Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin

Abstract

Beetle hyperactive antifreeze protein (AFP) has a unique ability to maintain a supercooling state of its body fluids, however, less is known about its origination. Here, we found that a popular stag beetle Dorcus hopei binodulosus (Dhb) synthesizes at least 6 isoforms of hyperactive AFP (DhbAFP). Cold-acclimated Dhb larvae tolerated −5 °C chilled storage for 24 h and fully recovered after warming, suggesting that DhbAFP facilitates overwintering of this beetle. A DhbAFP isoform (~10 kDa) appeared to consist of 6−8 tandem repeats of a 12-residue consensus sequence (TCTxSxNCxxAx), which exhibited 3 °C of high freezing point depression and the ability of binding to an entire surface of a single ice crystal. Significantly, these properties as well as DNA sequences including the untranslated region, signal peptide region, and an AFP-encoding region of Dhb are highly similar to those identified for a known hyperactive AFP (TmAFP) from the beetle Tenebrio molitor (Tm). Progenitor of Dhb and Tm was branched off approximately 300 million years ago, so no known evolution mechanism hardly explains the retainment of the DNA sequence for such a lo­ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.

The Spruce Budworm Genome: Reconstructing the Evolutionary History of Antifreeze Proteins

“…However, HGT between Notocelia and Choristoneura could explain the location of AFP-containing regions on non-homologous chromosomes and the apparent absence of AFP genes in C. pomonella , A. turbidana , and A. honmai . HGT has recently been proposed as a potential explanation for the presence of very similar AFPs in two distantly related coleopteran taxa ( Arai et al 2021 ). In insects, virus-mediated gene transfer between species has been well documented ( Gilbert et al 2016 ), sometimes involving portions of DNA large enough to harbor genes such as those encoding AFPs (see Drezen et al 2017 ; Zakharov 2016 ).…”

Subzero Nonfreezing Hypothermia with Insect Antifreeze Protein Dramatically Improves Survival Rate of Mammalian Cells

“…The solution was then warmed to nearly 0 °C to retain a single ice crystal. This ice crystal changed in appearance from a rounded, disk-like shape to a lemon-like morphology or a hexagonal bipyramid when we slightly lowered the stage temperature, indicating AFP-binding [ 19 , 50 ]. For such an ice crystal in the AFP-bound state, we measured the temperature at which the crystal started to melt ( T m ).…”

Advances in Antifreeze Molecules: From Design and Mechanisms to Applications

“…45 They possess similar repeat motifs, which are thought to be gene-transferred from TmAFP. 45 Radically different from TmAFPs, CfAFPs possess a left-handed helix conformation with triangular cross sections. Besides, it also contains regular rectangle sides consisting of TxT motifs to form IBSs, 46 which can bind to the basal and prism planes of ice crystals, 47 resulting in a TH activity of 4.7 °C at 0.05 mM.…”

“…These motifs are arrayed on the conserved side to form ice-binding sites (IBSs), accounting for 5.5 °C TH activity at 1 mg/mL . Besides, many other beetle AFPs have been found highly homologous to Tm AFPs, such as Dendroides canadensis AFPs ( Dc AFPs), Microdera dzhungarica punctipennis AFPs ( Mdp AFPs), Anatolica polita AFPs ( Ap AFPs), and Dorcus hopei binodulosus AFPs ( Dhb AFPs) . They possess similar repeat motifs, which are thought to be gene-transferred from Tm AFP .…”