|Sažetak rada|| |
Bone homeostasis is secured by a combined action of bone forming osteoblasts and bone resorbing osteoclasts. When this balance is impaired, osteopenia is induced, which in time develops into osteoporosis. Antibody glycosylation influences osteoclast differentiation. It is known that immunoglobulin G (IgG) immune complexes positively stimulate osteoclast differentiation, resulting in subsequent inflammatory bone loss. In order to further investigate this process, a method was developed to isolate IgG from serum and to investigate the changes in IgG glycosylation patterns in osteoporotic rats treated with clinoptilolite, a natural zeolite with great detoxification and ion exchange properties. An experimental rat model was set up, and following groups of animals were analysed: healthy control, sham control, ovariectomized control (OVX), OVX supplemented with synthetic zeolite, OVX supplemented with natural clinoptilolite and OVX supplemented with micro activate clinoptilolite. Using affinity chromatography by use of monolithic supports with immobilized immunoglobulin binding ligands as a robust tool for isolation of immunoglobulins, IgG can be purified from rat sera. Compared to proteins A and G, recombinant protein L binds by far the largest number of isoforms of all immunoglobulins. For this reason, this ligand immobilized on a monolithic column has been used in this work. To procure a highly enriched IgG preparation, the fraction with proteins eluted from the column was analysed on a 1D polyacrylamide gel, after which in-gel tryptic digestion of this protein was performed. The resulting peptides were successfully identified by MALDI-TOF/TOF mass spectrometry as parts of IgG heavy and light chains. In the next step, the IgG glycan structure was analysed by use of the same technique, after the glycans have been removed from the protein using deglycosylation enzyme PNGase F. Next to the IgG analysis, liver proteomes of healthy, sham operated and ovariectomized (OVX) rats treated with zeolites were fractionated according to hydrophobicity and each fraction was separately analysed by SDS PAGE. Finally, liver cryo slices were examined by Synchrotron radiation. Present results give us the evidence that the developed high throughput protocols for analysis of glycosylation of rat immunoglobulins, namely IgG, IgA and IgM, as well as the protocol for quantitative proteomic investigations of rat liver proteome, give us the fundament for further investigations by use of a larger number of experimental animals. We suggest that clinoptilolite positively affects bone status in osteoporotic rats as a consequence of signalling changes in the body, particularly those initiated by the liver and the systemic spread of IgG molecules.