Biochemistry PETER

of fish antifreeze

L. DAVIES*l

AND

CHOY

proteins

L. HEW1

#{176}Department of Biochemistry, Queen’s University, Kingston, Ontario, for Sick Children, Toronto, and Departments of Clinical Biochemistry Ontario,

Canada M5G

Canada K7L 3N6 and tResearch Institute, Hospital and Biochemistry, University of Toronto, Toronto,

1L5

water (-0.45 -

Four distinct macromolecular antifreezes have been isolated and characterized from different marine fish. These include the glycoprotein antifreezes (Mr 2.5 33 K),

which

are

(Ala-Ala-Thr) threonyl

made

residues,

types.

Type

up

of a repeating

with a disaccharide and

I is an

three

tripeptide

attached

antifreeze

alanine-rich,

to

protein

the

(AFP)

amphiphilic,

cr-helix

K); type II is a larger

its freezing

M) depresses a typical

-1.9#{176}C, whereas -0.7#{176}C(1). This

ABSTRACT

points

means

north

temperate

teleost

discrepancy

that

down

will

to at

freeze

of

-1#{176}Cin freezing in polar and be at risk of freezing to falls below -0.7#{176}C.

unprotected

Waters

point

serum teleosts

would

death when their temperature Although there is evidence that some fish can survive at these temperatures in deep water in a supercooled state (2), this is not possible in shallow water where contact with ice negates supercooling. When Scholander

protein (Mr 14 K) with a high content of reverse turns and five disulfide bridges;and type III is intermediate in size (Mr 6-7 K) with no distinguishing features of secondary structure or amino acid composition. Despite their marked struc-

and co-workers (3) first investigated this problem using inshore Arctic fish, they observed unusually low serum freezing temperatures - 1.4#{176}C). The nature of the

tural differences, all four antifreeze function in the same way by binding

types appear to to the prism faces

Wohlschlag of nototheniid

of ice crystals

along

that was soluble

(Mr

3-5

and

It is suggested the

prism

faces

helix

macrodipole

cules

in the

gen

inhibiting

that

and lattice. and

helix results ing exposed

I

as a result

ice

bonding,

type

growth

AFP

binds

of interactions

the dipoles Binding

the

the

to

between

the

on the water

is stabilized

amphiphilic

in the hydrophobic to the solvent. When

a-axes.

preferentially

mole-

by hydro-

character

of

the

phase of the helix bethe solution tempera-

ture is lowered further, ice crystal growth occurs primarily on the uncoated, unordered basal plane resulting in bipyramidal-shaped crystals. The structural features of type I AFP that could contribute to this mechanism of action are reviewed. Current challenges lie in solving the other antifreeze structures and

interpreting

common

C. L.

Words:

amphiphilicity tionships

WHY

SOME

in light of action.

of what

appears

-DAVIES,

of fish antifreeze

Biochemistry

4: 2460-2468; Key

them

mechanism

proteins.

helix

FISH

protein/glycoprotein ice crystal givwth macrodipole . structure/function rela-

HAVE

2460

these researchers.

freezing

However,

(4) reported fish was

point

depression

in 1969 DeVries

and

that the antifreeze in the blood a proteinaceous macromolecule

in 10% trichloroacetic

characterization revealed a set of glycoproteins that

that this are each

acid.

Further

antifreeze comprised made up of a tripep-

tide repeat (Ala-Ala-Thr)n with a disaccharide moiety attached to the threonyl residues (5). As more fish have been surveyed for antifreeze activity, three distinct antifreeze protein (AFP) types have been characterized in addition to the antifreeze glycoprotein (AFGP) (6). These are the alanine-rich, a-helical AFP of righteye flounders and sculpins (type I), the cystine-rich AFP of

the sea raven (type II), and an AFP (type III) found in eel pouts,

which

lacks

sition

and sequence

THE

INTERACTION

WITH

features

distinctive 1).

in its compo-

(Fig.

OF

AFP

AND

AFGP

ICE

Despite the marked differences in amino acid composition and protein structure between these macromolecular antifreeze types, they all appear to interact with ice in the same way. They have no untoward effect on the melting point of ice formed in their presence, and any depression of the melting point is entirely explained by

‘To whom correspondence of Biochemistry, Queen’s

of marine

ity of solutes.

eluded

for this

MACROMOLECULAR

ANTIFREEZES The sera to seawater,

responsible

to be a

P. L.; HEW, FASEBJ.

1990. antifreeze

(-

antifreeze

teleosts are hypoosmotic in relation having approximately one-third its molarThe colligative effect of the solutes in sea-

should University,

be addressed, at: Department Kingston, Ontario, Canada,

K7L 3N6. 2Abbreviations: AFP, antifreeze protein; AFGP, antifreeze glycoprotein; AF(G)P, both AFP and AFGP; CD, circular dichroism;

HPLC,

high

performance

liquid

chromatography.

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

m www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on August 02, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()

AFGP [-AIa-AIa-Thr-]

absence

4-.4O

CH2OH1

>60

mol % Ala

HOtO\J CH2OH A

HO

0

NH Mr

OH

of AF(G)P, ice grows most rapidly along the to give a hexagonal-shaped crystal (Fig. 3). It is this growth that is markedly inhibited by AF(G)P. When the temperature of the solution is lowered, ice crystal growth eventually recommences, but at an accelerated rate and primarily along the c-axis, to give bipyramidal crystal forms. At high AF(G)P concentraa-axes

2,600

-

33,000

CH3

tions,

TYPE

needle-shaped

crystals

are

formed

(11).

I AFP

OH

AFP mol % Ala -helix

N

The type I AFP of righteye flounders and sculpins is the most extensively characterized AFP. It is the only one for which an x-ray crystal structure is known, and for which detailed structure/function relationships have been proposed.

37 aa

Presence of a macromolecular Circular

II

129

aa

suggest

dichroism that

(CD)

peptide

measurements

it is an a-helical

structure,

dipole of type

I AFP

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

Figure

1. Schematic

AFGP:

showing

representations

the glycopeptide

of the four AF(G)P repeating

structure;

64

aa

structures.

type

00

I: winter

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

(-

I -J

4 the colligative properties of the protein in solution. It is the freezing point of their solutions that is lowered beyond the value predicted from the colligative effect (7-9). The difference between freezing and melting

points function

is termed

thermal

of antifreeze

hysteresis,

OF FISH ANTIFREEZE

I

I-

and its value is a

protein/glycoproteins

(AF(G)P)

concentration. The relationship between thermal hysteresis and AF(G)P concentration approaches linearity only at very low values; thereafter the shape of the curve becomes hyperbolic (Fig. 2). Thermal hysteresis values for most fish AF(G)P approach a plateau value of greater than 1#{176}C at saturating concentrations. Observation of ice crystal growth under the microscope shows that the presence of AF(G)P not only lowers the freezing point of the solution but also alters the growth habits and growth rates of ice (10). In the BIOCHEMISTRY

LU

PROTEINS

I

2

3

AFP

4

5

6

(mM)

Figure 2. Comparison of thermal hysteresis curves on a molar basis of AF(G)P obtained from sea raven (SR) (Mr 14000), ocean pout (OP) (Mr 6000), shorthorn sculpin (SH) (Mr 4000), winter flounder (F) (M. 3300), and Atlantic cod (C) (Mr 2600). The AFGP-5 (Mr 10,500) curve was taken from Schrag et al. (64).

2461

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A

C

B

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

shape.

the temperature is raised, being 47% at 25#{176}C and approaching random coil at above 70#{176}C (12). The x-ray crystal structure of Winter flounder AFP component A

tively charged, they may also

shows that the protein state (14). Ho! et al. (15) have

Amphiphilicity

macrodipoles.

Associated

is a single pointed

a-helix out

that

in the a-helices

with each peptide

solid are

bond in the

peptide backbone is a significant electric dipole. When ordered in the a-helix, these dipoles become aligned close to the helix axis, leading to a resultant macrodipole along the helix axis. This dipole is equivalent to an isolated half-unit charge at either end of the helix, orientated such that the positive charge is at the NH2terminus and the negative charge at the COOH terminus. However, the electric field strength of helices greater than 15 residues is only marginally dependent on helix length. These investigators suggested that the

peptide

macrodipoles

binding of charged NAD, NADP, and pounds, long-range and the acceleration

Recent the

role

studies

play

an

important

role

in the

substrates or coenzymes such as other phosphate-containing comattraction of charged substrates, of several enzymic reactions.

of model peptides

have helped

define

dipole in helix stability, as well as some features that contribute to the strength of the dipole (16, 17). Based on these findings, the type I AFP of the righteye flounders have several structural features that might stabilize their helix conformation by interacting favorably with the helix dipole. These include a negatively charged NH2-terminal amino acid (Asp) and a positive charge on the COOH terminal residue (Arg), which is enhanced by amidation of the COOH terminal carboxyl group arising from processing of proAFP to AFP (18). Intramolecular salt bridges, such as those between Lysis and Glu22 in winter flounder AFP (HPLC-6) and between Lysig and Aps23, and Lysao, and Asp34 in yellowtail flounder AFP (Fig. 4), are known to strengthen the a-helix (19), but their

of the

polarity

of the

structural

(positive

NH2-terminal

2462

crystal grown in the absence of AFP. B) Crystal shape. C) Crystal grown in high [AFP] showing

Vol. 4

charge Asp;

toward the negatively

negative

May 1990

charge

toward

charged, the posi-

The

of the

two major

winter tain

COOH-terminal reinforce the helix

and

are each 37 amino

11-amino-acid

ThrX2AsxX7,

suggests

that

helix

AFPs-A(HPLC-6)

flounder

three

Arg) dipole.

tandem

B (HPLC-8)-in

acids long and conof the sequence

repeats

where X is usually alanine

or some other

amino acid that favors a-helix formation repeat structure is obvious when winter yellowtail flounder AFPs are compared

(20-22). This flounder and (Fig. 4). The

latter protein contains an additional 11-amino-acid repeat (23). At the DNA level there is evidence for AFP sequences

in winter

flounder

that

contain

four

or even

five of the repeats but no evidence that their gene products, if expressed, make a significant contribution to antifreeze levels in the blood (24, 25). The effect of this tandem, repeating structure is to

generate a helix with amphiphilic characteristics. helix structure is stabilized by dipolar interaction

This with

terminal amino acids and by intrachain salt bridge formation, as indicated above. Because of the presence of some alanyl residues on the hydrophilic side of the helix

and some

hydrophilic

the AFP are not phobic moments

residues

strictly

on the hydrophobic

amphiphilic.

Indeed,

side,

the hydro-

(j ranges from 0.1 to 0.27) are rather small. A helical wheel projection indicates that Ser4, Lys18, and Glu22 in HPLC-6 are the residues with hydrophilic side chains projecting from the hydrophobic

side of the helix. The intrachain salt bridge, presently unknown.

Ice-binding From

binding Thr2, Arg37

The FASEB journal

amino

model

amino

building

acid

latter two whereas

acid

side there

side the

chains are

side chains

chains form the role of Ser4 is

and nine

AFP

length

potential

in HPLC-6:

ice-

Asp1,

Asp5, Thr13, Asn16, Thr24, Asn27, Thr35, and (Fig. 4). X-ray crystallographic studies indicate

DAVIES AND

HEW

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Sculpin AFP (SS-8) K,

Flounder AFP (HPLC-6)

T2 08

A12

123 K

-

822

N16

113

T24

N27 Tu

K3oK