Molecular basis of ice-binding and cryopreservation activities of type III antifreeze proteins

https://doi.org/10.1016/j.csbj.2021.01.016Get rights and content
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Highlights

  • The QAE2ACT and SP ACT mutants showed full TH and IRI activities.

  • Active AFPs effectively preserved intact follicle and prevented DSB damage.

  • Active AFPs exhibited unique structural feature in the first 310 helix of the IBS.

  • Unique structure of the IBS determines TH, IRI, and cryopreservation activities.

Abstract

Antifreeze proteins (AFPs) can inhibit the freezing of body fluid at subzero temperatures to promote the survival of various organisms living in polar regions. Type III AFPs are categorized into three subgroups, QAE1, QAE2, and SP isoforms, based on differences in their isoelectric points. We determined the thermal hysteresis (TH), ice recrystallization inhibition (IRI), and cryopreservation activity of three isoforms of the notched-fin eelpout AFP and their mutant constructs and characterized their structural and dynamic features using NMR. The QAE1 isoform is the most active among the three classes of III AFP isoforms, and the mutants of inactive QAE2 and SP isoforms, QAE2ACT and SPACT, displayed the full TH and IRI activities with resepect to QAE1 isoform. Cryopreservation studies using mouse ovarian tissue revealed that the QAE1 isoform and the active mutants, QAE2ACT and SPACT, more effectively preserved intact follicle morphology and prevented DNA double-strand break damage more efficiently than the inactive isoforms. It was also found that all active AFPs, QAE1, QAE2ACT, and SPACT, formed unique H-bonds with the first 310 helix, an interaction that plays an important role in the formation of anchored clathrate water networks for efficient binding to the primary prism and pyramidal planes of ice crystals, which was disrupted in the inactive isoforms. Our studies provide valuable insights into the molecular mechanism of the TH and IRI activity, as well as the cryopreservation efficiency, of type III AFPs.

Abbreviations

AFP
Antifreeze protein
TH
thermal hysteresis
IBP
ice-binding protein
IRI
ice recrystallization inhibition
OT
ovarian tissue
QAE
quaternary-amino-ethyl
SP
sulfopropyl
IBS
ice-binding surface
H-bond
hydrogen bond
wt
wild-type
nfeAFP
notched-fin eelpout AFP
DSB
double-strand break
TUNEL
terminal deoxynucleotidyl transferase dUTP nick end labelling
EG
ethylene glycol
DMSO
dimethyl sulfoxide
D-PBS
Dulbecco’s phosphate-buffered saline
RT
room temperature
CPA
cryoprotective agent

Keywords

Antifreeze protein
Cryopreservation
Ice crystallization inhibition
NMR
Thermal hysteresis

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1

These authors contributed equally to this work.