Evolution of Antifreeze Glycoproteins

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The Evidence of Convergent Evolution of Antifreeze Glycoproteins in Fish

Since millions of years ago, animals, insects, plants and many other organisms have gone through series of evolutions by selective pressure to gain unique mechanisms in order to survive and adapt in harsh environments at extreme temperatures. Nearly two-third of the earth's surface is compromised of sea water and the temperatures vary according to latitude, from between -2ºC at polar regions to 36ºC at the Persian Gulf (http://www.windows.ucar.edu/tour/link=/earth/Water/temp.html&edu=high). Sea water temperatures within the polar regions are constantly below the freezing point of pure water, due to the high concentration of salinity in its surroundings (http://nsidc.org/seaice/intro.html). The effect of these very low temperatures is usually extremely harmful and deadly to cells of majority of organisms.

Many researchers have investigated and obtained numbers of results suggesting the different mechanisms present in many species of fish living in the polar regions which enables them to survive in the freezing water. By analysis of these fish blood plasma and their hemoglobin molecular structure, functional features and also phylogeny, it was showed that they have a slightly higher concentration of small ions and salts in comparison to fish in temperate regions. Due to this factor, and the presence of certain distinctive series of glycoproteins and proteins, enables the fish to have a defensive effect, or the serum freezing point depression against the sub-zero temperatures.

Antifreeze glycoproteins (AFGPs) and antifreeze proteins (AFP) have been identified in various polar fish species and they have been evolved to a total of four classes of structurally distinct types of AFPs, categorized as type I, type II, type III and type IV and a single class of AFGP. Figure 1 summarizes the classification and key structural differences between AFPs and AFGP. These compounds were found unusual as they caused a freezing point depression far greater than predicted to come from their colligative properties alone. They were hence concluded to exhibit thermal hysteresis, a behavior which creates difference between melting point and freezing point which causes inhibition of ice crystals growth within their body. This property is advantageous for fish to survive in subzero waters as their blood and internal fluids are prevented from crystallization and at the same time, their cell membranes are protected from any damages caused by the cold.

Unexpectedly, most fish related phyletically do not possess the same AFP types and vice versa for unrelated fish. The best example is the phylogenatically distant Antartic notothenoid and northern cods having produced near-identical AFGPs through evolution. The study of their AFGPs gives molecular evidences that strongly support convergent evolution to have had occur independently to each species during a period of time million years ago.

Antifreeze glycoprotein widely refers to a family of at least eight compositionally related glycoproteins that are found at high proportion in proteins in blood plasmid of Antartic notothenoids and Arctic cods. In Antartic notothenoids, an AFGP is composed of (Ala-Ala-Thr)n repeating units with small sequence variations and a glycoside, disaccharide β-D-galactosyl-(1→3)-α-N-acetyl-D-galactosamine joined to the hydroxyl oxygen of Thr residues, such as shown in Figure 2. The glycopeptides are divided into eight classes, ranging by their relative molecular mass from small AFGP 2.6 kDa (n = 4) to large AFGP 33.7 kDa (n = 50). Another variation of these proteins is the difference in the composition of amino acids in small AFGPs on which the first Ala is replaced by Pro in some repeats. Antartic notothenoids AFGPs basically have a straightforward primary structure, although they differ slightly in molecular size and amino acids constitution. On the other hand, Arctic cods have glycoproteins surprisingly similar to notothenoids', eventhough with the occasional replacement of an Arg residue on Thr, causing the lack of disaccharides at the positions. Further different amino acids substitutions could be tolerated, such as found in Antarctic fish species Pleuragramma antarcticum. As there are now concluded that different AFGPs have different molecular sizes, just the generic term AFGP has produced many confusion to people as it is not clear whether it has the pure glycopeptides or a mixture of different glycopeptides. Also, due to the importance nowadays for the exact amino acids compositions to be specified in order to develop further molecular understanding and research, abbreviations such as AFGP-Pro and AFGP-Arg (if there is a substitution of amino acid in the Ala-Ala-Thr tripeptide backbone) are used.

Other than Antactic notothenioid, AFGPs were also isolated from the rock cod, Gadus odac and other northern cods from the family Gadidae. As of date, the most studied AFGPs are from Dissostichus mawsoni and Trematomas borgrevinki from Antarctic and Boreogadus saida from the northern parts of the ocean. In both of the Antarctic fish, it was found that they have a total concentration of 25 mg•mL-1 of AFGPs with 75% of them consisting of the small AFGPs. From the hard works of Chen and Chang et al., the issue concerning AFGPs' evolutionary origins was lastly resolved. There were major similarities in structures of AFGPs found in two different species of fish, the Antarctic notothenioids (Family: Nototheniidae, Artedidraconidae, Bathydraconidae and Channichthyidae) and Arctic cods (Gadidae). From the Antarctic fish Dissostichus mawsoni, it was showed that the AFGP genes were derived from a pancreatic trypsinogen encoding gene through a special mechanism which does not include recycling of the existing gene proteins.

The distinct portion of AFGP gene which encodes the function of ice-binding arises from the enrolment and the repetition of a small region within the boundary between the first intron and second exon of trypsinogen gene. Replication, amplification and repeated duplication of this portion resulted to producing 41 tandemly repeated segments which have sequences nearly identical to trypsinogen at either end. This divergence between the small sequence of nothothenioid AFGP and trypsin genes signifies that the conversion to ice-growth inhibition gene from protein gene had occurred approximately when Antarctic Ocean started to go to the freezing point. This unique conversion is a good example on how an old protein gene brought into existence a new gene with an entirely new protein function.

The notothenioid AFGP gene was compared to another study on the sequence of Arctic cod Boreogadus saida and similarities were found in their polyprotein structures which have multiple AFGP coding sequences copies which are linked by small spacers that could be cleaved. Despite this, by detailed analysis, molecular evidences strongly argue on complete independent evolution of the AFGP genes from the two fish species. The evidences include a difference in their signal peptide sequences, difference in their mechanisms of processing their polyprotein precursors due to dissimilar spacer sequences on the linkage of the AFGP molecules and polyprotein, discrete codon bias for the AFGP tripeptide on its nine nucleotide sequence, and difference on the genomic AFGP loci in nototheninoid and cod. Hence, the higly-identical AFGPs on these two completely unrelated fish is one of the few examples of protein change by convergent evolution, as similar proteins were developed through similar environmental pressure. Also, the various lengths of AFGPs produced were found out not to be caused by cleaving large ones into smaller sizes or by small AFGPs splicing, but every AFGPs are clearly encoded as individual copies within the genes.