Cloning, expression, and activity of type IV antifreeze protein from cultured subtropical olive flounder (Paralichthys olivaceus)

Abstract

Antifreeze proteins (AFPs) lower the freezing point but not the melting point of aqueous solutions by inhibiting the growth of ice crystals via an adsorption-inhibition mechanism. However, the function of type IV AFP (AFP IV) is questionable, as its antifreeze activity is on the verge of detectable limits, its physiological concentration in adult fish blood is too low to function as a biological antifreeze, and its homologues are present even in fish from tropic oceans as well as freshwater. Therefore, we speculated that AFP IV may have gained antifreeze activity not by selective pressure but by chance. To test this hypothesis, we cloned, expressed, and assayed AFP IV from cultured subtropical olive flounder (Paralichthys olivaceus), which do not require antifreeze protein for survival. Among the identified expressed sequence tags of the flounder liver sample, a 5′-deleted complementary DNA (cDNA) sequence similar to the afp4 gene of the longhorn sculpin was identified, and its full-length cDNA and genome structure were examined. The deduced amino acid sequence of flounder AFP IV shared 55, 53, 52, and 49 % identity with those of Pleuragramma antarcticum, Myoxocephalus octodecemspinosus, Myoxocephalus scorpius, and Notothenia coriiceps, respectively. Furthermore, the genomic structure of this gene was conserved with those of other known AFP IVs. Notably, the recombinant AFP IV showed a weak but distinct thermal hysteresis of 0.07 ± 0.01°C at the concentration of 0.5 mg/mL, and ice crystals in an AFP IV solution grew star-shaped, which are very similar to those obtained from other polar AFP IVs. Taken together, our results do not support the hypothesis of evolution of AFP IV by selective pressure, suggesting that the antifreeze activity of AFP IV may have been gained by chance.

Characteristics of Antifreeze Proteins

“…Gauthier et al (2008) proposed that, although type IV has the potential to develop into a functional AFP, it has not been selected for this purpose due to the presence of other functional AFPs. This is supported by the presence of type IV AFP in temperate, subtropical and tropical species, including species living in fresh water (Liu et al 2009;Xiao et al 2014;Lee et al 2011;Lee and Kim 2016). These species have no need for any freeze protection, and type IV AFP may instead be involved in embryogenesis, since several of its homologues are essential in this process.…”

“…Its proposed structure consists of four amphipathic α-helixes of similar length folded in a four-helix bundle (Deng and Laursen 1998). Type IV AFP has been found in many species, including Arctic longhorn sculpin (Myoxocephalus octodecemspinosus) and shorthorn sculpin (M. scorpius) (Deng and Laursen 1998;Gauthier et al 2008) and two Antarctic nototheniids, Pleuragramma antarcticum and Notothenia coriiceps (Lee et al 2011;Lee and Kim 2016). However, its role as a functional AFP has been questioned, since it is a very weak AFP, causing only 0.07 C thermal hysteresis at a concentration of 0.5 mg/mL, and is present in blood in concentrations less than 100 μg/mL, far too low to protect these fishes against freezing in icy waters (Gauthier et al 2008;Lee and Kim 2016 AU1 ).…”

Molecular Characterization and mRNA Expression of ISP2 and ISP4 in the Large Yellow Croaker (Larimichthys Crocea) Under Acute Cold Stress

“…Based on these ndings, Gauthier et al (2008) speculated that ISP4 may not be a key antifreeze protein in the blood of polar sh when other types of antifreeze protein are present. Surprisingly, in addition to polar sh, ISP4 is also found in warm-temperate sh and freshwater sh (Nishimiya et al, 2008;Zhang et al, 2009;Kim, 2015;Lee and Kim, 2016). These reports indicate that ISP4 may have other effects as well as antifreeze activity in warm-temperate sh.…”

“…Ice structure proteins (ISPs), also known as antifreeze protein, play a role in inhibiting the growth of ice crystals, modifying ice morphology and inhibiting the recrystallization of ice via adsorption-inhibition (DeVries and Wohlschlag, 1969; Lee and Kim, 2016). ISPs lower the point of freezing non-colligatively but do not change the point of melting through adsorption on the surface of ice crystals and then inhibit ice growth, which increases the temperature gap between melting point and freezing point, and this temperature gap is termed thermal hysteresis (TH) (Barrett, 2001).…”

Transcriptomics of a Greenlandic Snailfish Reveals Exceptionally High Expression of Antifreeze Protein Transcripts

“…Deng and Laursen 30 showed that the concentration of the LS-12 gene in the longhorn sculpin, M. octodecimspinosis , is so low that without concentration of the protein, antifreeze activity could not be detected. A protein homologlous to the M. octodecimspinosis LS-12/TypeIV AFP from the subtropical olive flounder ( Paralichthys olivaceus ), 31 which does not require AFPs for survival, has been cloned, heterologously expressed (in E. coli ) and assayed for antifreeze activity. Based on its comparatively “very weak” thermal hysteresis activity (0.07 ± 0.01°C at 0.5 mg/mL), the P. olivaceus LS-12 homolog would not prevent freezing at subzero temperatures.…”