Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period

Abstract

Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago.

Figure 1

Comparison of ice-shaping in collembolan homogenates. Freeze-dried Collembola were gently homogenized in buffer (8:1 v/w) and the supernatants were assayed for antifreeze activity (TH). For each species (left column), an image was captured from the video during the TH measurement (middle column) and just as the freezing point was exceeded (right column). The positive control is type I AFP from winter flounder (Pseudopleuronectes americanus). Negative control is buffer (50 mM Tris–HCl (pH 7.8), 150 mM NaCl, 1 mM phenylthiocarbamide and 1 × EDTA-free Roche protease inhibitor cocktail).

Figure 2

MALDI spectra of purified collembolan AFP extracts. Proteins from: (A) Protaphorura pseudovanderdrifti, (B) Cryptopygus antarcticus, (C) Ceratophysella denticulata, (D) Megaphorura arctica, (E) Folsomia candida, and (F) Granisotoma rainieri were purified with four rounds of ice-affinity purification and subjected to MALDI-MS. Major peak masses are labelled.

Figure 3

Sequence alignment of the 8.5-kDa CaAFP isoforms. (A) The amino acid sequences of CaAFP isoforms from ESTs and the generated transcriptome were aligned. Identical amino acid residues are highlighted in grey. Glycine residues are coloured blue. Cysteine residues are coloured red and highlighted yellow. The signal peptides are shown in lowercase and the coding sequence is shown is uppercase. The rectangles below show putative PPII helices for the IBS (blue) and non-IBS (red) faces. CaAFPa-1, OQ445586; CaAFPa-2, GR870234; CaAFPa-3, OQ445587; CaAFPa-4, FF279148; CaAFPa-5, OQ445583; CaAFPa-6, GR869204; CaAFPa-7, FF278983. (B) Schematic of six polyproline type II helical bundle. The antiparallel helices are connected by loop regions (not shown) and arrange into two layers. The ice-binding surface (blue) and non-ice-binding surface (red) are shown. Hydrogen bonding between helices stabilizes the fold.

Figure 4

Sequence alignment of the 15-kDa CaAFP isoforms. The amino acid sequences of CaAFP isoforms from the generated transcriptome were aligned for the (A) first and (B) second groups. (C) The third CaAFP isoform (CaAFPb-8) is aligned to one sequence from the first group (CaAFPb-3) and the second group (CaAFPb-6). The colouring is the same as in Fig. 3. CaAFPb-1, OQ445590; CaAFPb-2, OQ445584; CaAFPb-3, OQ445585; CaAFPb-4, OQ445589; CaAFPb-5, OQ445588; CaAFPb-6, OQ445592; CaAFPb-7, OQ445593; CaAFPb-8, OQ445591.

Figure 5

Polyproline type II helical bundle schematics. The protein sequences of FcAFP and CcAFP arranged into individual polyproline helices. (A) FcAFP can be arranged into 9 helices and (B) CcAFP can be arranged into 12 helices Colouring is the same as in Fig. 3.

Figure 6

Taxonomic tree of antifreeze-protein-producing Collembola. A taxonomic tree for species from the four orders of Collembola (Entomobryomorpha, Poduromorpha, Symphypleona, and Neelipleona) was generated based on NCBI taxonomy. Species with and lacking TH activity are coloured in red and blue, respectively, and groups not tested are in black. Species assayed in this paper are bolded and all other species not bolded were tested by Zettel, except Gomphiocephalus hodgsoni and Cryptopygus terranovus (syn. Gressittacantha terranova). Species with a star produced glycine-rich AFPs. Gomphiocephalus hodgsoni with a red square is a proposed cystine- and histidine-rich AFP.

Figure 7

The relationship between collembolan phylogeny and ice ages. A phylogenetic tree showing the timeline of divergence in Collembola. The shaded green, purple, and yellow show estimated times for divergence of orders, families, and genera/ species, respectively. Number of AFP-producing species are shown below the order name. The red arrow indicates the emergence of fish AFPs during the Cenozoic era. Icebergs display the timespan of each respective ice age.