Tài liệu Báo cáo khoa học: Type I antifreeze proteins expressed in snailfish skin are identical to

Type I antifreeze proteins expressed in snailfish skin are

identical to their plasma counterparts

Robert P. Evans and Garth L. Fletcher

Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada

Teleost fish that inhabit icy seawater synthesize anti￾freeze proteins⁄ polypeptides (AFPs) or antifreeze glyco￾proteins (AFGPs) for protection against freezing.

Diverse species from numerous taxonomic groups pro￾duce AFPs that are grouped into four distinct classes

(types I, II, III and IV) based on their primary and

secondary structural characteristics [1–3]. Regardless of

protein structure, all fish AFPs lower the solution

freezing point noncolligatively by binding to certain

surfaces of ice crystals, modifying their structure and

inhibiting further growth. The difference between the

lowered freezing point and unaltered melting point is

termed thermal hysteresis and is used as a measure of

antifreeze activity [1,3,4].

Of the four classes of AFPs described thus far, the

simplest is type I AFP found in right-eye flounders

(Pleuronectes) and a few sculpin species (e.g. Myoxo￾cephalus). These polypeptides have high alanine con￾tent (> 60 mol%), have an amphipathic a-helical

secondary structure, and are usually quite small (3.3–

4.5 kDa) [2,5]. Until the past decade, it was generally

accepted that the synthesis of AFPs was confined

solely to liver tissue (termed liver type) for secretion

into blood for extracellular freeze protection. How￾ever, more recently, a novel subclass of type I

AFPs was isolated and characterized from the skin of

winter flounder Pseudopleuronectes americanus (for￾merly Pleuronectes americanus) [6]. These AFPs, which

Keywords

antifreeze; cDNA; protein expression;

snailfish; type I

Correspondence

R. P. Evans, Department of Biochemistry,

University of Alberta, Edmonton, Alberta

T6G 2H7, Canada

Fax: +1 780 492 0886

Tel: +1 780 492 3481

E-mail: [email protected]

(Received 13 June 2005, revised 8 August

2005, accepted 22 August 2005)

doi:10.1111/j.1742-4658.2005.04929.x

Type I antifreeze proteins (AFPs) are usually small, Ala-rich a-helical poly￾peptides found in right-eyed flounders and certain species of sculpin. These

proteins are divided into two distinct subclasses, liver type and skin type,

which are encoded by separate gene families. Blood plasma from Atlantic

(Liparis atlanticus) and dusky (Liparis gibbus) snailfish contain type I AFPs

that are significantly larger than all previously described type I AFPs. In

this study, full-length cDNA clones that encode snailfish type I AFPs

expressed in skin tissues were generated using a combination of library

screening and PCR-based methods. The skin clones, which lack both signal

and pro-sequences, produce proteins that are identical to circulating plasma

AFPs. Although all fish examined consistently express antifreeze mRNA in

skin tissue, there is extreme individual variation in liver expression – an

unusual phenomenon that has never been reported previously. Further￾more, genomic Southern blot analysis revealed that snailfish AFPs are

products of multigene families that consist of up to 10 gene copies per

genome. The 113-residue snailfish AFPs do not contain any obvious amino

acid repeats or continuous hydrophobic face which typify the structure of

most other type I AFPs. These structural differences might have implica￾tions for their ice-crystal binding properties. These results are the first to

demonstrate a dual liver⁄skin role of identical type I AFP expression which

may represent an evolutionary intermediate prior to divergence into distinct

gene families.

Abbreviations

AFGPs, antifreeze glycoproteins; AFPs, antifreeze proteins ⁄ polypeptides; IBM, ice-binding motif; ORF, open reading frame;

UTR, untranslated region.

FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS 5327