Nonhepatic origin of notothenioid antifreeze reveals
pancreatic synthesis as common mechanism in polar
fish freezing avoidance
Chi-Hing C. Cheng*†, Paul A. Cziko*, and Clive W. Evans‡
*Department of Animal Biology, University of Illinois, Urbana, IL 61801; and‡Molecular Genetics and Development, School of Biological Sciences,
University of Auckland, Auckland, New Zealand
Communicated by George N. Somero, Stanford University, Pacific Grove, CA, May 9, 2006 (received for review March 30, 2006)
Phylogenetically diverse polar and subpolar marine teleost fishes
have evolved antifreeze proteins (AFPs) or antifreeze glycopro-
teins (AFGPs) to avoid inoculative freezing by internalized ice. For
over three decades since the first fish antifreeze (AF) protein was
discovered, many studies of teleost freezing avoidance showed
hepatic AF synthesis and distribution within the circulation as
pivotal in preventing the blood, and therefore the fish, from
freezing. We have uncovered an important twist to this long-held
paradigm: the complete absence of liver synthesis of AFGPs in any
life stage of the Antarctic notothenioids, indicating that the liver
plays no role in the freezing avoidance in these fishes. Instead, we
found the exocrine pancreas to be the major site of AFGP synthesis
and secretion in all life stages, and that pancreatic AFGPs enter the
intestinal lumen via the pancreatic duct to prevent ingested ice
from nucleating the hyposmotic intestinal fluids. AFGPs appear to
remain undegraded in the intestinal milieu, and the composition
and relative abundance of intestinal AFGP isoforms are nearly
identical to serum AFGPs. Thus, the reabsorption of intact pancre-
as-derived intestinal AFGPs, and not the liver, is the likely source of
circulatory AFGPs in notothenioid fishes. We examined diverse
northern fish taxa and Antarctic eelpouts with hepatic synthesis of
bloodborne AF and found that they also express secreted pancre-
atic AF of their respective types. The evolutionary convergence of
this functional physiology underscores the hitherto largely unrec-
ognized importance of intestinal freezing prevention in polar
teleost freezing avoidance, especially in the chronically icy Antarc-
antifreeze glycoprotein-null liver ? antifreeze paradigm shift ? evolutionary
adaptation ? intestinal freeze avoidance ? functional convergence
(20–35 mg?ml) of protein antifreeze (AF) (a family of AF glyco-
proteins; AFGPs) in their blood and other body fluids (4, 5). The
synthesis of blood AFGPs in Antarctic notothenioids has histori-
cally been attributed to the liver (6, 7), because the vertebrate liver
is well known as the major source of secreted plasma proteins (8,
9), and thus there were no a priori reasons to invoke a different
source for the abundant plasma AFGPs. Also contributing to the
prevailing notion of universal hepatic AF synthesis is the readily
demonstrable liver expression of AF mRNA by Northern blots
and?or cDNA cloning in all other AF protein (AFP)-bearing fish
AFGP-bearing polar cod (15). However, definitive verification of
liver biosynthesis of AFGPs in Antarctic notothenioids has been
lacking. Early radioactive-tracer investigations of notothenioid
AFGP biosynthesis could not rule out a nonhepatic synthesis site,
because the appearance of labeled AFGPs and non-AFGP plasma
proteins in the blood was drastically asynchronous (AFGPs lagged
nototheniid Notothenia coriiceps showed AFGP mRNA hybridiza-
tion, but an unusually large amount (50 ?g) of polyA? RNA was
he predominant endemic Antarctic marine teleost group, the
notothenioid fishes (1), inhabit the world’s coldest and iciest
required (16), contradictory to liver being a strong expression site.
If there is little or no hepatic AFGP biosynthesis, the tissue origin
of the abundant bloodborne AFGPs in Antarctic notothenioids
returns as an unsolved fundamental question ?30 years after the
discovery of the protein (5). In our prior elucidations of the
evolution of the notothenioid AFGP gene from a pancreatic
TLP cDNAs were obtained from exocrine pancreas RNA, indicat-
ing that exocrine pancreas is an AFGP expression site (17, 18). In
this comprehensive study, we confirm that the exocrine pancreas is
the major AFGP synthesis site in Antarctic notothenioid fishes
from hatching through adulthood, whereas the liver is AFGP-
expression null in all life stages. We show that pancreatic AFGPs
are secreted into the intestine, and, with additional AFGP contri-
bution from the anterior portion of the stomach (the only other
icy Antarctic waters. The apparently undegraded intestinal AFGPs
raise the possibility that plasma AFGPs in the notothenioids are
derived from reabsorption of these intact macromolecules, which
despite the absence of liver AF synthesis and secretion. In addition,
we examine diverse AF-bearing species from both north- and
south-polar and subpolar regions and confirm that they have
which attests to intestinal freezing prevention as a vital and integral
component of the repertoire of teleost freeze-avoidance strategies.
AFGP mRNA Expression in Adult Notothenioid Tissues. To determine
the tissue site or sites of AFGP synthesis in adult notothenioids,
Northern blots of total RNA from different tissues were hybridized
to an AFGP gene (coding region only) probe (Fig. 1). Like the
phyletically basal AFGP-null New Zealand notothenioid Bovichtus
each of the five endemic Antarctic notothenioid families (Noto-
theniidae, Artedidraconidae, Harpagiferidae, Bathydraconidae,
and Channichthyidae; detailed species information in Table 1,
showed no hybridization, as opposed to the intense hybridization in
pancreatic RNA sampled from the Antarctic nototheniid Dissos-
tichus mawsoni included in the blot (Fig. 1A Upper). As a control,
Conflict of interest statement: No conflicts declared.
Abbreviations: AF, antifreeze; AFGP, antifreeze glycoprotein; AFP, antifreeze protein; GI,
gastrointestinal; IF, intestinal fluid; PF, pancreatic fluid; TLP, trypsinogen-like protease.
Data deposition: The 26 sequences reported in this paper have been deposited in the
GenBank database (accession nos. DQ062435–DQ062459 and DQ394083). Details listed in
Table 4, which is published as supporting information on the PNAS web site.
†To whom correspondence should be addressed at: Department of Animal Biology,
515 Morrill Hall, 505 South Goodwin Avenue, University of Illinois, Urbana, IL 61801.
© 2006 by The National Academy of Sciences of the USA
July 5, 2006 ?
vol. 103 ?
no. 27 ?
Our proposal of reabsorption of intestinal AFGP molecules as the
source of blood AF in Antarctic notothenioids is an experimentally
testable hypothesis that may further our knowledge of transport
physiology of macromolecules. Although freeze avoidance of the
blood remains crucial, our finding that divergent AF-bearing fish
of secreted AF regardless of AF type attests to freezing prevention
of the hyposmotic GI fluids as a vital component of teleost freeze
avoidance strategies. The universal pancreatic AF mRNA expres-
expectedly stimulate further investigations into the details of GI
freeze avoidance across other fish taxa and life stages.
Materials and Methods
Animals and Sampling. Fish were collected by traps or line through
sea ice or in open water, or by trawls from research vessels.
Blood was obtained from the caudal vein by using a needle and
syringe, and tissues were dissected, frozen in liquid nitrogen, and
stored at ?80°C. Intestinal fluid from unfed fish was drained
and collected from ligated intestines after they were blotted dry of
AF-bearing peritoneal fluid and removed from the abdomen.
Pancreatic fluid (up to ?400 ?l) from unfed D. mawsoni was
sampled with an insulin syringe from the pancreatic duct, which
distends into a visible small reservoir when filled with pancreatic
secretion. All animal handling was carried out in accordance with
institutionally approved protocols.
Northern Blot and RT-PCR Amplification. Total RNA was isolated
from tissues and 5–15 ?g per sample were used in Northern blot
analyses as described (15). Blots were stripped of hybridized probe
by using 0.1% SDS at 100°C before hybridization with a second
probe. Sequences of the primers used to RT-PCR amplify the
cDNA of each type of AF protein, elongation factor 1-? subunit,
and pancreatic trypsinogen are given in Table 3, which is
published as supporting information on the PNAS web site.
Amplified cDNA products were cloned into pGemTeasy (Pro-
mega) and sequenced with BigDye Terminator v.3 cycle se-
quencing (Applied Biosystems).
Osmolarity, Melting Point, and Freezing Point Determinations of Fish
pressure osmometer, and melting and freezing points were deter-
mined by using single crystal seed ice in a Clifton nanoliter
osmometer as described (25).
AFGPs were treated with trichloroacetic acid (5% final concen-
tration), and the acid-resistant AFGPs were purified from the
soluble fraction by dialysis and lyophilization. About 400 ?g of
purified AFGPs were fluorescently labeled with fluorescamine
polyacrylamide gel as described (15). Type III AFP from fluid
samples of Antarctic eelpouts were purified by G75 Sephadex
(Amersham Pharmacia) gel filtration column chromatography.
The AFP-containing column fractions were lyophilized and ?30
?g were electrophoresed on 15% SDS?polyacrylamide gel as
Immunodetection of AFGPs. Whole larvae or dissected tissues were
fixed immediately in cold 4% paraformaldehyde prepared in no-
tothenioid PBS (0.1 M sodium phosphate, pH 7.6, and adjusted to
450 mOsm with NaCl). Frozen sections (5–15 ?m) were pretreated
with PDB (notothenioid PBS containing 1% vol?vol DMSO and
1% wt?vol Ig-free BSA) for 20 min at 20°C, washed, and then
four times for 5 min with PDB and incubated for 1 h at room
temperature with an Alexa Fluor 546-labeled secondary goat
anti-rabbit IgG (Molecular Probes) diluted 1:1000 with PDB. After
0.2 ?g ml?1DAPI for 5 min at 20°C between the first and second
washes, the sections were mounted under a coverslip by using
FluoroGuard (Bio-Rad). Control sections were treated with pre-
immune serum instead of the primary antibody and were consis-
tently negative (results not shown).
We thank Clarabelle DeVries for assistance with the AFGP character-
ization and Vivian Ward with the immunodetection images. This work
was supported by U.S. National Science Foundation Office of Polar
Programs Grants OPP0002654 and OPP0231006 (to C.-H.C.C.). C.W.E.
acknowledges additional support from the University of Auckland
1. Eastman, J. T. (2005) Polar Biol. 28, 94–107.
2. Dayton, P. K., Robilliard, G. A. & DeVries, A. L. (1969) Science 163, 273–274.
3. Hunt, B. M., Hoefling, K. & Cheng, C.-H. C. (2003) Antarct. Sci. 15, 333–338.
4. DeVries, A. L. (1983) Annu. Rev. Physiol. 45, 245–260.
5. DeVries, A. L. (1971) Science 172, 1152–1155.
Physiol. B Biochem. Mol. Biol. 62, 179–183.
7. O’Grady, S. M., Clarke, A. & DeVries, A. L. (1982) J. Exp. Zool. 220, 179–189.
8. Miller, L. L., Bly, C. G., Watson, M. L. & Bale, W. F. (1951) J. Exp. Med. 94,
9. Haschemeyer, A. E. V. & Smith, M. A. (1979) Biol. Bull. (Woods Hole, Mass.)
10. Ewart, K. V., Rubinsky, B. & Fletcher, G. L. (1992) Biochim. Biophys. Res.
Commun. 185, 335–340.
11. Ewart, K. V. & Fletcher, G. L. (1993) Mol. Mar. Biol. Biotechnol. 2, 20–27.
12. Gong, Z., Ewart, K. V., Hu, Z., Fletcher, G. L. & Hew, C. L. (1996) J. Biol.
Chem. 271, 4106–4112.
13. Wang, X., DeVries, A. L. & Cheng, C.-H. C. (1995) Mol. Mar. Biol. Biotechnol.
14. Deng, G., Andrews, D. W. & Laursen, R. A. (1997) FEBS Lett. 402, 17–20.
15. Chen, L., DeVries, A. L. & Cheng, C.-H. C. (1997) Proc. Natl. Acad. Sci. USA
(1990) Proc. Natl. Acad. Sci. USA 87, 9265–9269.
17. Chen, L., DeVries, A. L. & Cheng, C.-H. C. (1997) Proc. Natl. Acad. Sci. USA
18. Cheng, C.-H. C. & Chen, L. (1999) Nature 40, 443–444.
19. Eastman, J. T. & DeVries, A. L. (1997) Polar Biol. 17, 1–13.
20. Smith, L. S. (1989) in Fish Nutrition, ed. Halver, J. E. (Academic, San Diego),
21. Kirsch, R. & Meister, M. F. (1982) J. Exp. Biol. 98, 67–81.
22. DeVries, A. L. & Cheng, C.-H. C. (2005) in The Physiology of Polar Fishes, eds.
Farrell, A. P. & Steffensen, J. F. (Elsevier, San Diego), Vol. 22, pp. 155–201.
23. Fletcher, G. L., Hew, C. L., Li, X., Haya, K. & Kao, M. H. (1985) Can. J. Zool.
24. Wang, X., DeVries, A. L. & Cheng, C.-H. C. (1995) Biochim. Biophys. Acta
25. Cziko, P. A., Evans, C. W., Cheng, C.-H. C. & DeVries, A. L. (2006) J. Exp.
Biol. 209, 407–420.
26. Hernandez-Blazquez, F. J. & Cunha da Silva, J. R. M. (1998) Can J. Zool. 76,
27. Sire, M. F. & Vernier, J.-M. (1992) Comp. Biochem. Physiol. A Physiol. 103,
28. Eastman, J. T. & DeVries, A. L. (1986) J. Fish Biol. 29, 649–662.
29. Eastman, J. T. (1993) Antarctic Fish Biology: Evolution in a Unique Environment
(Academic, San Diego).
30. Coleman, R. (1987) Biochem. J. 244, 249–261.
31. LaRusso, N. F. (1984) Am. J. Physiol. 247, G199–G205.
32. Eurell, J. A. & Haensly, W. E. (1982) J. Fish Biol. 21, 113–125.
33. Smith, H. W. (1930) Am. J. Physiol. 93, 480–505.
34. O’Grady, S. M., Ellory, J. C. & DeVries, A. L. (1982) J. Exp. Biol. 98, 429–438.
35. O’Grady, S. M., Ellory, J. C. & DeVries, A. L. (1983) J. Exp. Biol. 104, 149–162.
36. Præbel, K. & Ramløv, H. (2005) J. Exp. Biol. 208, 2609–2613.
www.pnas.org?cgi?doi?10.1073?pnas.0603796103Cheng et al.