Intracellular antifreeze polypeptides and nucleic acids

Details Intracellular antifreeze polypeptides and nucleic acids Intracellular antifreeze polypeptides and nucleic acids

1998-11-20

2001-10-23

Carlson, Karen Cochrane (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

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

C530S350000, C530S300000, C435S069100, C435S068100, C435S069700, C435S091100, C435S320100, C435S419000, C435S252300, C536S023500

Reexamination Certificate

active

06307020

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 which 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, three 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 half cystine and are found in sea raven, smelt and herring. Type III AFPs are globular proteins found in several Zoarcoid families including eelpout and wolfish. The AFGPs, which are found in three families of Antarctic fish and polar cods, largely consist of a tripeptide repeat (Ala-Ala-Thr) with a disaccharide attached to the threonyl residue.
Although the different AFPs and AFGPs are structurally distinct, they share the ability to inhibit ice crystal growth by binding to the ice surface. At present, the AFGPs and AFPs have been isolated from serum, and their DNA sequences deduced by cDNA cloning from the liver. All of the proteins described to date are synthesized as larger precursor polypeptides containing the signal peptides, indicating a secretory role for the polypeptides.
AFPs in the liver (liver type AFPs) from the winter flounder,
Pleuronectus americanus
, has been studied extensively in terms of its protein 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).
SUMMARY OF THE INVENTION
A new class of AFPs is discovered and described herein. The new class of AFPs, termed “skin-type” AFPs have several features which are different from all previously described AFPs, including the fact that the AFPs are not secreted, i.e., the skin-type AFPs are intracellular. Genomic Southern analysis shows that like the liver-type AFP genes, there are multiple copies of the skin type AFPs (30-40 copies in winter flounder). While the previously described liver-type AFP genes are specifically expressed in the liver, and, to a less extent, in intestine, the skin-type AFP genes are expressed in all tissues examined (including the liver) and abundantly in exterior tissues, i.e. skin, scales, fin and gills, showing that skin type AFPs have a protective role in these exterior tissues.
In one preferred class of embodiments, the invention provides isolated skin-type intracellular antifreeze polypeptides (AFPs). In this embodiment, the AFPs have an N terminal MDAP subsequence (SEQ ID NO:1), and an internal AATAAAAKAAA subsequence (SEQ ID NO: 2). More preferably, the N terminal sequence is an MDAPA sequence (SEQ ID NO:9). The AFPs do not have a signal sequence (i.e., the polypeptides do not have a pre sequence). The AFPs have the physical ability to induce a concentration-dependent decrease in the freezing point of an aqueous solution such as H
2
O.
In some preferred embodiments, skin type AFPs comprise the sequence MDAPAX
1
AAAATAAAAKAAAEATX
2
AAAAX
2
AAAX
3
T (SEQ ID NO:3); wherein X
1
is selected from the group consisting of R, K, and A; X
2
is selected from the group consisting of K and A; and X
3
is selected from the group consisting of A, D and a bond.
Particularly preferred polypeptides have a M
r
of about 3400, but the M
r
. can vary substantially, for instance due to multiple skin-type antifreeze polypeptide domains being connected into a single polypeptide, or due to the addition of non-functional amino acids, e.g., at the carboxyl terminus of the protein. Typically the M
r
of skin-type AFPs will be about 2500 to about 13,000, more typically about 3,000 to about 6,000, yet more typically about 3200 to about 4,000. Typically, the skin-type AFPs are between about 30 and about 100 amino acids in length, more typically between about 35 and 55 amino acids in length, and usually about 35-45 amino acids in length. However, specific examples are provided herein which exceed the usual 35-45 amino acids in length. Example skin type AFP polypeptides include sAFP1, sAFP2, sAFP3, sAFP4, sAFP5, sAFP6, sAFP7, sAFP8, F2 and 11-3.
Preferred Skin type AFPs of the invention are optionally assessed by examining the secondary structure of the polypeptides. In one class of embodiments, the polypeptides of the invention, as measured by circular dichroism, are between about 55% and about 75% &agr; helical, more typically about 60% and 70% &agr; helical, often about 65% &agr; helical. Certain polypeptides of the invention optionally do not meet this criteria, e.g., where the polypeptide is a fusion protein which includes subsequences which are unrelated to a skin-type AFP. Fusion proteins comprising skin-type AFP subsequences are a feature of the invention. Preferred fusion proteins include a skin-type AFP subsequence and a subsequences from GST, keyhole limpet hemanocyanin and other common fusion polypeptide domains.
Skin type AFPs of the invention are optionally defined by their immunological characteristics. Preferred AFPs bind polyclonal antibodies raised against any (or all) of the polypeptides sAFP1, sAFP2, sAFP3, sAFP4, sAFP5, sAFP6, sAFP7, sAFP8, F2 and 11-3. Preferred polypeptides also bind to polyclonal antibodies raised against any one (or all) of the polypeptides sAFP1, sAFP2, sAFP3, sAFP4, sAFP5, sAFP6, sAFP7, sAFP8, F2 and 11-3, where the polyclonal antisera are subtracted with a liver-type polypeptide such as HPLC 6 or HPLC 8.
Isolated polypeptides are optionally present as purified lyophilized powders, in aqueous solutions (e.g., comprising water, for instance with salts at physiological concentrations), in recombinant cells, plants, animals, bacteria, prokaryotes, cell extracts and the like. The polypeptides are optionally present in foods such as ice cream or frozen yoghurt.
Isolated skin-type antifreeze polypeptides are preferably encoded by a coding nucleic acid (RNA or DNA) which hybridizes to a skin type antifreeze nucleic acid selected from the group consisting of sAFP1, sAFP2, sAFP3, sAFP4, sAFP5, sAFP6, sAFP7, sAFP8, F2 and 11-3 in a northern blot or Southern blot under high stringency wash conditions. An example high stringency wash condition is 0.015M NaCl at 72° C. Preferably, the skin-type antifreeze polypeptides do not significantly hybridize to liver type AFP coding nucleic acids, such as a nucleic acid encoding HPLC6 (e.g., PENCE 17, see
FIG. 7
) under the same high stringency wash conditions. The liver type AFP nucleic acid does not significantly hybridize to a skin-type nucleic acid if the signal to noise ratio on a Southern or northern blot is reduced 75% or more as compared to the binding of a fully complementary skin type AFP. For example, if a radiolabeled liver type probe and a radiolabeled skin type probe with the same specific activity are used to probe duplicate Southern blots, and an autoradiogramii shows that the liver specific probe signal has less than 25% the intensity of the skin specific probe after a high stringency wash, then the detected nuc

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