Báo cáo khoa học: ‘Antifreeze’ glycoproteins from polar fish docx

In contrast to the more widely studied antifreeze proteins, little is known about the mech-anism of ice growth inhibition by AFGPs, and there is no definitive model that explains their pr

R E V I E W A R T I C L E

‘Antifreeze’ glycoproteins from polar fish

Margaret M Harding1, Pia I Anderberg1and A D J Haymet2

1

School of Chemistry, The University of Sydney, New South Wales, Australia;2CSIRO Marine Research, Hobart,

Tasmania, Australia

Antifreeze glycoproteins (AFGPs) constitute the major

fraction of protein in the blood serum of Antarctic

noto-thenioids and Arctic cod Each AFGP consists of a varying

number of repeating units of (Ala-Ala-Thr)n, withminor

sequence variations, and the disaccharide b-D

-galactosyl-(1fi3)-a-N-acetyl-D-galactosamine joined as a glycoside to

the hydroxyl oxygen of the Thr residues These compounds

allow the fish to survive in subzero ice-laden polar oceans by

kinetically depressing the temperature at which ice grows in a

noncolligative manner In contrast to the more widely

studied antifreeze proteins, little is known about the

mech-anism of ice growth inhibition by AFGPs, and there is no

definitive model that explains their properties This review

summarizes the structural and physical properties of AFGPs

and advances in the last decade that now provide

oppor-tunities for further research in this field

Highfield NMR spectroscopy and molecular dynamics

studies have shown that AFGPs are largely unstructured in

aqueous solution While standard carbohydrate degradation

studies confirm the requirement of some of the sugar

hydroxyls for antifreeze activity, the importance of following

structural elements has not been established: (a) the number

of hydroxyls required, (b) the stereochemistry of the sugar

hydroxyls (i.e the requirement of galactose as the sugar),

(c) the acetamido group on the first galactose sugar, (d) the stereochemistry of the b-glycosidic linkage between the two sugars and the a-glycosidic linkage to Thr, (e) the require-ment of a disaccharide for activity, and (f) the Ala and Thr residues in the polypeptide backbone The recent successful synthesis of small AFGPs using solution methods and solid-phase chemistry provides the opportunity to perform key structure-activity studies that would clarify the important residues and functional groups required for activity Genetic studies have shown that the AFGPs present in the two geographically and phylogenetically distinct Antarctic notothenioids and Arctic cod have evolved independently,

in a rare example of convergent molecular evolution The AFGPs exhibit concentration dependent thermal hysteresis withmaximum hysteresis (1.2C at 40 mgÆmL)1) observed with the higher molecular mass glycoproteins The ability to modify the rate and shape of crystal growth and protect cellular membranes during lipid-phase transitions have resulted in identification of a number of potential applica-tions of AFGPs as food additives, and in the cryopreserva-tion and hypothermal storage of cells and tissues

Keywords: antifreeze; ice; hysteresis; glycoproteins; ice/ water interface; fish; glycosylation

Introduction

Many plants, insects, animals and other organisms have

evolved with unique adaptive mechanisms that allow them

to survive in harsh environments at the extremes of

temperature [1–4] Nearly two-thirds of the surface of the

earthis comprised of water, withthe average surface

temperature of seas and oceans varying from )2 C to

30C depending on latitude [1] Within the polar regions,

seawater temperatures are consistently below the freezing

point of physiological solutions, which themselves have

freezing points below the freezing point of pure water, 0C

at 1 atmosphere, due to dissolved sugars and salts The

effect of these subzero temperatures on the cells of plants, animals, bacteria and fungi can be extremely harmful, if not deadly [5]

Scholander [6,7] and DeVries [8,9] were the first to investigate the mechanisms by which species of fish inha-biting the polar oceans at temperatures that are frequently below that of the freezing point of pure water, are able to survive Analysis of the blood plasma of these fish showed that while the concentrations of salts and small ions in the body fluids are somewhat higher relative to fish in temperate waters, these salts are only responsible for 40–50% of the observed freezing point depression The remainder of the protective effect was attributed to the presence of a series of relatively high molecular mass glycoproteins and proteins [10–13]

‘Antifreeze’ proteins (AFPs) and ‘antifreeze’ glycopro-teins (AFGPs) have since been identified in the body fluids

of many species of polar fish Four classes of structurally diverse AFPs, classified as type I [14,15], type II [16,17], type III [18,19] and type IV [20,21] have now been identified along witha single class of glycosylated protein denoted AFGP [22–24] The principal characteristics of these

Correspondence to M M Harding, School of Chemistry,

The University of Sydney, NSW 2006, Australia.

Fax: + 61 29351 6650,

E-mail: harding@chem.usyd.edu.au

Abbreviations: AFGP, antifreeze glycoprotein; AFP, antifreeze

protein.

(Received 5 December 2002, accepted 28 January 2003)

compounds, which are compared in a number of articles

[3,25–27], are summarized in Fig 1 In contrast to many

solutes, these compounds kinetically depress the

tempera-ture at which ice grows in a noncolligative manner, and

hence exhibit thermal hysteresis, i.e a positive difference

between the equilibrium melting point and the ice growth

temperature (the temperature at which seed ice crystals will

grow in the solution) This property allows fish to survive in

the subzero waters at temperatures colder than the

equili-brium freezing point of their blood and other internal fluids,

by modifying or suppressing ice crystal growthand by

protecting cell membranes from cold-induced damage

[3,28] These versatile properties have attracted significant

interest for their potential applications in medicine and

industry where low temperature storage is required and ice

crystallization is damaging [29] Applications include

improved protection of blood platelets and human organs

at low temperatures [30], increasing the effectiveness of the

destruction of malignant tumors in cryosurgery [31], and

improvement of the smooth texture of frozen foods [32]

Most researchhas focused on the type I AFPs and a

number of reviews summarizing progress in this area have

been published [3,25–27,33–35] Studies of the more

com-plex type II and III AFPs are now being addressed [19,

36–40] In contrast to the AFPs, the AFGPs present in cold

water fish have been much less studied This is due to their

structural complexity compared to AFPs (Fig 1), and the

difficulties in accessing sufficient quantities of pure material

to allow detailed studies to be performed

This review will focus on new research published in the

last decade on AFGPs Several reviews have already

summarized the AFGP literature published in the 1970s

and 1980s [1,41–44] and hence this work will be only

briefly mentioned in this article Recent new insights into

the mechanism of action of type I AFPs, as well as studies

on type II and III AFPs, have provided new clues about

the crucial interactions that occur between AFPs and the

ice/water interface, which need to be considered in the

mechanism of action of AFGPs Other recent progress

that is significant in the field includes detailed

characteri-zation of the solution conformation of AFGPs, the

development of methodology to allow the production of

synthetic AFGPS, and molecular evolutionary studies on the origin of AFGPs

Structure and classification of glycoproteins

Antifreeze glycoprotein is a collective name that has been used widely in the literature to refer to a group of at least eight structurally related glycoproteins that constitute the major fraction of protein in the blood serum of Antarctic notothenioids and Arctic cod Each AFGP consists of a number of repeating units of (Ala-Ala-Thr)n, with minor sequence variations and the disaccharide b-D -galactosyl-(1fi3)-a-N-acetyl-D-galactosamine joined as a glycoside to the hydroxyl oxygen of the Thr residues (Fig 2A) The glycoproteins isolated from the notothenioids [22] have been further classified as AFGP1–8 on the basis of their relative rates of electrophoretic migration [45] There are eight distinct classes of glycopeptides, which range in relative molecular mass from 33.7 kDa (n¼ 50) to 2.6 kDa (n ¼ 4) (Fig 2A) For convenience these are generally further classified as large (AFGP1–5) and small (AFGP6–8)

In addition to these molecular mass size variations, there

is some minor difference in the amino-acid composition in AFGPs 6–8 in which the first Ala in some of the repeats is replaced by Pro (Fig 2B) [11,46,47] Thus, while the notothenioid AFGPs have a simple primary structure, they exhibit significant size and some amino-acid variation AFGPs have also been identified in several Arctic and northAtlantic cods [48–50] These glycoproteins are remarkably similar to those present in the unrelated notothenioids, with the exception that Thr is occasionally replaced by an Arg residue (Fig 2C) and hence the glycopeptide lacks a disaccharide at this position

While Fig 2 shows the most common AFGP structures, there is evidence that further amino-acid substitution can be tolerated A novel AFGP containing the carbohydrate residue N-acetylglucosamine and the amino acids Asn, Gln, Gly, Ala and traces of Arg, Val, Leu and Thr has been isolated from the Antarctic fish species Pleuragramma antarcticum[51]

The general abbreviation AFGP has been widely used

in the literature although many other intermediate sizes

Fig 1 Summary of classification and key structural differences between antifreeze proteins and glycoproteins.

of glycoproteins than those shown in Fig 2 have been

identified as a result of better protein resolution

tech-niques [52] This fact has been highlighted in a recent

study in which AFGPs were isolated and purified from

the blood plasma of the rock cod Gadus ogac with

additional purification and characterization using

electro-spray mass spectrometry [53] This allowed more accurate

mass identification and showed multiple isoforms for

AFGPs within a particular mass range For example,

glycoproteins classified as AFGP6 on the basis of their

overall molecular mass, were further subdivided into two

mass fractions of 6026–9784, containing 14 different

isoforms and 3865, which contained a single sequence

Thus, the abbreviations AFGPx (x¼ 1–8) does not

always refer to a single compound, but in many cases a

mixture of glycopeptides in an approximate mass range

The use of the generic term AFGP to refer to all of the structures represented in Fig 2 has led to confusion in some literature reports where it is not clear whether a pure glycoprotein or a mixture of different molecular mass glycoproteins have been used In addition, as studies are now addressing the molecular level mechanism of ice growth inhibition, the exact amino-acid composition is also important, and the presence of any minor sequence variations in the Ala-Ala-Thr backbone needs to be established Hence we propose an expanded list of abbre-viations (Fig 2) in order to clarify the amino-acid compo-sition of the glycoprotein being studied As the exact number and positions of the Pro and Arg residues in AFGP-Pro and AFGP-Arg are frequently unknown, these abbreviations simply subclassify whether the tripeptide repeat is constant Ala-Ala-Thr or not For example, AFGP-Arg8 would refer to a tripeptide repeat where

n¼ 4 with Arg substituted for some of the Thr residues, and an approximate molecular mass of 2.7 kDa In glycoproteins in which the exact number and positions of the Pro or Arg residues are known, a full sequence and unique abbreviation is required

Origin and evolution of glycoproteins

Table 1 summarizes the phylogenetic relationship of teleost fish that produce AFGPs, adapted from Cheng [54] AFGPs have been isolated from both Antarctic notothenioid fish

as well as from a northern gadid in the Labrador, the rock cod, Gadus ogac and other high-latitude northern cods belonging to the family Gadidae [43,55] The most studied AFGPs are from the Antarctic fish, Trematomas borgrevinki and Dissostichus mawsoni, and from a northern fish, Boreogadus saida In both Trematomas borgrevinki and Dissostichus mawsoni the total AFGP concentration is about 25 mgÆmL)1of which approximately 25% is due to AFGP1–5 with the remaining 75% containing the smaller AFGP6–8

A long standing issue regarding the evolutionary origin of AFGPs was recently resolved in elegant work by Chen, DeVries and Cheng [52,56,57] The high degree of structural similarity between AFGPs found in the two geographically and phyologenetically distinct Antarctic notothenioids and Artic cods (Table 1) has been noted for many years Chen

et al sh owed th at th e AFGP gene from th e Antarctic notothenioid Dissostichus mawsoni derives from a gene encoding a pancreatic trypsinogen via a unique mechanism that does not involve the more common recycling of existing

Fig 2 General structures of antifreeze glycoproteins and abbreviations.

(A) AFGP the most common structural motif with n ¼ 4–50 (B)

AFGP-Pro in which Pro replaces Ala and (C) AFGP-Arg in which

Arg replaces Thr, with the loss of a disaccharide group, frequently at

the C-terminus of the sequences AFGP-Pro and AFGP-Arg

consti-tute <5% of the naturally occurring glycoproteins.

Table 1 Summary of phylogenetic relationship of teleost fish that pro-duce AFGPs adapted from Cheng [54].

Species Northern cods Antarctic notothenioids Family Gadidae Nototheniidae

Artedidraconidae Bathydraconidae Channichthyidae Order Gadiformes Perciformes Superorder Paracanthopterygii Acanthopterygii Division Teleosti Teleosti

protein genes The novel portion of the AFGP gene which

encodes the ice-binding function derives from the

recruit-ment and iteration of a small region spanning the boundary

between the first intron and second exon of the trypsinogen

gene Expansion and iterative duplication of this new

segment produces 41 tandemly repeated segments, with

sequences at either end that are nearly identical to

trypsi-nogen The small sequence divergence between notothenioid

AFGP and trypsin genes indicates that the transformation

of the protein gene into the novel ice-growth inhibition gene

occurred about 5–15 million years ago, which is consistent

withthe estimated times of freezing of the Antarctic Ocean

This conversion is unique and shows how an old protein

gene spawned a new gene for an entirely new protein witha

new function

In a related study, the sequence for the Arctic cod,

Boreogadus saidawas compared with the notothenioid gene

[57] While the Boreogadus saida AFGP genes have a similar

polyprotein structure to the notothenioid genes in which

multiple copies of the AFGP coding sequences are linked by

small cleavable spacers, molecular evidence from detailed

comparative analyses argue strongly for independent

evo-lution of the cod AFPG genes This evidence includes (a)

different signal peptide sequences, (b) different spacer

sequences that link the encoded AFGP molecules in the

polyprotein, invoking different mechanisms of processing of

the polyprotein precursors, (c) distinct codon bias of the

nine nucleotide sequence for the AFGP tripeptide, and (d)

different genomic loci of the AFGP gene loci in the cod and

notothenioid AFGPs Thus, the near-identical AFGPs of

these two unrelated fish is a rare example of protein

sequence convergence, i.e the development of a similar

protein from different parents under similar environmental

pressure Furthermore these studies established that every

AFGP isoform is distinctly encoded as individual copies

within polyprotein genes, i.e the various lengths of AFGPs

shown in Fig 2A are not due to protein processing through

splicing small AFGPs or cleaving large ones into small ones

The high concentration of the AFGPs in blood

(35 mgÆmL)1) also suggest that a large family of polyprotein

genes must escalate the gene dosage

Properties

AFGPs accumulate at certain faces of the ice/water

interface, and modify the rate and shape of crystal growth

The terms ‘antifreeze’ activity, ice growth inhibition and

hysteresis, and definitions and labelling of the different ice

planes are illustrated in our earlier review of type I AFPs

[27]

A characteristic property of AFPs and AFGPs is thermal

hysteresis, which is determined by measurement of the

kinetic ice growthpoint and subtraction of the equilibrium

melting (¼ freezing) point of a solution [43] In the presence

of an AFGP, the measured melting point depression is as

expected on the basis of colligative properties, i.e it is

proportional to the molar fraction of molecules in solution

The depression of the ice growth point (the temperature at

which ice starts to grow from a seed ice crystal) is, however,

very much greater than this Figure 3 shows the

concentra-tion-dependent thermal hysteresis exhibited by AFGPs, the

magnitude of which depends on the length of the polymer

chain Maximum hysteresis is observed with AFGP1–5, compared withthe lower molecular mass AFGP6–8 [58] These values are comparable to the thermal hysteresis exhibited by many type I AFPs [27]

Other phenomena associated with ice growth inhibition include accumulation at specific faces of the ice crystal, detected by hemisphere etching [59], and modification of the crystal habit when ice is grown in a thermal gradient Ice may exist in many polymorphic forms, withice 1 hthe most stable form at 1 atmosphere below zeroC The hexagonal ice 1 hlattice unit may be characterized by four axes, a1, a2,

a3and c with the surface of the hexagonal unit comprising eight faces, two basal faces normal to the c-axis and six prism faces [27] As it is normal to the c-axis, the basal face is known as the c-face or (0001) Directions and vectors within the ice lattice are also described in terms of the four axes and are distinguished by the types of brackets that encloses the coordinates For example, 2 0  22 1

designates the group

of 12 equivalent surfaces of a hexagonal bipyramid of which ð2 0 22 1Þ is one specific surface

Raymond et al showed that single ice crystals suspended

in solutions of AFGP1–5 at temperatures within the hysteresis gap form hexagonal pits on the basal plane, while

in the presence of AFGP7–8, c-axis growthoccurred to a greater extent and the edges of the basal plane formed bipyramidal faces [60] Figure 4 illustrates the effect of blood serum from Dissostichus mawsoni on ice crystal growth, showing the formation of the ‘pits’ on a flat basal surface of a growing seed crystal of ice 1 h The equilibrium melting/freezing point of the solution is measured to be )1.21 C The three images are taken approximately 30 s apart, in order to show the growth of hexagonal pits These pits eventually cover the entire exposed surface of ice, which then stops growing (even though below the equilibrium melting/freezing temperature of the solution) until the temperature is decreased even further, well below temper-atures the fish encounter in the ocean This modification of the ice crystal habit by AFGPs is quite different to the AFPs which typically inhibit growth along the a-axis resulting in

Fig 3 Measured thermal hysteresis for AFGP1–5 (diamonds) and AFGP7, from Knight, DeVries and Oolman [58], as a function of con-centration The lines are our two-parameter Langmuir fits to the data

of the form (DT/DT max ) ¼ (c/d)/[(c/d) + 1], where for AFGP1–5

DT max ¼ 1.40 and d ¼ 10.7 mgÆmL)1, and for AFGP7 DT max ¼ 0.78 and d ¼ 11.2 mgÆmL)1.

accelerated growth primarily along the crystallographic

c-axis to give bipyramidal crystal forms [27]

Using hemisphere etching, a simple test to determine

which crystal planes (if any) are recognized by a compound,

AFGP7 and AFGP8 were shown to accumulate at the

primary prism planesð1 0 11 0Þ by Knight [61,62], while at

very low concentrations (<0.03 mgÆmL)1) AFGP1–5

accumulate at the ð4 1 55 0Þ plane, changing to the

ð1 0 11 0Þ plane at higher concentrations [59] Elegant

ellipsometry measurements withAFGP7 and AFGP8 have

shown that that the AFGPs accumulate at the basal and

prism planes of single ice crystals [63] The particular faces at

which specific AFGPs accumulate were determined

ele-gantly by Knight and colleagues [59]

Effect of molecular mass

As shown in Fig 3, the molecular mass of the different

AFGPs is important withthe longer polymers (AFGP1–5)

having enhanced thermal hysteresis properties compared to

the shorter polymers (AFGP6–8) The small molecular mass

forms (AFGP7 and 8) comprise most of the circulating

antifreeze [64] but show only two-thirds of the antifreeze

activity of the larger molecular mass AFGPs [65] However,

comparison of the effect of a synthetic dimer of AFGP6 to

the monomeric AFGP6 did not show substantially greater

activity when the molecular mass was doubled [66] The

dimer was prepared by carbodiimide coupling of methylated

AFGPs, followed by HPLC purification and cleavage of the

O-acyl bonds The synthetic dimer contains a different

peptide sequence to the natural AFGP with a Pro following

three Ala residues This sequence may affect the AFGP

conformation and hence the ability to inhibit ice growth

resulting in no increase in thermal hysteresis

A more rigorous study of the hysteresis values of a

series of highly purified AFGPs from the rock cod Gadus

ogac showed that they could be grouped into two

distinct classes AFGPs withmolecular mass >13 kDa

gave approximately three to four times higher hysteresis

values than the smaller Pro–containing AFGPs (mole-cular mass <10 kDa) [53]

The hysteresis values of AFGP from cod has been compared withthe values obtained for different AFPs [67] Due to the structural differences between the AFGPs and AFPs (see Fig 1) these results are not directly comparable, but in terms of molecular mass it was noted that the type I AFPs from the winter flounder and shorthorn sculpin had greater activity than did glycoproteins of similar size However AFGPs witha molecular mass of 10 kDa or higher had activities which exceeded those of any known AFP

Structural modification of sugars

There are limited studies on the structural requirements of the disaccharide that are required for activity This is directly related to difficulty in the synthesis of AFGPs and derivatives (discussed in a later section) Hence the only data available is on standard carbohydrate degradation studies The key derivatives that have been prepared, and the effects

of these structural modifications are summarized in Fig 5 However, it should be noted that in most cases the derivatives were not isolated and purified

The glycopeptide structure is important as b-elimination

of the saccharides and loss of the Thr hydroxyl function-ality removes all antifreeze activity [23,68] Acetylation of the sugar hydroxyls to give derivative 1, or periodate oxidation of the terminal galactose sugar to give derivative

2, removed the hysteresis properties of the AFGP consistent withthe requirement of at least some, if not all, of the hydroxyls on the galactose sugar [23] Oxidation withD-galactose oxidase to give the bisaldehyde 3 had no effect on hysteresis showing that the hydroxyl group at C6

of galactose is not essential for activity However, conversion of the newly formed aldehydes to negatively charged groups by oxidation to the acid 5 or by addition

of bisulfite to give 4 removed activity [22,69] Thus the type of functional groups present on the sugars are

Fig 4 Series of photographs of ice growing from Dissostichus mawsoni blood serum, which contains AFGPs The equilibrium melting/freezing point

of the solution is ) 1.21 C, and the magnification is 15 x The photographs are taken 30 s apart, from right to left The pits are growing on a flat basal surface of ice 1 h, which is advancing slowly towards the camera, at a temperature of approximately )2.0 C The symbol ‘V’ indicates a fixed position between two pits, which to grow substantially Eventually, the entire surface is covered, no basal surface is exposed, and the ice stops growing (data not shown) Upon lowering the temperature further, beyond the hysteresis gap, the ice grows ‘explosively’, shooting out spicules through the entire remaining solution (data from D J Haymet, unpublished results).

important and the loss of activity of both 4 and 5

compared with 3 suggest that the negative charge is not

tolerated Addition of 0.15Msodium borate to the AFGP

eliminated hysteresis This reagent complexes cis-hydroxyl

groups and hence gives rise to a mixture of products

including 6a and 6b This reaction is pH dependent and

could be reversed to give fully active AFGP [22,70]

More recently, oxidation of the C-6 hydroxyls of a

mixture of AFGPs from Pagtothenia borchgrevinki to the

aldehyde with galactose oxidase and catalase, produced

peptides with an average of 75% of the activity of the native

AFGP, but withsome batches dropping to 30% activity

[71] These peptides were then reductively alkylated with a

variety of amino acids or short peptides and

cyanoboro-hydride and the antifreeze activity reported relative to the

oxidized starting material Glycopeptides withGly to (Gly)4

substitution all contained activity >60% that of the

oxidized starting material, indicating that bulky substitution

at the C-6 position is not detrimental to activity while the

lowest activities were reported for the Gly-Glu (13%),

Gly-Gly-Phe (30%) and Arg (30%) derivatives

Taken together, these degradation results support a

requirement for at least some of the sugar hydroxyl groups

for activity The C-6 hydroxyl group does not appear to be

required for activity, and the C6-position tolerates a range

of substituents with the exception of charged groups The

importance of the following structural elements for

anti-freeze activity has not been established: (a) the number of

hydroxyls required, (b) the stereochemistry of the sugar

hydroxyls (i.e the requirement of galactose sugars), (c) the

acetamido group on the first galactose sugar and (iv) the

stereochemistry of the b-glycosidic linkage between the two

sugars and the a-glycosidic linkage to Thr, and (d) the

requirement of a disaccharide for activity

Modification of the peptide backbone

As discussed above and shown in Fig 2, the most common tripeptide in AFGPs is Ala-Ala-Thr, while in the smaller glycopeptides Pro or Arg substitutes occasionally for Ala A series of glycopeptides of approximately the same molecular mass but containing different amounts of Pro and Arg were prepared by Edman degradation of AFGPs isolated from different species of fish [72] The very similar hysteresis values measured on solutions of these different AFGPs at a range of concentrations indicate that the amino-acid composition does not have a significant effect on noncol-ligative freezing point depression Of note is the fact that substitution of Arg for Thr removes the disaccharide from one of the tripeptide units, but this structural modification does not affect antifreeze activity However, a systematic study of the number of substitutions of Ala for Pro or Thr for Arg that can be tolerated in a given molecular mass AFGP has not been carried out Cleavage of the peptide backbone withsubtilopeptidase A, as expected, removed activity [9]

There have been no systematic investigations into the role of the Ala and Thr residues in activity Outstanding questions include whether Ser could be substituted for Thr, which would simplify synthetic production of AFGP analogues, and the role of the Ala sidechains The evolution

of Ala in the tripeptide could be due to the hydrophobic nature of the sidechain, or the small sidechain, which has unique secondary structure preferences It is interesting to note that in the type I AFPs, Thr and Ala are critical amino acids required for activity in these a-helical proteins In particular, the b-methyl group of Thr, along with sur-rounding hydrophobic residues, including Ala, provide a hydrophobic face of the helix which is oriented towards the

Fig 5 Summary of key degradation studies on AFGPs With the exception of oxidation of the primary alcohols on the galactose sugars to give derivative 3, all other modifications give derivatives that lack antifreeze activity.

ice/water interface [27] The role of hydrophobicity in the

mechanism of action of AFGPs has not yet been

consid-ered (discussed in a later section) but in this context, the

effect of mutation of the Thr and Ala residues in the

Ala-Ala-Thr tripeptide repeats of AFGPs would be highly

informative

Synthesis of antifreeze glycoproteins

AFGPs are presently only available from natural sources in

limited amounts Difficulties in isolation from natural

sources in analytically pure quantities for commercial

development, as well as the fact that harvesting of fish is

necessary, require the development of an alternative source

of compounds Hence recent research has focused on the

development of an efficient synthetic route to AFGPs and

analogues The synthesis of glycoproteins and

carbo-hydrates is significantly more demanding than for protein

synthesis While automated solid-phase peptide synthesis or

molecular biology techniques allow the routine production

of AFPs, as well as the incorporation of mutations and

isotopic labels into AFP sequences (see for example [27,73]),

these methods are not widely applicable to the preparation

of AFGPs

The first and only synthesis of a naturally occurring

AFGP was reported in 1996 and is summarized in Fig 6A

[74] The key glycotripeptide was polymerized using

diphenylphosphoryl azide to give a polymer with an

estimated molecular mass of 6000–7300, i.e n¼ 10–12 A

full paper describing the experimental details and testing of

the synthetic AFGPs for activity has not been reported In

principle, modification of this synthetic scheme should allow

the production of synthetic AFGP analogues in which the

number and relative stereochemistries of the hydroxyls are

varied in eachsugar, and hence provide access to

compounds which would allow key structure activity studies

to be performed Other potential synthetic routes to AFGPs

which involve glycosidation of Thr as the last step in the

synthesis [75,76] are currently restricted to model tripeptides

An alternate route to high molecular mass AFGPs using

solid-phase peptide synthesis has recently been reported

(Fig 6B) using Fmoc-chemistry and standard protecting

groups to produce AFGPs where n¼ 4 and 8 [77] Related

solid-phase synthesis [78,79] of AFGPs containing single

sugars should allow access analogues for structure-activity

studies

The advantage of using solid-phase synthesis (Fig 6B) is

the ability to generate oligomers of defined length and

sequence variation, including mutations of the Ala residues

at one or more sites in the sequence and modification of the

structure of eachsugar by the preparation of a different

Fmoc-protected building block In contrast, the solution

phase route (Fig 6A) will always produce mixtures of

oligomers which need to be separated, and require the use of

a single tripeptide unit for the polymerization reaction

Given the synthetic difficulties outlined above,

ana-logues of AFGPs that are synthetically more accessible by

the replacement of Thr with Lys and the formation of the

more stable C-glycosides in place of O-glycosides have

been reported [80,81] However, the effect of these drastic

structural modifications on hysteresis has not been

published

Solution conformation

A detailed knowledge of the solution conformation of AFGPs is clearly essential in understanding the molecular mechanism of ice growth inhibition A range of techniques have been used to study the solution conformation of different AFGPs including CD, Raman spectroscopy, light scattering measurements and NMR spectroscopy

Fig 6 Synthesis of AFGPs Comparison of th e key steps in th e syn-thesis of low molecular mass AFGPs by (A) solution phase methods, withglycosylation of a tripeptide followed by polymerization, and (B) solid phase methods, with utilization of a glycoslyated threonine pre-cursor in elongation of the peptide backbone.

Early CD studies of one AFGP [9] concluded that the

compound had a random coil conformation Due to the

similarity of the CD spectrum of a random coil and a

left-handed 3-residue-per-turn helix, the temperature

depend-ence of the CD spectra of the AFGP from Trematomus

borchgrevinkiand Eliginus gracilis were measured [82] The

lack of a sharp transition in the spectra was consistent with

an unordered conformation in solution Independent CD

studies [83], quasielastic light scattering [84] and Raman

spectroscopy measurements [85] all suggested the presence

of some folded structure

Natural abundance 13C NMR spectroscopy of an

aqueous solution of AFGP3–6 from Dissostichus mawsoni,

including measurement of relaxation times, nOes and

variable temperature experiments were consistent withthe

AFGPs existing as predominantly flexible random coil

polymers [86] Early 1H NMR data (300 MHz) of

AFGP1–4 [87] provided a more detailed picture of the

conformation and, along withconformational energy

calculations, it was proposed that the hydrophobic surfaces

of the disaccharide side chains are wrapped closely against a

threefold left handed helical backbone A comparison of the

solution conformation of AFGP1–4 and AFGP8 suggested

that both AFGPs adopt similar conformations [88], and

hence the differences in their ice growth inhibition properties

(see Fig 3) are not due to a structural difference 2D NMR

studies (300 MHz) [89] allowed further refinement of this

data and measurement of amide exchange rates which ruled

out significant strong hydrogen bonding involving the

amide protons in aqueous solutions Comparison of AFGP

amide vibrational frequencies withthose observed and

calculated for beta and gamma-turns in other peptides

suggests that AFGPs contain substantial turn structure [90]

while NMR studies on model glycopeptides showed an

intramolecular hydrogen bond between the amide proton of

N-acetylgalactosamine and the carbonyl oxygen of the Thr

to which the sugar is attached [91]

The most detailed insight into the global conformation of

AFGPs has been provided from two recent complementary

papers from the same group [92,93] A combination of high

field NMR (500 MHz) and IR spectroscopies, along with

molecular dynamics calculations were performed on the 14

amino-acid residue Thr-Pro-Ala glycoprotein AFGP8 (i.e

AFGP-Pro8 in Fig 2B), and a mixture of AFGP1–5, which

contains no Pro residues While AFGP-Pro8 has no

long-range order, it displays significant local order In contrast,

AFGP1–5 was reported to be a dynamically disordered

molecule that shows neither significant long or short range

order The somewhat unexpected result that AFGP-Pro8

lacks long range order [92,93], has prompted a closer study

of this Pro-containing AFGP Using an initial model

derived from 10 NMR structures, molecular dynamics

simulations along withfree energy calculations using a

continuum solvation model were performed to gain insight

into the nature of the conformations and motions in this

AFGP-Pro8 [94] While the presence of the Pro residues

does induce adoption of a poly proline helix, the

glycopro-tein exists in a number of structurally distinct, but

energeti-cally equivalent conformers Hydrogen bonding between

the N-acetyl groups and the peptide backbone were also

identified as making a significant contribution to the overall

stability of the AFGP [94]

13C NMR spectroscopy and FTIR spectroscopy have been used to probe the dynamics and conformations of an N,N-dimethylated AFGP from the Greenland cod in the presence of ice [95] Overall the study concluded that the AFGP adopts a similar type of three-dimensional fold in the presence of ice and in a freeze-dried state but, as in related studies, the molecule is highly flexible accessing a large number of conformers

Despite these recent NMR studies, no three-dimensional solution structure of any AFGP has been published The torsional flexibility in the sequence, as well as the fact that a large number of conformers are available do not allow a definitive structure to be produced This contrasts with the well-defined secondary and tertiary structures present in the type I-IV AFPs (Fig 1)

Mechanism

There is currently no mechanism that explains the ice growthinhibition properties of AFGPs Just as withearly proposed models for the mechanism of type I AFPs (summarized in [27]), in the case of AFGPs many erroneous conclusions were drawn from vacuum/ice models at the absolute zero of temperature, which have little or nothing in common withice/water interfaces at or near the melting point A hydrogen-bonding dominated mechanism, that involves insertion of the disaccharide hydroxyls of AFGPs into the vacuum/ice lattice, was proposed by analogy with a model for the type I AFP from the winter flounder that relied on hydrogen bonding involving the hydroxyl groups

in the Thr residues [62] However, structure-activity studies have now shown conclusively that hydrophobic interactions provided by the b-methyl group of the Thr residues are crucial to the ice growth inhibition mechanism in type I AFPs [73,96–99] Lavalle, DeVries and colleagues [100] have recently studied adsorption of AFGP1–5 on surfaces other than ice, namely two silicate minerals While not directly relevant to the behavior in water, they conclude that their results argue ‘against a crucial role of hydroxyl matching in the antifreeze action’ [100], and cite the companion story in type I AFPs [27]

To date, apart from the work of Lavalle, DeVries and colleagues [100], the ice/vacuum mechanism for AFGPs involving hydrogen bonding has not been revisited In light

of the recent new insights into the mechanism of action of type I AFPs, including the important role of hydrophobic interactions, new mechanisms for the molecular action of AFGPs need to be considered The chemistry of modifica-tion of the hydroxyl groups (including stereochemistry), as well as the hydrophobic amino-acid sidechains, which illuminated the interactions of type I AFPs with the ice/ water interface, is obviously more difficult for the AFGPs,

as described in the Synthesis section above It will be of interest to see whether hydrophobicity is a dominant interaction in the mechanism of action of both AFGPs and AFPs While AFGPs are unstructured in solution, it has been noted that in a three-fold left-handed helical conformation, the glycoprotein contains a hydrophilic side face and a hydrophobic face in which most of the Ala side-chains are located [3,34,87] Whether this conformation is a significant contributor to the alignment of AFGP molecules withspecific surfaces at the ice/water interface is unknown

and will need to be tested through structure-activity

relationship studies

Applications

BothAFGPs and AFPs exhibit a number of unique

properties which protect biological systems in vitro and have

been investigated for potential applications in medicine,

biotechnology and the food industry A comprehensive

review summarizing th e effects of AFPs and AFGPs on low

temperature preservation processes has recently been

pub-lished [31] The ability to change the normal growth habit of

ice, the capacity to inhibit recrystallization and the

protec-tion of cell membranes are all properties of AFPs and

AFGPs that may be tailored for a range of low temperature

processes

The ability of AFGPs to aid in the cryopreservation and

hypothermal storage of cells and tissues was noted by

Rubinsky et al [101] The effect of the addition of a mixture

of AFGP1–8 (one part AFGP1–5 to three parts AFGP7–8),

or separate solutions of AFGP1–8 and AFGP7–8, on the

storage of pig oocytes which cannot survive hypothermic

temperatures as high as 10C, was evaluated [102]

Protection of the oocytes was monitored by measurements

of the membrane potential across the oolemma, and it was

proposed that AFGP1–8 protect the cell membranes and

inhibit ion leakage Later studies proposed a more detailed

mechanism of cellular protection by both AFPs and AFGPs

involving blocking of the potassium and calcium ion

channels during cooling [103,104] In contrast, AFGPs

failed to enhance storage of isolated rat hearts at

hypother-mic temperatures and caused increased damage under

freezing conditions regardless of AFGP concentration [105],

and samples of ram spermatozoa were not stabilized in the

presence of AFGPs when chilled and rewarmed [106]

In an effort to understand the apparent different

pro-perties of AFGPs discussed above, Crowe and coworkers

performed a series of studies on liposomes as a model for

studying the effects of lipid-phase transitions The effects of

AFGPs on the leakage of a trapped marker from liposomes

during chilling were monitored [107] While cooling of these

liposomes through the transition temperature resulted in

leakage of approximately 50% of their contents, addition of

less than 1 mgÆmL)1of AFGP prevented up to 100% of th is

leakage, both during chilling and warming through the

phase transition Thus it was concluded that the stabilizing

effects of AFGPs on intact cells during chilling reported in

earlier studies [103,104] was possibly be due to a nonspecific

effect on the lipid components of native membranes

[107,108] The importance of performing studies with

purified AFGPs was also highlighted, with contaminants

from other blood proteins present shown to also associate

with liposomes, leading to defects in the bilayer and thus

leakage [108] An independent study on the effect of AFGPs

from the rod cod Gadus ogac showed that all AFGPs with

molecular mass 2.6–24 kDa prevented leakage from model

liposomes as they were cooled through their phase transition

temperature, withthe larger molecular mass compounds

being about four times as effective as the smaller ones [53]

In support of the hypothesis that AFGPs protect cellular

membranes during lipid-phase transitions, improved

stor-age of chilled blood platelets was demonstrated [30,108] In

contrast to liposomes, only the AFGPs provided a protect-ive mechanism with nonglycosylated AFPs and ovotrans-ferrin having no beneficial effects The internal calcium concentration of human platelets was shown to increase during chilling [109] but AFGPs did not eliminate this rise in concentration

More recently the effects of AFGPs on different mem-brane compositions has been studied [110] The effects of freezing spinachthyalkoloid membranes and model mem-branes of varying lipid compositions in the presence of AFGPs showed that the lower molecular mass AFGP8 offers a limited degree of protection during freezing and does not induce membrane fusion at concentrations up to

10 mgÆmL)1 This behavior is quite distinct from that exhibited by AFPs [111], or the larger molecular mass AFGP1–5 or AFGP3–4, which are cryotoxic to thyalka-loids and liposomes

AFGPs and AFPs have been identified as useful in cryosurgery, increasing the destruction of solid tumors through mechanical damage to cells caused by the growth of bipyramidal ice crystals [112] However specific applications are limited to AFPs [113,114] and the effectiveness of AFGPs in this field has yet to be demonstrated

BothAFPs and AFGPs have attracted significant interest

as potential food additives that inhibit ice recrystallization and hence the formation of large ice crystals in the storage

of frozen foods [29,32,115,116] Unfortunately the use of the generic term antifreeze proteins or compounds to refer to bothAFPs and AFGPs in many papers and reviews makes

it difficult to establish exactly which AFGPs have been tested Studies of the effect of AFGP1–8 (Dissostichus mawsoni) in the quality of frozen meat have shown reduced tissue damage due to freezing [117], and improved drip loss and sensory properties of thawed meat from lambs that had been administered AFGPs prior to slaughter [118] How-ever, efficient and cost-effective methods of using these compounds as additives are required for commercial applications

Conclusions

While significant progress had been made in the structural characterization and properties of AFPs and AFGPs from cold water fish, the molecular level detail of how each class

of compounds is able to inhibit ice growth is still not fully understood The key structural features required for ‘anti-freeze’ activity by type I AFPs have been identified through structure-activity studies on analogues accessible using either synthetic or molecular biology techniques In contrast, the lack of a feasible synthetic route to AFGP analogues has hampered progress with this class of compound, and the understanding of the accumulation of AFGPs at certain ice/ water interfaces stands at roughly the same point as type I AFPs were in the early 1990s A concerted attempt at the routine production of AFGP analogues is warranted, as difficult as this may be, to provide essential data regarding the mechanism of ice growth inhibition In addition, such studies have the potential to identify simpler AFGP analogues that are less difficult to produce

A second avenue ripe for exploration is the interaction of AFGPs (and AFPs) withmembranes, bothsynthetic and natural [110–112] While potential applications in the

storage and preservation of low temperature biological

samples has been demonstrated, systematic studies are still

required to establishhow eachclass of compound interacts

withmembranes and other biomolecules in order to tailor

new AFPs and AFGPs for specific applications

Acknowledgements

M M H acknowledges financial support from the University of

Sydney Sesqui Research and Development Scheme and the Australian

ResearchCouncil, and travel funds from the Australian Academy of

Science A D J H thanks the Welch Foundation for support at the

University of Houston where part of this review was written, and NSF

for use of the Crary Research Laboratory, McMurdo Sound, where the

data for Fig 4 were collected A D J H acknowledges many helpful

conversations on this topic over the years with Drs Art DeVries, Chris

Cheng, Charlie Knight and Peter Wilson.

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