Sculpin-type antifreeze polypeptides and nucleic acids

Details Sculpin-type antifreeze polypeptides and nucleic acids Sculpin-type antifreeze polypeptides and nucleic acids

1999-06-24

2002-08-06

Martin, Jill D. (Department: 1632)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C426S321000, C530S300000, C530S327000

Reexamination Certificate

active

06429293

ABSTRACT:

BACKGROUND OF THE INVENTION
The survival of cellular organisms is dependent on the physical properties of water. The freezing point of liquid water sets the lower limit for the survival of most cells, because the formation of ice causes dehydration and osmotic damage to the cell. Organisms that inhabit sub-zero environments, have special adaptations which permit the organism to survive. For example, Arctic and Antarctic fish which live in cold seawater have various macromolecular antifreeze polypeptides in the serum of their blood. Such antifreeze polypeptides are a mixture of glycoproteins having a range in relative molecular mass (M
r
) from about 2,500 to 34,000 (antifreeze glycoproteins, or “AFGPs”) and antifreeze polypeptides (AFPs) with M
r
from about 3,300 to 12,000. Ananthanarayanan (1989)
Life Chemistry Reports
7:1-32 provides an overview of AFPs and AFGPs. See also DeVries (1983)
Annu. Rev. Physiol
45: 245-260; Davies et al., (1990)
FASEB J
4: 2460-2468 and Warren et al., U.S. Pat. No. 5,118,792.
At present, four distinct types of AFPs have been characterized from a variety of cold water fish See, Davies et al., (1990)
FASEB J.
4: 2460-2468; and Griffith and Ewart et al. (1995)
Bioteca Adv.
13(3): 375-402, and references therein. Type I AFPs are alanine-rich, &agr;-helical polypeptides, found in many right-eye flounders and sculpins. Type II AFPs are enriched with cysteine and are found in sea raven, smelt and herring. Type III AFPs are globular proteins found in several Zoarcoid families including eelpout and wolffish. Type IV AFPs are characterized by a helix bundle and have been found in longhorn sculpin,
Myoxocephalus octodecimspinosis
(see, G. Deng et. al. (1997)
FEBS Letters
402: 17-20. Although the different AFPs and AFGPs are structurally distinct, they share the ability to inhibit ice crystal growth by binding to the ice surface.
AFPs in the liver (liver-type AFPs; Type I) from the Winter flounder,
Pleuronectus americanus
, have been studied extensively in terms of their structure and function, gene organization, gene expression and regulation. The genome of the Winter flounder contains multiple copies of liver or serum type AFP genes, most of which are arranged as regular tandem repeats (Scott et al., (1985)
Proc. Natl. Acad Sci. USA.
82: 2613-2617).
WO97/28260 describes the presence of new isoforms of AFPs in the Winter flounder,
Pleuronectes americanus
that are synthesized in the peripheral tissues, such as the skin and gills. These AFPs are referred to as “skin-type AFPs” and are encoded by a distinct set of AFP genes that lack a signal peptide which is indicative of their intracellular location. Examples of skin isotypes from the Winter Flounder are wfsAFP-1 and wfsAFP-8. The presence of extracellular and intracellular AFPs with differential tissue expression within a single fish species has raised questions about the relative roles of these AFP isoforms in cold protection, function, and evolutionary relationship.
There exists a need in the art for new AFPs with unique physiologic function and in situ location that inhibit ice recrystallization and induce a concentration-dependent decrease in the freezing point of aqueous solutions. The present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
The present invention relates to a new intracellular AFP found in the shorthorn sculpin (
Myoxocephalus scorpius
) (“sculpin-type AFP”). These new sculpin-type AFPs aid the fish in its defense against the dangers of freezing in the ice-laden, sub-zero sea water. The shorthorn sculpin synthesizes AFPs that serve to depress the freezing temperature of its intracellular fluids. These sculpin-type AFPs thus function as protectors of intracellular components.
As such, the sculpin-type AFPs of the invention are generally useful in protecting solutions against freezing. This improves the shelf-life of many refrigerated foods, making the foods more palatable. The sculpin-type AFP, when added, inhibits ice recrystallization during cold storage, improving the texture and palatability of the food. In addition, cells that express the sculpin-type AFPs of the invention are more cold-tolerant than counterpart cells which do not express sculpin-type AFPs. Thus, the sculpin-type AFPs of the invention are used to improve the cold tolerance of bacteria, cell cultures, plants and animals. The sculpin-type AFPs of this invention can also be expressed in commercially farmed fish such as catfish, Atlantic salmon and talipia to improve the freeze tolerance of the fish. Sculpin-type AFPs also have certain antibacterial properties, providing a means of reducing unwanted bacteria in foods such as recombinant fruits expressing sculpin-type AFPs, and in blended food stuffs such as ice cream. This improves shelf life, food quality, and makes such products safer for consumption. These and other commercial applications are aspects of the present invention.
In one aspect, the present invention relates to an isolated intracellular antifreeze polypeptide (sculpin-type AFPs). In this embodiment, the polypeptide typically comprises four or more Pr-X
2
-Pr-X
7
subsequences, where Pr is a polar amino acid and X is a naturally occurring or synthetic amino acid. The polypeptide is alanine rich, with X being predominately alanine. The sculpin-type AFPs of the present invention have the physical ability to induce a concentration-dependent decrease in the freezing point of an aqueous solution such as water.
The polypeptide typically comprises at least six of these 11 amino acid subsequences (note that the subsequences are overlapping) where the polar amino acids are N, D, E, and K. In addition, the preferred polypeptides have a MW of about 7900 DA to about 9700 DA. Typically, the sculpin-type AFPs are between about 45 and about 100 amino acids in length, more preferably between about 60 and 100 amino acids in length, and most preferably about 80-100 amino acids in length.
Preferred sculpin-type AFPs of the present invention are optionally assessed by examining the secondary structure of the polypeptides. In certain aspects, the polypeptides of the present invention, as measured by circular dichroism, are at least 70% &agr;-helical and, preferably at 0° C., essentially entirely &agr;-helical. Certain polypeptides of the invention optionally do not meet these criteria, e.g., where the polypeptide is a fusion protein that includes subsequences that are unrelated to a sculpin-type AFP. Fusion proteins comprising sculpin-type AFP subsequences are a feature of the present invention.
Sculpin-type AFPs of the present invention are optionally defined by their immunological characteristics. Preferred sculpin-type AFPs bind polyclonal antibodies raised against the polypeptide shorthorn sculpin skin-type (sssAFP-2; SEQ ID NO:2). Preferred polypeptides also bind to polyclonal antibodies raised against the polypeptide sssAFP-2, where the polyclonal antisera are first subtracted with a skin-type polypeptide, such as wfsAFP-1, from the Winter Flounder.
In certain embodiments, polyclonal antisera for use in immunoassays are generated using sssAFP-2 as described herein. The polyclonal antisera is then tested for its cross-reactivity against skin-type AFPs from Winter flounder (e.g. wfsAFP-1 i.e., a skin isotype from the Winter flounder) using a competitive binding immunoassay. For example, the immunogenic polypeptide is immobilized to a solid support. wfsAFP-1 added to the assay competes with the binding of the antisera to the immobilized antigen. The ability of the skin-type AFPs from Winter flounder (wfsAFP-1) to compete for binding of the antisera to the immobilized protein is compared to the immunogenic polypeptide. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with skin-type wfsAFP- 1 are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immuno absorbtion (subtraction) with wfsAFP-1. Preferred polypeptides are those that bind to the ant

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