Biochemistry of fish antifreeze proteins

PETER L. DAVIES*l AND CHOY L. HEW1

#{176}Department
of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6 and tResearch Institute, Hospital
for Sick Children, Toronto, and Departments of Clinical Biochemistry and Biochemistry, University of Toronto, Toronto,
Ontario, Canada M5G 1L5

water (-0.45 M) depresses its freezing point down to

ABSTRACT - -1.9#{176}C,
whereas a typical teleost serum will freeze at
-0.7#{176}C(1). This discrepancy of
-1#{176}Cin freezing
Four distinct macromolecular antifreezes have been points means that unprotected in polar and
teleosts
isolated and characterized from different marine fish. north temperate Waters would be at risk of freezing to
These include the glycoprotein antifreezes (Mr 2.5 death when their temperature falls below -0.7#{176}C.
33 K), which are made up of a repeating tripeptide Although there is evidence that some fish can survive
(Ala-Ala-Thr) with a disaccharide attached to the at these temperatures in deep water in a supercooled
threonyl residues, and three antifreeze protein (AFP) state (2), this is not possible in shallow water where con-
types. Type I is an alanine-rich, amphiphilic, cr-helix tact with ice negates supercooling. When Scholander
(Mr 3-5 K); type II is a larger
protein (Mr 14 K) with
and co-workers (3) first investigated this problem using
a high content of reverse turns and five disulfide inshore Arctic fish, they observed unusually low serum
bridges;and type III is intermediate in size (Mr 6-7 K) freezing temperatures - 1.4#{176}C).
The nature
(- of the
with no distinguishing features of secondary structure antifreeze responsible for this freezing point depression
or amino acid composition. Despite their marked struc- eluded these researchers. However, in 1969 DeVries and
tural differences, all four antifreeze types appear to Wohlschlag (4) reported that the antifreeze in the blood
function in the same way by binding to the prism faces of nototheniid fish was a proteinaceous macromolecule
of ice crystals and inhibiting growth along the a-axes.
that was soluble in 10% trichloroacetic acid. Further
It is suggested that type I AFP binds preferentially to characterization revealed that this antifreeze comprised
the prism faces as a result of interactions between the a set of glycoproteins that are each made up of a tripep-
helix macrodipole and the dipoles on the water mole- tide repeat (Ala-Ala-Thr)n with a disaccharide moiety
cules in the ice lattice. Binding is stabilized by hydro- attached to the threonyl residues (5). As more fish have
gen bonding, and the amphiphilic character of the been surveyed for antifreeze activity, three distinct anti-
helix results in the hydrophobic phase of the helix be- freeze protein (AFP) types have been characterized in
ing exposed to the solvent. When the solution tempera- addition to the antifreeze glycoprotein (AFGP) (6).
ture is lowered further, ice crystal growth occurs These are the alanine-rich, a-helical AFP of righteye
primarily on the uncoated, unordered basal plane flounders and sculpins (type I), the cystine-rich AFP of
resulting in bipyramidal-shaped crystals. The struc- the sea raven (type II), and an AFP (type III) found in
tural features of type I AFP that could contribute to
eel pouts, which lacks distinctive features in its compo-
this mechanism of action are reviewed. Current
sition and sequence (Fig. 1).
challenges lie in solving the other antifreeze structures
and interpreting them in light of what appears to be a
common mechanism of action. -DAVIES, P. L.; HEW, THE INTERACTION OF AFP AND AFGP
C. L. Biochemistry of fish antifreeze proteins. FASEBJ. WITH ICE
4: 2460-2468; 1990.
Despite the marked differences in amino acid composi-
tion and protein structure between these macromolecu-
Key Words: antifreeze protein/glycoprotein ice crystal givwth
lar antifreeze types, they all appear to interact with ice
amphiphilicity helix macrodipole . structure/function rela-
in the same way. They have no untoward effect on the
tionships
melting point of ice formed in their presence, and any
depression of the melting point is entirely explained by

WHY SOME FISH HAVE MACROMOLECULAR

‘To whom correspondence should be addressed, at: Department
ANTIFREEZES
of Biochemistry, Queen’s University, Kingston, Ontario, Canada,
K7L 3N6.
The sera of marine teleosts are hypoosmotic in relation 2Abbreviations: AFP, antifreeze protein; AFGP, antifreeze
to seawater, having approximately one-third its molar- glycoprotein; AF(G)P, both AFP and AFGP; CD, circular dichro-
ity of solutes. The colligative effect of the solutes in sea- ism; HPLC, high performance liquid chromatography.

2460 0892.6638/90/0004-2460/$01.50. © FASEB

 absence of AF(G)P, ice grows most rapidly along the
AFGP [-AIa-AIa-Thr-] 4-.4O
a-axes to give a hexagonal-shaped crystal (Fig. 3). It is
this growth that is markedly inhibited by AF(G)P.
CH2OH1 >60 mol % Ala When the temperature of the solution is lowered, ice
HOtO\J
crystal growth eventually recommences, but at an ac-
CH2OH celerated rate and primarily along the c-axis, to give
A 0 bipyramidal crystal forms. At high AF(G)P concentra-
HO NH
Mr 2,600 33,000 tions, needle-shaped crystals are formed (11).
OH -

CH3
TYPE I AFP
OH
The type I AFP of righteye flounders and sculpins is the
AFP most extensively characterized AFP. It is the only one
mol % Ala for which an x-ray crystal structure is known, and for
-helix which detailed structure/function relationships have
N been proposed.
37 aa
Presence of a macromolecular peptide dipole
Circular dichroism (CD) measurements of type I AFP
II 129 aa suggest that it is an a-helical structure, at least at the
low temperatures where AFP is operative (12, 13). At
-1#{176}C,
flounder AFP was reported to have 85% or
more helix context, but this value decreases sharply as

III 62 - 64 aa

Figure 1. Schematic representations of the four AF(G)P structures. 00

AFGP: showing the glycopeptide repeating structure; type I: winter
flounder AFP emphasizing its helix content, charges on Asp1, U)
Arg3i, and the internal salt bridge, and potential hydrogen bonding
U)
interactions with ice (---); type II: emphasizing its tertiary struc- w
ture, high content of reverse turns (<),
and disulfide bridges
w
(-S-S-); type III: emphasizing its tertiary structure. The sizes of I-
the AF(G)Ps are indicated by the number of amino acids (aa) or U)
>-
Mr range.
I

-J
4
the colligative properties of the protein in solution. It is
the freezing point of their solutions that is lowered be- LU
yond the value predicted from the colligative effect I
I-
(7-9). The difference between freezing and melting
points is termed thermal hysteresis, and its value is a
function of antifreeze protein/glycoproteins (AF(G)P)
concentration. The relationship between thermal hyste-
resis and AF(G)P concentration approaches linearity
only at very low values; thereafter the shape of the
curve becomes hyperbolic (Fig. 2). Thermal hysteresis I 2 3 4 5 6
values for most fish AF(G)P approach a plateau value AFP (mM)
of greater than 1#{176}C at saturating concentrations. Figure 2. Comparison of thermal hysteresis curves on a molar basis
Observation of ice crystal growth under the micro- of AF(G)P obtained from sea raven (SR) (Mr 14000), ocean pout
scope shows that the presence of AF(G)P not only (OP) (Mr 6000), shorthorn sculpin (SH) (Mr 4000), winter
lowers the freezing point of the solution but also alters flounder (F) (M. 3300), and Atlantic cod (C) (Mr 2600). The
the growth habits and growth rates of ice (10). In the AFGP-5 (Mr 10,500) curve was taken from Schrag et al. (64).

BIOCHEMISTRY OF FISH ANTIFREEZE PROTEINS 2461

 A B C

Figure 3. Ice crystal growth patterns in the presence and absence of AFP. A) Hexagonal crystal grown in the absence of AFP. B) Crystal
grown in the presence of AFP showing inhibition of a-axis expansion to give a bipyramidal shape. C) Crystal grown in high [AFP] showing
needle-like shape.

the temperature is raised, being 47% at 25#{176}C

and ap- tively charged, COOH-terminal Arg) suggests that
proaching random coil at above 70#{176}C
(12). The x-ray they may also reinforce the helix dipole.
crystal structure of Winter flounder AFP component A
shows that the protein is a single a-helix in the solid Amphiphilicity of the helix
state (14).
Ho! et al. (15) have pointed out that a-helices are The two major AFPs-A(HPLC-6) and B (HPLC-8)-in
macrodipoles. Associated with each peptide bond in the winter flounder are each 37 amino acids long and con-
peptide backbone is a significant electric dipole. When tain three 11-amino-acid tandem repeats of the sequence
ordered in the a-helix, these dipoles become aligned ThrX2AsxX7, where X is usually alanine or some other
close to the helix axis, leading to a resultant macro- amino acid that favors a-helix formation (20-22). This
dipole along the helix axis. This dipole is equivalent to repeat structure is obvious when winter flounder and
an isolated half-unit charge at either end of the helix, yellowtail flounder AFPs are compared (Fig. 4). The
orientated such that the positive charge is at the NH2- latter protein contains an additional 11-amino-acid
terminus and the negative charge at the COOH termi- repeat (23). At the DNA level there is evidence for AFP
nus. However, the electric field strength of helices sequences in winter flounder that contain four or even
greater than 15 residues is only marginally dependent five of the repeats but no evidence that their gene
on helix length. These investigators suggested that the products, if expressed, make a significant contribution
peptide macrodipoles play an important role in the to antifreeze levels in the blood (24, 25).
binding of charged substrates or coenzymes such as The effect of this tandem, repeating structure is to
NAD, NADP, and other phosphate-containing com- generate a helix with amphiphilic characteristics. This
pounds, long-range attraction of charged substrates, helix structure is stabilized by dipolar interaction with
and the acceleration of several enzymic reactions. terminal amino acids and by intrachain salt bridge for-
Recent studies of model peptides have helped define mation, as indicated above. Because of the presence of
the role of the dipole in helix stability, as well as some some alanyl residues on the hydrophilic side of the helix
of the structural features that contribute to the strength and some hydrophilic residues on the hydrophobic side,
of the dipole (16, 17). Based on these findings, the type the AFP are not strictly amphiphilic. Indeed, the hydro-
I AFP of the righteye flounders have several structural phobic moments (j ranges from 0.1 to 0.27) are rather
features that might stabilize their helix conformation small. A helical wheel projection indicates that Ser4,
by interacting favorably with the helix dipole. These in- Lys18, and Glu22 in HPLC-6 are the residues with hy-
clude a negatively charged NH2-terminal amino acid drophilic side chains projecting from the hydrophobic
(Asp) and a positive charge on the COOH terminal side of the helix. The latter two side chains form the
residue (Arg), which is enhanced by amidation of the intrachain salt bridge, whereas the role of Ser4 is
COOH terminal carboxyl group arising from process- presently unknown.
ing of proAFP to AFP (18). Intramolecular salt bridges,
such as those between Lysis and Glu22 in winter Ice-binding amino acid side chains and AFP length
flounder AFP (HPLC-6) and between Lysig and Aps23,
and Lysao, and Asp34 in yellowtail flounder AFP (Fig. From model building there are nine potential ice-
4), are known to strengthen the a-helix (19), but their binding amino acid side chains in HPLC-6: Asp1,
polarity (positive charge toward the negatively charged, Thr2, Asp5, Thr13, Asn16, Thr24, Asn27, Thr35, and
NH2-terminal Asp; negative charge toward the posi- Arg37 (Fig. 4). X-ray crystallographic studies indicate

2462 Vol. 4 May 1990 The FASEB journal DAVIES AND HEW
Sculpin AFP (SS-8)
K, + 822

Flounder AFP (HPLC-6) #{149}

NH, 0

T2 05 113 N16 T24 N27 Tu

08 A12 123 K 028 K3oK<

Sculpin APP (SS-3) Flounder AFP (HPLC-8)

M1N2 p4 S4

- -
0840 OH
OH O
NH2 NH2 NH2
T2 05 113 N,5 124 N27 T
T11 K10 0j4 Ifl K21 025
Yellowtail APP K,
Grubby AFP (GS-5) 2 NH 03
M,02 p4
-

QOJflO H)#{176}
NH2
bH)#{176},
NH2
NH2
o OH OH OH OH
12 05 113 124 T T45
K, 0j4 122 K2, 025 K32

Figure 4. Secondary structure of type I AFP from sculpins and righteye flounders.

enough torsional freedom of the side chains to accom- C

modate the binding of these amino acid side chains to <211>

BASAL PLANE
different ice surfaces (14). Solid-phase synthesis of
shortened forms has shown that Thr2-Arg37 and Asps-
Arg37 are active antifreezes, although they are less
effective in inhibiting a-axis ice growth (26, 27). The 1. A binds preferentially toprism
latter form is lacking two potential ice-binding resi- faces (parallel 10<211>). through
- bpolar and hydrogen bond irleractions.
dues, Aspi and Thr4. Shorter forms of HPLC-6 with
only two 11-amino-acid repeats (26 amino acids) are in- 2. This results in ordering of
water-d,o4es (shaded area) that lie
active, which is consistent with previous observations within the field of AFt’ heIix.do4ea.
that limited proteolysis destroyed antifreeze activity.
The AFP from yellowtail flounder (Fig. 4) is longer
unordered
than HPLC-6 by one 11-amino-acid repeat but it con- basal plane
tains one fewer (eight) potential ice-binding side chain. PRISM FACE
APP
Its antifreeze activity is similar to the three repeat AFPs
of winter flounder when calculated on a molar basis
(23). 3. Ice still grows on unordered basal plane.

4. AFP binds tonewicefronts.

Mechanism of action
The occurrence of AFP as a single a-helix has
prompted Yang et al. (14) to postulate that the dipole
moment created from the helix is the initial driving
force for the specific recognition of the AFP-ice inter-
action. When AFP approaches the ice nuclei via ran-
dom diffusion, the peptide dipole induces an anti-
parallel alignment of water molecules on the lattice
5. Continued ice growth on basal plane, and
(Fig. 5). Examination of a model ice crystal indicates continued binding of APP toprism faces results In
that the dipoles of all water molecules are orientated in bipyramidal ice crystals.
one of two directions: either inclined at 55#{176}to the c-axis
and at 30#{176}
to the a-axis, or inclined at 55#{176}
to the c-axis
and at 90#{176}
to the a-axis. The resultant of these two vec-
tors is inclined at 51#{176}
to the c-axis and at 60#{176} to the Figure 5. Schematic representation of type I AFP interaction with
a-axis (<211>, Fig. 5). The only naturally occurring ice.

BIOCHEMISTRY OF FISH ANTIFREEZE PROTEINS 2463

ice planes that are parallel to the resultant dipole vector a-N acetyl-D-galactosamine through the hydroxyl oxy-
(<211>) are the prism faces. The AFP can maintain gen of the threonyl residue (32). The purification of
its alignment with the <211> dipole vector and lie flat AFGPs has been facilitated by their superabundance in
on the prism face. This preferential alignment directs nototheniid serum and their solubiity in 10% trichlo-
the binding of AFP to the prism face, and its absorption roacetic acid. As many as eight components have been
inhibits a-axis growth by the adsorption inhibition resolved by gel electrophoresis (33). They have been
model (28). The amphiphilicity of the helix, with its listed as components 1 to 8 in order of increasing elec-
alignment of hydrophilic side chains, provides the H- trophoretic mobility and decreasing size. Their molec-
bonds necessary for interaction with the ice lattice, ular masses range from 2,600 to 33,000. In some of the
whereas the cluster of hydrophobic side chains serves to tripeptide repeats of the smaller AFGP components,
impede further growth of the ice nuclei. proline replaces alanine on the COOH-terminal side of
The water dipoles of the interior of the ice crystal and threonine (34). Detailed sequence analysis shows that
the surfaces of the basal planes would be unordered, component 8 from Pagothenia borchgrevinki is itself a mix-
and nucleation and ice growth on the basal plane could ture of sequences in which the proline substitutions oc-
continue to occur. The newly formed unordered ice cur in different sets of tripeptide repeats. The smaller,
front on the basal plane would be retained when it proline-containing AFGPs are less active as antifreezes
either advances into the ordered outer layer or is bound than the larger ones.
by other AFPs in solution. This growth habit alteration This same antifreeze type is found in cods of the tem-
causes the development of bipyramidal ice crystals (Fig. perate and polar regions of the Northern hemisphere
3 and Fig. 5). The altered growth habit of ice in AFP (35-37). Even more surprising, in view of the evolu-
solutions suggests that the AFP bind to prism faces with tionary distance between nototheniids and cods, is that
greater affinity than to basal planes. The AFP-ice prism the number and size of the AFGP components is simi-
face complex is stabilized by both dipolar and hydrogen lar in both groups of fish. The alanine/proline substitu-
bond interactions, whereas the basal plane complex is tion also occurs in the smaller AFGPs of the cods. In
stabilized primarily by hydrogen bonding (27). two of these species, Microgadus tomeod (frostfish) and
Eleginus gracilis (saffron cod), arginine appears to
Type I AFP from sculpins replace threonyl residues in some of the tripeptide
repeats (38). It is not clear if these amino acid substitu-
The other type I AFP that has been described comes tions are responsible for the lower antifreeze activity of
from sculpins (Fig. 4) (29). Based on its amino acid the smaller components. In this regard it is perhaps
composition, in particular an alanine content of more significant that enzymatic cleavage of the larger AFGP
than 60 per 100 residues, sequence, and an a-helical components to fragments of 11 or fewer glycotripeptide
secondary structure, there is no problem in classifying units results in considerable loss of activity (39). Other
it with the flounder AFP. However, it is not clear if manipulations that result in loss of activity are modifi-
these proteins are similar because they are homologous cations of the disaccharide moieties by acetylation,
or because they have evolved convergently (30). The periodate oxidation, complex formation with borate, or
sculpin AFP components are even more amphiphilic their removal by f3-elimination (39, 40). These re-
than the flounder AFPs. For example, sculpin AFP actions result in the loss of hydrogen-bonding groups
SS-8 has a hydrophobic moment of 0.26 compared with that have been postulated by DeVries, Feeney, and co-
a value of 0.13 for flounder AFP HPLC-6. Salt bridge workers (28, 32) to be involved in the binding of AFGP
formation of side chains in SS-8 is feasible at Lys22 and to the ice-lattice.
Asp26, and at appropriate residues in SS-3 and GS-5 Although numerous physical studies have been car-
(Fig. 4), although proof of their existence would require ried out with AFGP since their discovery (reviewed re-
confirmation by physical techniques. Sculpin AFPs cently by Ananthanarayanan, ref 41), the conformation
differ significantly from flounder AFPs at their NH2- of these glycoproteins has not been unequivocally
terminal ends. Also, in sculpin AFPs an internal repeat defined. The most plausible model for their structure is
structure is not as well demarcated as it is in the one in which the polypeptide backbone forms a poly-
flounder AFPs. The smallest naturally occurring type I proline Il-like left-handed helical structure with three
AFP is SS-3 from the shorthorn sculpin (31). This pep- residues per turn, and the disaccharides are organized
tide is 33 amino acids long with a helix content of 45% in a planar conformation with their hydrophilic groups
at 4#{176}C.It has a helix breaking proline at position 4, exposed to the aqueous solvent and their hydrophobic
which may reduce the effective length of the helix to 29 faces adjacent to the polypeptide chain (42, 43). This
residues (44 A). model is remarkably similar in principle to that of the
amphiphiic a-helix of the type I AFP, and might ac-
count for the functional similarity of such very different
ANTIFREEZE GLYCOPROTEINS
antifreeze types.
The first macromolecular antifreezes to be character-
ized biochemically were the AFGP from nototheniids TYPE II AFP
of the Antarctic Ocean (4). This antifreeze type is built
up of a tripeptide repeating unit (Ala-Ala-Thr)0, to Type II AFP is characterized by its high content of half-
which is linked the disaccharide 13-D-galactosyl-(1 -p3)- cystine (8 per 100 residues) and inactivation by suif-

2464 Vol. 4 May 1990 The FASEB Journal DAVIES AND HEW
hydryl reagents (44). At present this AFP type has been limited heterogeneity exists in the form of one or
reported in only one species of fish, the sea raven several amino acid replacements.
(Hemitripierus arnericanus). Although cysteine/cystine- Secondary structure predictions (51) indicated a pau-
rich thermal hysteresis proteins have been found in city of a-helix and f3-sheet but a high content of reverse
some insects, not enough is known about their proper- turns (48). Circular dicroism studies confirm the low a-
ties to indicate if they should be classified as type II helix content and the enrichment in f3-structure/reverse
AFPs (45-47). turns (44). They also suggest the presence of aromatic
The presence of five cystines in a relatively small pro- residues in an asymmetric environment, which is con-
tein has complicated its biochemical characterization, sistent with a folded tertiary structure.
and a combination of protein and DNA sequencing has
been needed to provide definitive structural informa- TYPE III AFP
tion (48-49). The primary translation product of sea
raven AFP mRNA was sequenced by Edman degrada- The type III AFPs do not contain cysteine, and their
tion to reveal the locations of the first four leucyl amino acid compositions do not have an imbalance of
residues. With reference to the cloned AFP cDNA se- any one amino acid. They were first isolated from the
quence (48), the AFP precursor is therefore 163 amino eel pout, Macrozoarces americanus, of the family Zoarcidae
acids long. Amino acid analysis and sequencing of the (52), and more recently from three other family mem-
deblocked NH2-terminal peptide from the mature cir- bers, Rhigophila dearborni and Austrolycicthys brachycep/za-
culating form of the AFP indicated that glutamine at lus, both antarctic eel pouts, and Lycodes polaris, an arc-
position 35 is the NH2-terminal residue (49). Thus the tic eel pout (53, 54). Recent work by G. L. Fletcher et
mature form is 129 amino acids long and contains no al. (unpublished results) has shown that the type III
free cysteine. According to the algorithm developed by AFP is even more widely distributed, appearing in at
von Heijne (50), the most likely site of signal peptide least three other families of the Zoarcoidei besides the
cleavage is after alanine at position 17. If so, the type Zoarcidae.
II AFP would have a proprotein precursor form, the AFP from the Newfoundland ocean pout comprise a
function of which is unknown. set of related proteins, the major components of which
The degree of heterogeneity in sea raven AFP is not are separable by ion exchange chromatography and
yet clearly established. There is some evidence for mul- reverse phase HPLC. AFPs from 8 of the 12 defined
tiple, similar components from high performance liquid HPLC peaks, HPLC 1, 4, 5-7, 9, 11, and 12, have been
chromatography (HPLC) analysis (48). Comparison of sequenced by conventional protein sequencing (55, 56)
a cDNA sequence (48) with that of a genomic clone (Fig. 6). The NH2-terminal residue of HPLC-4, 5 and
(49) shows that if both were expressed, the mature pro- 6 is glutamine, which has been cyclized to form pyroli-
teins would differ by a single amino acid at position 4 dine carboxylic acid, and is therefore resistant to
(Gly-* Pro). Altogether there are 12-15 copies of the Edman degradation. These proteins have from 62 to 66
AFP gene in the sea raven genome. Those that have amino acids. Although there are some differences in the
been mapped show no evidence of major differences in extent of NH2- and COOH-terminal processing, only
the coding region. It seems likely, therefore, that sea one component (HPLC-5) could be the product of
raven AFP is produced by multiple genes and that another (HPLC-6) (55). All the other components have

Type III Antifreeze Protein from Eel Pouts

HPLC 1 SQ*SVVATQLIPMNTALTPVMMEGKVTNPIGIPFAE*MSQIVGKQVNTPVAKGQTIMPNMVKTYAA
HPLC 4 -* I A *

HPLC 6 -* I A * L V-G
HPLC 7 --* R A * RI L
HPLC 9 A * RI L
HPLC 11 I A * RI L
cDNA dO S *---M R K
cDNA c7 I *
genomic I * L
woiffish 1.5 I I--K-Q-V--A * RA---DE-L R-A-
woiffish 1.9 I I--K-Q-V--A * R----DE-L R-A-
HPLC 12 L---RSE-VT-V---*--DIPRL-SM---RA-PL-T-L--D---G-PPA
genomic A3 I L---TTR-IY-T---*--DIPRL-SM---QA-PM-T-L--D---F-CLCAPLN
L.P. NKA----N----I L---RAE-VT-A---*--DIPRL--L---RA-LI-T-L--D---G--PQ
R.D. NKA----N----I LI--KAE-VT-M---*--DIPR-I-M---RA-PL-T-L--D---N-E
AB1 TK*----S----I A--KA-EVS-K---*--E--K---M---RA-NDLE-L--D---T-Q
AB2 L---KAEEVS-K---*--EIPRL--M---RA-YLDE-L--D---N-E

Figure 6. Compendium of sequences for type III AFP. Sequences were derived from ocean pout AFP components (HPLC 1, 4, 6, 7,
9, 11, and 12), ocean pout AFP cDNA clones (dO, c7), ocean pout AFP genomic clones (X5, X3), woiffish AFP genomic clones (1.5, 1.9),
Lycodes polaris AFP (L.P.), Rhigophila dearborni AFP (R.D.), and Austrolycicthys brachycep/zalus AFP (AB1 and AB2). (See refs55, 56, 66, 53,
and 54, respectively.)

BIOCHEMISTRY OF FISH ANTIFREEZE PROTEINS 2465

internal amino acid differences (Fig. 6). Based on their bonding to ice, as is proposed for the threonine and
amino acid sequences, the type III AFPs can be divided asparagine side chains along the hydrophilic face of the
into two groups. The larger group comprises the com- a-helical type I AFP, and the carbohydrate groups of
ponents that bind to SP-Sephadex. Their sequence the AFGP. If so, there are a number of highly con-
identity is 90%, and most of the amino acid substitu-
- served hydrophilic residues in the type III AFP se-
tions are conservative. The other, rarer set of type III quences (Fig. 6) that might fulfill this role (56). Prelimi-
AFPs bind to QAE-Sephadex. Although they show nary studies using site-directed mutagenesis have shown
- 75% sequence identity within the group, this falls to that there is a 25% decrease in activity when Glu21 or
-50% when they are compared with the SP series. Glu34 is substituted by alanine (X. Li and C. L. Hew,
This is in accordance with the immunological studies of unpublished results).
Hew et al. (52), which indicated that the two groups,
although cross-reactive, were immunologically distinct.
Ocean pout AFP genomic and cDNA clones picked
at random for sequencing encode proteins that fit into PROJECTIONS FOR THE FUTURE:
the SP or QAE series, and either confirm existing pro- STRUCTURE/FUNCTION RELATIONSHIPS
tein sequences or show minor variations (Fig. 6) (56).
Obviously there are active genes other than those that Even at the protein level there are many unanswered
give rise to the major protein components, which in questions about macromolecular antifreezes.One of
turn might be major components because they are en- the most fascinating observations is that four com-
coded by multiple gene copies. Indeed, two cDNA pletely different structures appear to interact with ice in
clones that differ slightly in their nucleotide sequences a similar manner, and if so, might operate by the same
at silent base positions both encode HPLC-6 (55). Con- mechanism of action to lower the freezing point (60).
sistent with these observations, the ocean pout from How could such different structures present fundamen-
Newfoundland waters has been estimated to have -150 tally similar binding sites that interact specifically with
copies of the AFP gene. The same species from New the prism faces of ice? Characterization of the struc-
Brunswick waters has far fewer copies (between 30 and tures of type I AFP and AFGP has led to the hypothesis
40) of the gene (56) and produces proportionally less that an amphipathic structure might be required to
AFP (57). bind to ice on the hydrophiic side and exclude H2O on
Despite the wealth of amino acid sequence data on the hydrophobic side (28). These features are recogniz-
the type III AFPs, very little is known about its struc- able in both antifreezes. Details of the model, such as
ture or mechanism of action. A CD spectral analysis of the importance of the helix macrodipole or the spacing
both the QAE- and SP-components from ocean pout between hydrogen bonding groups, remain to be estab-
showed that this antifreeze has a well-defined secondary lished. They can be tested and refined, at least for the
and tertiary structure (58). In the far ultraviolet region type I AFP, by a structure/function study in which vari-
(190-250 nm), the CD spectra are qualitatively similar ous amino acids are changed through either solid-phase
to those produced by the AFGP. However, in the near- peptide synthesis (26) or site-directed mutagenesis in
ultraviolet region (250-300 nm), there are bands in- conjunction with biosynthesis.
dicative of asymmetrically arranged aromatic amino Type II and type III AFPs are more complex struc-
acids. Also, the sigmoidal thermal transition observed tures that appear to have an element of tertiary struc-
on heating the AFP suggest the presence of cooperative ture. The solution of these structures by x-ray crystal-
interactions acting to stabilize its secondary structure. lography and/or 2-dimensionai-NMR is an obvious
These are features not seen with the AFGP. A CD spec- priority to see if they too have an amphipathic structure
tral study on the QAE-type component from L. polaris and perhaps a recognizably regular spacing of hydro-
(53) confirmed the paucity of a-helix and f3-sheet origi- gen bonding groups. The recognition of a common
nally indicated by secondary structure predictions (55), structural motif in all four AF(G)P structures will go a
and the presence of a folded tertiary structure. long way to confirming the model for interaction with
There is variability in the NH2- and COOH- ice and will complement structure/function studies.
terminal residues of the type III AFPs. This is not seen The discovery of four different antifreeze structures
in the type I AFPs from the flounders, where these in teleosts with very little logic to their distribution
residues and their modifications are believed to play a within the currently accepted phylogenetic scheme has
key role in the structure and hence activity of the anti- led to the suggestion that their elaboration as anti-
freeze. Recently, expression studies have shown that a freezes is a relatively recent event on a geological time
type III AFP produced in E. coli with an NH2-terminal scale (30). Since they are clearly not monophyletic in
Met-Lys extension and one produced in Drosophila origin, the possibility exists that additional antifreeze
melanogaster with a seven-amino-acid NH2-terminal ex- types will be discovered in fish. At the present time
tension are fully active (59; X. -M. Li, K. Y. Trinh, and cysteine-rich antifreeze proteins are being character-
C. L. Hew, unpublished results). ized in smelt and herring (G. L. Fletcher et al., unpub-
All fish AF(G)Ps appear to interact with ice in a simi- lished results). It is not yet clear if they belong in the
lar manner (60). It is therefore likely that the structure type II category or represent a separate type. Also,
of type III AFP presents a set of hydrophiic residues on other types of AFP might be discovered as more species
one side of the molecule that are capable of hydrogen are surveyed.

2466 Vol. 4 May 1990 The FASEB Journal DAVIES AND HEW
 Antifreeze proteins, more appropriately called ther- 12. Ananthanarayanan, V. S., and Hew, C. L. (1977) Structural
studies on the freezing point-depressing protein of the winter
mal hysteresis proteins, have also been described in
flounder Pseudopleuronectes americanus. Biochem. Biophys. Res. Corn-
insects (61). These proteins are not yet well enough mun. 74, 685-689
characterized to begin their classification. One of the 13. Raymond,J. A., Radding, W., and DeVries, A. L. (1977) Circu-
difficulties has been in obtaining sufficient material to lar dichroism of protein and glycoprotein fish antifreeze. Bio-
work with. However, with the advent of capillary elec- polymers 16, 2575-2578
14. Yang, D. S. C., Sax,Chakrabartty,
M., A., and Hew, C. L.
trophoresis, microbore HPLC, and the increased sensi-
(1988) Crystal structure
of an antifreeze polypeptide and its
tivity of gas-phase protein sequencing, these difficulties mechanistic implications. Nature (London) 333, 232-237
are unlikely to delay characterization of the insect anti- 15. Hol, W. 0. J., van Duijnen, P. T., and Berendsen, H. J. C.
freezes much longer. Since overwintering terrestrial in- (1978) The a-helix dipole and the properties of proteins. Nature
sects usually face lower temperatures than fish, it will (London) 273, 443-446
16. Fairman, R., Shoemaker, K. R., York, E. J., Stewart, J. M., and
be interesting to see if their thermal hysteresis proteins
Baldwin, R. L. (1989) Further studies of the helix dipole model:
can cause a greater lowering of the freezing point than effects of a free a-NH34 or a-C00 group on helix stability. Pro-
the 1#{176}C+that appears to be the maximum value for teins 5, 1-7
fish antifreezes (Fig. 2). Thermal hysteresis values of 17. Shoemaker, K. R., Kim, P. S., York, E. J., Stewart, J. M., and
Baldwin, R. L. (1987) Tests of the helix dipole model for stabili-
several degrees have been reported for crude insect
zation of a-helices. Nature (London) 326, 563-567
hemolymph, but it remains to be seen if these values 18. Hew, C. L., Wang, N. C., Yan, S., Cai, M., Sclater, A., and
can be attributed to a single, pure component. If they Fletcher, G. L. (1986) Biosynthesis of antifreeze polypeptides in
can, then their mechanism of action might be different the winter flounder. Eur. j Biochem. 160, 267-272
from that of the fish AF(G)Ps. 19. Marqusee, S., and Baldwin, R. L. (1987) Helix stabilization by
Gly ..Lys salt bridges in short peptides of de novo design. Proc.
Ultimately, the knowledge about structure/function Natl. Acad. Sci. USA 84, 8898-8902
relationships may allow the design of an improved or 20. DeVries, A. L., and Lin, Y. (1977) Structure of a peptide anti-
more efficient macromolecular antifreeze, that is, one freeze and mechanism of absorption to ice. Biochim. Biophys. Acta
with a higher thermal hysteresis value per unit concen- 495, 380-392
21. Davies, P. L., Roach, A. H., and Hew, C. L. (1982) DNA se-
tration. An AFP with this property would be particularly
quence coding for an antifreeze protein precursor from winter
useful for transgenic studies designed to confer im- flounder. Proc. NatI. Acad. &i. USA 79, 335-339
proved freeze resistance through gene transfer (62, 63). 22. Pickett, M., Scott, 0., Davies, P., Wang, N., Joshi, S., and Hew,
C. L. (1984) Sequence of an antifreeze protein precursor. Eur.
We would like to thank our many co-workers and collaborators j Biochem. 143, 35-38
who have contributed to the characterization of antifreeze proteins. 23. Scott,G. K., Davies,P. L.,Shears,M. A., and Fletcher, G. L.
(1987) Structural variations in the alanine-rich antifreeze pro-
We also thank the Medical Research Council of Canada for grant
teins of the Pleuronectinae. Eur. J. Biochem. 168, 629-633
support and Dianne Blair for typing the manuscript. 24. Lin, Y., and Gross, J. K. (1981) Molecular cloning and charac-
terization of winter flounder antifreeze cDNA. Proc. Nat!. Acad.
Sci. USA 78, 2825-2829
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2468 Vol. 4 May 1990 The FASEB Journal DAVIES AND HEW