Date Published: November 7, 2012
Publisher: Public Library of Science
Author(s): Shuaiqi Guo, Christopher P. Garnham, John C. Whitney, Laurie A. Graham, Peter L. Davies, Michael Hensel. http://doi.org/10.1371/journal.pone.0048805
A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.
Antifreeze proteins (AFPs) were initially characterized in marine fishes ,  where they protect their hosts from freezing by binding to, and preventing the growth of, seed ice crystals . AFPs lower the freezing temperature of a solution containing ice below the melting point of the ice. This difference between the freezing and melting temperatures is called thermal hysteresis (TH) and is used as a measure of antifreeze activity. AFPs were subsequently found in freeze-tolerant organisms ,  where, rather than preventing freezing, they stop ice crystals in frozen tissues from growing larger through the process of ice recrystallization (IRI) .
Here we report the purification of wild-type MpAFP, its full amino acid sequence, and show that its tremendous size (ca. 1.5 MDa) is the result of ca. 120 tandem copies of an identical 104-aa repeat that is predicted to form an Ig-like beta-sandwich domain. The region is flanked by one or two non-identical repeats (<65% identity) on each side. All of the other repeats are 100% identical at the DNA level, as shown by sequencing of numerous genomic clones. This suggests that their expansion in M. primoryensis, likely by duplication followed by multiple rounds of unequal recombination , is a relatively recent event. Each of the ∼120 Ig-like domains is expected to fold as an independent unit, forming a chain as observed for a combinatorial model of mouse cadherin based on electron tomography  and X-ray crystallography of five repeats . As the monomers of cadherin and those predicted for MpAFP are spaced approximately 50 Å apart, MpAFP could be over 0.6 µm long, or 20% of the length of the cell. This implies that extreme length is a necessary property of this protein. Source: http://doi.org/10.1371/journal.pone.0048805