Convergent evolution of antifreeze glycoproteins in Antarctic notothenioid fish and Arctic cod

Abstract

Antarctic notothenioid fishes and several northern cods are phylogenetically distant (in different orders and superorders), yet produce near-identical antifreeze glycoproteins (AFGPs) to survive in their respective freezing environments. AFGPs in both fishes are made as a family of discretely sized polymers composed of a simple glycotripeptide monomeric repeat. Characterizations of the AFGP genes from notothenioids and the Arctic cod show that their AFGPs are both encoded by a family of polyprotein genes, with each gene encoding multiple AFGP molecules linked in tandem by small cleavable spacers. Despite these apparent similarities, detailed analyses of the AFGP gene sequences and substructures provide strong evidence that AFGPs in these two polar fishes in fact evolved independently. First, although Antarctic notothenioid AFGP genes have been shown to originate from a pancreatic trypsinogen, Arctic cod AFGP genes share no sequence identity with the trypsinogen gene, indicating trypsinogen is not the progenitor. Second, the AFGP genes of the two fish have different intron-exon organizations and different spacer sequences and, thus, different processing of the polyprotein precursors, consistent with separate genomic origins. Third, the repetitive AFGP tripeptide (Thr-Ala/Pro-Ala) coding sequences are drastically different in the two groups of genes, suggesting that they arose from duplications of two distinct, short ancestral sequences with a different permutation of three codons for the same tripeptide. The molecular evidence for separate ancestry is supported by morphological, paleontological, and paleoclimatic evidence, which collectively indicate that these two polar fishes evolved their respective AFGPs separately and thus arrived at the same AFGPs through convergent evolution.

Figure 1

Polyacrylamide gel of fluorescently labeled AFGPs from Antarctic notothenioid D. mawsoni (Dm) and Arctic cod B. saida (Bs) and the amino acid compositions of the three size groups of Arctic cod AFGPs. The two polar fishes show comparable size heterogeneity, especially in the range of AFGPs 6–8.

Figure 2

Nucleotide sequence of an AFGP polyprotein gene and the encoded amino acids from B. saida subclone Bs3–1 aligned with a 5′ AFGP cDNA (A), and D. mawsoni subclone Dm3L (B). Dots represent identical nucleotides between the B. saida AFGP gene and cDNA sequences. The introns are shown in lowercase letters. The distinct spacer sequences linking multiple AFGP sequences are indicated in shaded boxes (Bs3–1) or open boxes (Dm3L). ▾, Putative cleavage site for the signal peptide in both genes. The underlined sequences in Dm3L (B) are regions that share high sequence identities with notothenioid trypsinogen gene (12); not all of the similar intron sequence is shown.

Figure 3

Exon–intron organizations of B. saida and D. mawsoni AFGP genes, showing also the dissimilar signal peptide sequences of the two AFGP polyproteins. The coding region for the signal peptide in each gene is indicated by the shaded areas. ▾, Putative cleavage site for the signal peptide.

Figure 4

(A) Northern blot of liver poly(A)⁺ RNA from Arctic cod (Bs) showing multiple AFGP messages. ◃, Predominant 1.8-kb mRNA. (B) Southern blot of genomic DNA digested with MboI from D. mawsoni (Dm) and B. saida (Bs) showing AFGP multigene family. Bracket indicates the range of DNA fragments recovered for construction of B. saida partial genomic library.