Tài liệu Báo cáo khoa học: Co-operative effect of the isoforms of type III antifreeze protein

Co-operative effect of the isoforms of type III antifreeze

protein expressed in Notched-fin eelpout, Zoarces

elongatus Kner

Yoshiyuki Nishimiya1

, Ryoko Sato1

, Manabu Takamichi2

, Ai Miura1 and Sakae Tsuda1,2

1 Functional Protein Research Group, Research Institute of Genome-based Biofactory (RIGB), National Institute of Advanced Industrial

Science and Technology (AIST), Sapporo, Japan

2 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan

Antifreeze protein (AFP) possesses the unique ability

to bind to the surface of ice crystals, which permits

growth of ice at limited open spaces on the surface,

leading to the formation of numerous convex ice surfa￾ces between the bound AFPs [1]. The growing ice sur￾face becomes energetically unfavorable for further

absorption of water molecules proportionately with the

surface curvature, leading to termination of ice growth

(Kelvin effect) [2]. This AFP-induced inhibition of ice

crystal growth can be detected macroscopically as a

depression in the freezing temperature (Tf) of the

solution without alteration of the melting temperature

(Tm) (|Tf ) Tm|). This is generally termed thermal hys￾teresis (TH).

AFPs categorized as type III (
7 kDa) have been

identified in blood serum of fish living in polar sea￾water with a year-round temperature of
)1.8
C:

Arctic and Antarctic eelpouts (Austrolycicthys brachy￾cephalus, Lycodes polaris, and Lycodichthys dearborni),

Atlantic ocean pout (Macrozoarces americanus), and

Atlantic wolffish (Anarhichas lupus) [3–6]. Type III

AFP forms a globular shape characterized by internal

Keywords

co-operative effect; Notched-fin eelpout;

type III antifreeze protein

Correspondence

S. Tsuda, Functional Protein Research

Group, Research Institute of Genome-based

Biofactory (RIGB), National Institute of

Advanced Industrial Science and Technology

(AIST), 2-17-2-1 Tsukisamu-Higashi,

Toyohira, Sapporo 062-8517, Japan

Fax: +81 11 857 8983

Tel: +81 11 857 8912

E-mail: [email protected]

Note

The nucleotide and protein sequences

reported here have been deposited in the

DDBJ database under the accession

numbers AB188389–AB188401.

(Received 27 August 2004, revised 9

November 2004, accepted 17 November

2004)

doi:10.1111/j.1742-4658.2004.04490.x

We found that Notched-fin eelpout, which lives off the north east coast

of Japan, expresses an antifreeze protein (AFP). The liver of this fish

contains DNAs that encode at least 13 type III AFP isoforms (denoted

nfeAFPs). The primary sequences of the nfeAFP isoforms were categor￾ized into SP- and QAE-sephadex binding groups, and the latter were fur￾ther divided into two subgroups, QAE1 and QAE2 groups. Ice crystals

observed in HPLC-pure nfeAFP fractions are bipyramidal in shape with

different ratios of c and a axes, suggesting that all the isoforms are able

to bind ice. We expressed five recombinant isoforms of nfeAFP and ana￾lyzed the thermal hysteresis (TH) activity of each as a function of pro￾tein concentration. We also examined the change in activity on mixing

the isoforms. TH was estimated to be 0.60
C for the QAE1 isoform,

0.11
C for QAE2, and almost zero for the SP isoforms when the con￾centrations of these isoforms was standardized to 1.0 mm. Significantly,

the TH activity of the SP isoforms showed concentration dependence in

the presence of 0.2 mm QAE1, indicating that the less active SP isoform

becomes ‘active’ when a small amount of QAE1 is added. In contrast, it

does not become active on the addition of another SP isoform. These

results suggest that the SP and QAE isoforms of type III AFP have dif￾ferent levels of TH activity, and they accomplish the antifreeze function

in a co-operative manner.

Abbreviations

AFGP, antifreeze glycoprotein; AFP, antifreeze protein; nfeAFP, type III AFP from Notched-fin eelpout; TH, thermal hysteresis.

482 FEBS Journal 272 (2005) 482–492 ª 2004 FEBS