engleski

Characterization of organic cation transporters in zebrafish (Danio rerio Hamilton, 1882)

Members of SLC (Solute carrier) superfamily are membrane transporters responsible for the uptake and extrusion of enormous variety of organic and inorganic substrates. The SLC22 family consists of members of major facilitator superfamily (MFS) clan which encompasses all SLCs, together with other membrane proteins and is one of the largest clans of membrane transporters found in humans. Regardless of the maximal amino acid sequence homology of 25%, all members of SLC22 family have similar secondary and tertiary protein structures. They are monomeric polypeptides that have 12 transmembrane α-helices which form transmembrane domains and are incorporated in plasma membranes of the cell. Order and steric arrangement of transmembrane helices define polyspecific active site clefts of transporters. Organic cation transporters (OCTs) are members of the SLC22A family within SLC superfamily. These internal membrane proteins are responsible for uptake of broad and overlapping range of endo- and xenobiotics. Despite their cricial role in the ADME (Absorption, Distribution, Metabolism, Excretion) and toxicological response of organisms, their detailed ecotoxicological relevance and characteristics are still unknown.Therefore, we focused our research on molecular characterization of organic cation transporters in zabrafish (Danio rerio) as newly emerged and valuable model organism. Using methods such as phylogenetic analysis, qRT-PCR, and transfection in heterologous expression system, we developed transport assays based on fluorescent probes in order to identify potential substrates and inhibitors of ecotoxicological relevance and elucidate transport mechanism of zebrafish Octs as key elements in physiological and ADME preocesses of organic cations. Phylogenetic analysis revealed specific clustering of vertebrate SLC22 genes. Zebrafish Octs clustered with other vertebrate OCTs/Octs, and with OCTN/Octn and OCT6/Oct6 gene groups of other vertebrates. The analysis of gene block conservation and changes during evolution provided an insight to syntenic relationships among fish and human OCT/Oct genes. Synteny analysis showed that zebrafish Oct1 ortholog is located on chromosome 20, and it showed conserved synteny with human OCT cluster. Neighboring genes of zebrafish Oct1 matched the neighboring genes of the human ortholog cluster. Another zebrafish ortholog Oct2 is located on chromosome 17. Synteny analysis of Oct2 showed conserved syntenic relationship with human OCT cluster which was confirmed by analysis of neighboring genes locations. Secondary structure analysis of Oct1 showed that Oct1 forms twelve transmembrane α-helices. Analysis of Oct2 revealed the differences between these two proteins. The major differences are in number of transmembrane helices, with nine transmembrane helices of Oct2. N-glycosylation analysis identified three potential N-glycosylation sites on Oct1 extracellular loop and analysis of Oct2 revealed total of seven N-glycosylation sites. Tissue expression analysis revealed the highest expression of Oct1 in kidney, with clear expression differences among genders. Very high expression of Oct1 was observed in zebrafish liver, with dominant expression in male liver. Tissue expression of Oct2 was highest in testes, following with kidney and other analyzed tissues in lower range of expression, starting from intestine and following with liver, gills, brain and the lowest observed expression in ovaries. High expression of zebrafish Oct1 in major toxicologically relevant tissues, with emphasis on the highest expression in kidney, point to crucial role of this transporter in absorption, reabsorption and excretion of organic cations in zebrafish. Inhibition assays revealed high affinity interactions of Oct1 with steroid hormones, indicating the potential role of Oct1 in mediation of steroid hormone homeostasis which could function as important transporter in gonads and hormone synthesizing organs, as well as in biliary and renal excretion end reabsorption. From the ecotoxicological aspect, Oct1 may be crucial target for xenobiotic disruption by environmental hormones and their synthetic analogs. Inhibition assays also revealed Oct1 as important element in zebrafish ADME processes, with the proposed key role in defense against hazardous environmental and endobiotic compounds through biliary and renal excretion. Finally, determined 3D model of zebrafish Oct1 and human Oct1 and OCT2 provided new insight in structure and complexity of substrate binding region of analysed transporters, and confirmed complex interactions with potential substrates and inhibitors which affects determination of type of interaction between the transporter and identified interactors.

SLC transporters ; Danio rerio ; phylogenetic analysis ; synteny analyisi ; tissue expression ; function ; ADME

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