important role for this enzyme in tumor metabolism. This result led us to conclude that CA VII might serve as a novel potential biomarker of poor prognosis in diffuse astrocytomas.

The other main goal was to investigate and compare acetaldehyde-derived modifications in five cytosolic CAs, including CA I, II, III, VII, and XIII. Acetaldehyde, the first metabolite of ethanol, has been shown to be a toxic compound in the body, because of its capablity to form stable and unstable adducts with various proteins and cellular components. The acetaldehyde binding can result in functional and structural changes in proteins, which can further lead to harmful effects, such as autoimmune and carcinogenic processes.

Human CA II, the most active enzyme among the CA gene family, showed a significant decrease in the enzyme activity levels when treated with high concentrations of acetaldehyde. The other isozymes showed milder changes in the activity levels after acetaldehyde treatment. This difference can be explained by the unique structural features of CA II isozyme as compared to the other enzyme forms. CA II contains the highest number of reactive lysine residues on the surface, which makes it a more sensitive target for acetaldehyde binding and adduct formation.

The data obtained in the present studies will hopefully help to better understand the mechanisms of acetaldehyde adduct formation at the molecular level. This information may also deepen our insight into the pathogenesis of alcohol-related injuries in different organs.