Magnetite in the teeth: sequencing of transcriptomes of the tissues of the radula of the shell of mollusc shell

If you believe the statistics, the most terrible doctors are dentists. These adherents of a drill and laughing gas are ready for anything to make beads out of your teeth. At least that's what people with dentophobia think. In fact, the work of dentists is important and necessary. But who doesn’t need a dentist so to the chitons - shell-clad sea mollusks. Their teeth are so strong that the phrase “gnawing the granite of science” takes on literal meaning. The teeth on the rainbow of chitons are covered with the magnetite mineral, which these marine creatures produce on their own. And so the scientists decided to study this process in more detail in the hope of obtaining new information that can help in the creation of new heavy-duty materials. How scientists conducted their research on the "smiles" of chitons what they were able to discover and how their discoveries can help people - the answers to these questions are in the report of the research group. Go.

Malacology

Chitons or clamshell mollusks are a class of sea clams covered with a shell. Here is such a tautology. At the moment, there are about 1,500 species that inhabit the most diverse geographic regions and water layers (from shallow water to depths of 2500 m). The first feature that catches your eye is their shell. It consists of 8 plates interconnected quite mobile, which allows the chitons to coil in case of danger.


Heaton Cryptochiton stelleri.

If we say that the genealogy of the chitons is very ancient, it will be an understatement, because some of the remains of the ancestors of the chitons (fossils) are about 400,000,000 years old. In this case, the lion's share of human history, these marine inhabitants were reclusive - the first descriptions of chitons date back to 1758 for the authorship of the Swedish scientist Carl Linnaeus.


Radula chiton.

The nightmare of any dentist is the mouth of a chiton, equipped with a radula with several rows of teeth of 17 pieces each. Once again, thanks to evolution, that these creatures grow to a maximum of 35 cm and feed on microscopic algae, not humans. Each of the teeth is covered with magnetite, which helps them to scrape their food from any surface.

And it was precisely this feature that interested scientists. How do chitons produce magnetite, making their teeth one of the most durable in nature?


Magnetite

Magnetite (Fe ₃ O ₄/ iron oxide), as you might guess from the name, has strong magnetic properties, in addition, it is a semiconductor. Rare this mineral can not be called, but one thing to get it in some mountains and quite another - to develop it with your own body to give strength to your teeth.

So, we met a little bit with the main character of this research and with his “superpower”. Now it's time to delve into the study itself and its results.

The basis of the study

The fact that some organisms can produce magnetite scientists have known for a long time. Among these organisms are bacteria, bees, salmon and even pigeons. However, since the discovery of such a feature in chitons in 1962, the mechanism for producing magnetite has not been fully described. Scientists note that interest in chitons increased many times after the magnetite produced by Cryptochiton stelleri (a type of chitons) was recognized as one of the most durable biominerals.

Another important feature of chitons is the replacement of a number of worn teeth with new ones formed inside the rada, which makes it possible to analyze the entire mineralization process. The first 8-12 rows of C. stelleri teeth are transparent and do not have magnetite, and consist mostly of α-chitin and proteins. After 2-5 rows of teeth darken to a reddish-brown color, which visually corresponds to partial mineralization by means of amorphous nanocrystalline iron oxide (ferrihydrite). And the last rows of teeth already have pronounced black protrusions, which indicates the completion of the mineralization process, that is, the transition of ferrihydrite to magnetite.

An important nuance is that the surface of the radula and the base of the teeth consist mostly of α-chitin, but the point of the teeth consists of a mineral. This means that there are a number of proteins that determine the specificity of the formation of iron oxide and the further coating of this area with magnetite.

To study these processes, scientists conducted a series of observations that made it possible to recreate the radula of the C. stelleri chiton tissue from both regions, with and without mineralization, with the transcript of the tissues.

Results


Image No. 1 The

teeth of the mollusk are formed inside the tissues of the radula, where they are surrounded by epithelial cells ( 1a ). Consequently, the proteins necessary for their formation are produced in the same cells.

Scientists have isolated RNA from radula tissues in order to construct a series of expressed sequences in these tissues (the transcript).

The samples from RNA selection were tissues from three sites ( 1a ): the first 8–12 rows of teeth (without mineralization), 2–5 rows (partial mineralization) and 5–6 rows (complete mineralization). Next, RNA sequencing was carried out, that is, the determination of the primary structure of RNA. Data from mineralized and non-mineralized sites were collected in 187980 transcripts. The average length of one transcript was 705 bp (paired bases / base pairs), and the maximum reached 16738 bp.

Figure 1b shows schematically the chiton's tooth: the membrane of the radula at the base, then the main part and the sharp tip of the tooth (outer part).


Table of results of C. stelleri RNA sequencing.

Among the 20 most expressed transcripts in the non-mineralized area of ​​the teeth, five transcripts are the isoforms of the Cs17717 protein | c0_g1 . Also, 2 peritrofin - like protein transcripts were detected - Cs79475 | c0_g1_i1 and Cs70642 | c0_g2_i1 , which possess a chitin-binding domain. It is also important to note that Cs25220 | c1_g1_i1 is similar to the Pif protein, which is associated with the biomineralization of calcium carbonate in mollusks. Thus, the above proteins have a type 2 chitin-binding domain.

A different picture was observed in the twenty expressed transcripts in the mineralized area of ​​the teeth. 35% of them were enzymes of the mitochondrial respiratory chain. The Cs12250 | c0_g1_i1 protein , which is involved in chitin binding, was also detected . Interestingly, this protein was previously found in the beak of a squid. About 25% of transcripts are protein isoforms Cs22243 | c0_g7 , whose functions have not yet been studied.

Scientists have identified a number of important in their opinion proteins involved in the mineralization process. But the most important they consider ferritin, which is necessary for the storage and transfer of iron. Ferritin forms a protein cell - a kind of trap, which holds up to 4500 iron atoms in the form of a mineral based on iron oxide.


Table of all proteins associated with the mineralization of C. stelleri's teeth.

And since the ferritin genes were found in the tissues of the raula of the tested mollusks, the scientists boldly suggested that he was involved in the process of teeth mineralization.

Scientists have analyzed 134993 transcripts for signs of homology (genetic similarity) with ferritin genes. As a result, 4 amino acid sequences of transcripts were isolated.

Analysis of the four showed that Cs90734 | c0_g1_i1 has homology with secreted ferritin, and the three remaining ( Cs22563 | c0_g1_i1 , Cs75144 | c0_g1_i1 and Cs17042 | c0_g1_i1 ) are more similar to cytoplasmic ferrites more similar to cytoplasmic ferrites.

Scientists also point out that ferritinCs90734 | c0_g1_i1 , unlike its “colleagues”, has certain structural properties inherent in ferritin of the secreted type, and not cytoplasmic.

First, it has an insert of about 40 amino acid residues (amino acids outside the peptide sequence). Secondly, the metal bonding residues are replaced by other amino acids. The only thing Cs90734 | c0_g1_i1 does not have of the secreted ferritin type is the signal sequences.

One particular ferritin had the highest expression index — Cs75144 | c0_g1_i1 , which was, interestingly, more pronounced in the non-mineralized area of ​​the teeth. This indicates its importance in the mineralization process.

A total of 31 proteins were identified from the base of the tooth and the radula membrane. Of these, 22 were identified during the first stage of analysis using tandem mass spectrometry. This technique also made it possible to understand that the peptide sequences from the mineralized areas of the tooth correspond to 232 transcripts, and the peptide sequences from the base of the tooth and the membrane to 114 transcripts.

77 proteins were identified from the region of the teeth tips (61 during the first test and 54 - second), among which stands out the following: myoglobin, actin, elongation factor-1 alpha and argininkinaza ( Cs77196 | c0_g2_i1 , Cs47470 | c2_g3_i1 , Cs24354 | c0_ g1_i1 and Cs82664 | c0_g1_i1, respectively). All of them are represented by the twenty most expressed transcripts in the non-mineralized part of the teeth.

During the search for proteins responsible for the mineralization, Mascot software was used, which operates on a mass spectrometry database and identifies proteins by peptide sequences. Among the studied proteins, special attention was given to previously unidentified protein Cs68435 | c0_g1_i1 , which had no similarities with the sequences of other proteins. Scientists gave it the name RTMP1 (matrix protein of the radial teeth 1).

Peptide sequence of the protein Cs68435 | c0_g1_i1contains glycine and serine residues. Since the five-stroke (5 ') of this protein was shortened, the total sequence length was determined using 5′-RACE.

RACE (fast amplification of cDNA ends) is a method for producing double-stranded terminal regions of cDNA homologous to the 3 ′ or 5 ′ ends of specific mRNA molecules.

The complete amino acid sequence of the new RTMP1 protein.

The overall picture of the sequence is as follows: areas enriched with glycine and serine (GS), then areas with tryptophan and phenylalanine (WF) and areas rich in histidine (H).

Scientists also found that alignment of WF sequences from RTMP1 sites to the chitin-binding domains of four bacterial chitinases (enzymes that activate the process of chitin degradation) showed that three out of five suspected residues that interact with chitin in the chitin-binding domains are also present in RTMP1.

Scientists have determined the expression profile of transcripts associated with mineralization proteins in different mineralization periods. Approximately 4 times the level of expression of some transcripts (for example, neuroglobin-like proteins, superoxide dismutase, peroxiredoxin-6, etc.) differs in areas of teeth with and without mineralization. The level of expression varies with the course of the mineralization process. In other words, the closer to the site of mineralization, the lower the expression of some transcripts and the higher the expression of others (for example, RTMP1 protein).

For more detailed acquaintance with the study I strongly recommend to look into the report of the research group and additional materials to it.

Epilogue

This study for the first time demonstrated a chiton radula tissue transcript. Scientists found out that the most expressed transcripts in the non-mineralized regions contain ferritin genes, and in the mineralized ones they contain enzymes of the mitochondrial respiratory chain.

The researchers also identified 22 proteins in the mineralized area, among which a completely new one was discovered - RTMP1. It is these proteins that will be tested in further studies of the mineralization of iron oxide in chitons.

This work is the first step in understanding how minerals are actually formed in biological systems. Such cases are not uncommon, but they are currently studied extremely superficially. In the future, such knowledge can help to create new methods of growing certain materials, giving them very useful properties (strength, electrical conductivity, magnetization, etc.).

Thank you for your attention, remain curious and have a great work week guys.

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