Lab Snapshots

by Marek Ples

Reading the Genetic Code

Molecular structure of ferroportin and its expression patterns in Common Carp Cyprinus carpio

The Eurasian carp Cyprinus carpio, known better as the common carp, is a widespread freshwater fish found in eutrophic waters of lakes and large rivers in Europe and Asia. It lends its name to the carp family, Cyprinidae. While the native wild populations are considered at risk of extinction by the International Union for Conservation of Nature, this species has also been domesticated and introduced into environments worldwide, often earning a reputation as a destructive invasive species [1]. The common carp is native to Europe and Asia and has been introduced to almost every corner of the globe. Carp serve as a food source, but in some regions, they can also be considered pests due to their competitive nature with native fish.

Fig.1 - The Eurasian carp Cyprinus carpio, mirror type

The original common carp, found about 2000 years ago in the inland delta of the Danube River, had a torpedo-shaped body and a golden-yellow coloration. It possessed two pairs of barbels and a mesh-like scale pattern. Initially kept as a captive for exploitation, the Romans later raised it in specially constructed ponds in south-central Europe [2]. Both European and Asian subspecies have been domesticated for various purposes. In Europe, monks played a significant role in spreading the domestication of carp as a food fish between the 13th and 16th centuries. Variants that emerged from domestication include mirror carp (Fig. 1, Fig.2), leather carp, and fully scaled carp. Koi carp is a domesticated ornamental variety originating in Japan in the 1820s, likely descended from the East Asian carp, possibly Cyprinus rubrofuscus.

Fig.2 - The Eurasian carp Cyprinus carpio, mirror type

Iron is vital for living organisms, playing key roles in biochemical processes like electron transfer, DNA functions, and hemoglobin formation. Its versatility arises from its ability to form complexes and favorable redox potential, aiding biochemical reactions. Animals primarily acquire iron from food but lack an excretion pathway, requiring a feedback mechanism for intestinal absorption regulation.

Ferroportin is crucial, acting as the sole known iron transporter, moving it from cells to the bloodstream. Its sequence is highly conserved across species. Ferroportin is a transmembrane protein with two helix bundles, and its expression is highest in iron-rich tissues. Hepcidin, produced in the liver, regulates ferroportin activity. It serves as an iron regulator and antimicrobial peptide, influencing ferroportin through a negative feedback loop.

I believe that every biologist would agree that this topic is very interesting, and I am glad that I had the opportunity to work in a research team focused on characterizing ferroportin in common carp Cyprinus carpio and evaluating its baseline transcription levels in various tissues [3].

Liver RNA was extracted using the SV Total RNA Isolation System (Fot.3) and converted to cDNA with the RevertAid RT Reverse Transcription Kit. After PCR amplification, products were analyzed on a 1.2% agarose gel, purified with a GenElute PCR Clean-up Kit, and cloned into p-GEM T-Easy Vector. Clones were propagated in JM109 High-Efficiency Competent Cells, and plasmid DNAs from at least 10 colonies were purified with GenoPlast Plasmid DNA extraction Kit and sequenced. Sequences were assembled, and coding DNA was compared with genomic DNA to identify intron/exon boundaries and flanking regions. Analyses were conducted using CLC Main Workbench software (ver. 7.0).

Fig.3 - Isolating genetic material from fish liver

The research conducted here harnessed the power of PCR, or Polymerase Chain Reaction, a cornerstone technique in molecular biology. Its ability to amplify specific DNA sequences enables the thorough examination of genetic material. This precision and versatility have always fascinated me (Fig.4). The meticulous nature of PCR making it one of my favorite techniques. PCR's impact is profound, allowing for the identification of genetic markers, the study of gene expression, and even the detection of pathogens. Its widespread application across various fields underscores its significance in advancing our understanding of genetics and biotechnology. The results it delivers are consistently outstanding, reaffirming PCR's status as an indispensable tool in molecular research.

Fig.4 - Preparing samples for PCR reactions

One of the results obtained in our work was the development of the protein structure encoded by the examined genetic material fragments. Using the known human 3D structure, homology modeling was performed for fpn47 and fpn42 with a Phyre2 web server. Out of a total of 562 residues, 429 were modeled with over 90% confidence, and the remaining 133 residues were modeled ab initio. The secondary structure was predicted to consist of twelve transmembrane helices arranged in two domains connected by a 78aa (fpn47) and 79aa (fpn42) cytoplasmic loop, with both N- and C-terminal ends located intracellularly (Fig. 5).

Fig.5 - Three-dimensional structure of common carp ferroportin;
A - 47; B - 42

As we can see, the structure of analized protein comprising 12 transmembrane helices with N-lobe (blue) and C-lobe (red) being placed intracellularly.

A comparison of ferroportin-encoding sequences in other fish and in humans was also conducted. Interestingly, a high degree of conservation was observed. It was possible to demonstrate significant similarities, for example, a clearly identifiable conservative motif in the extracellular hepcidin-binding loop region (Fig. 6). Evidently, the significance of this protein fragment is so crucial for living organisms that it exhibits no significant differences not only between fishes but also even among mammals, including humans.

Fig.6 - Fragment of alignment of ferroportin sequences;
arrow - hepcidin-binding loop region (based on [3])

Phylogenetic analysis revealed distinct clades within Cypriniformes ferroportins, separate from those in other fish orders, such as Salmoniformes, Perciformes, and Tetraodontiformes (Fig.7). Cypriniformes ferroportins further divided into Cobitidae and Cyprinidae subclades. In Cyprinidae, two clusters were identified, though with low support (<75). Tor tambra and Cyprinus carpio, both known tetraploids, exhibited more than two ferroportin variants [4]. In common carp, four coding sequence variants were found, indicating a double heterozygote condition for ferroportin. Within Cyprinus carpio, the 'fpn47-like' isoform group was closely related to 'fpn42-like isoforms' and Carassius spp. ferroportins. Additionally, the 'fpn47-like' isoform group was sister to Carassius auratus + Carassius gibelio, rendering the entire ferroportin clade paraphyletic.

Fig.7 - Phylogenetic (maximum likelihood) tree of alignment of ferroportin sequences,
high-res image

The research I had the pleasure to participate in presents the first identification and characterization of ferroportin, the primary iron transporter, in common carp. Ferroportin, a widely conserved protein in vertebrates, exhibited similarity in genomic structure, amino acid sequence, and protein conformation when compared to other fish and vertebrates. Both ferroportin genes in carp shared fundamental features, including vital functional domains, confirming their operational capability. Three-dimensional modeling showed structural resemblances with human ferroportin, indicating similar functionality in carp. Phylogenetic analysis discerned Cypriniformes ferroportins, consistent with prior studies on Danio rerio. Notably, a paraphyletic clade containing Cyprinus carpio and Carassius spp. was observed, reflecting their close evolutionary relationship. Although genomic data only predicted two variants, RNA-seq analysis revealed a double heterozygous specimen with four slightly distinct ferroportin isoforms, illuminating the intricacy of gene expression in common carp. Furthermore, specific tissue expression patterns and genomic sequence disparities were noted between duplicated ferroportin genes, hinting at potential functional divergence. Understanding such genetic variation and expression differences is vital for studying proteins involved in maintaining balance within an organism. This research enhances our understanding of the molecular mechanisms governing iron regulation in vertebrates, with potential implications for treating iron-related disorders. The common carp genome stands as a valuable model for investigating functional specialization and expression divergence [3].

That's not all

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