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• W2014580500 abstract "A cDNA clone encoding a presumptive antifreeze protein was isolated from a skin library from shorthorn sculpin, Myoxocephalus scorpius. The clone encodes a 92-residue mature polypeptide (sssAFP-2) without any signal and prosequence, which suggests an intracellular localization. It is the largest alanine-rich, α-helical type I antifreeze protein known. A recombinant fusion protein containing an N-terminal-linked His-tag was produced and purified from Escherichia coli. This protein is α-helical at 0 °C and exhibits significant antifreeze activity. Northern blot and reverse transcription-polymerase chain reaction analyses indicate that sssAFP-2 mRNA has limited tissue distribution and is present in peripheral tissues such as skin and dorsal fin, but is notably absent in the liver. These studies reinforce recent evidence that indicate that the external tissues of cold water marine fishes are major organs for antifreeze protein synthesis and are likely the first line of defense against the threat of freezing. A cDNA clone encoding a presumptive antifreeze protein was isolated from a skin library from shorthorn sculpin, Myoxocephalus scorpius. The clone encodes a 92-residue mature polypeptide (sssAFP-2) without any signal and prosequence, which suggests an intracellular localization. It is the largest alanine-rich, α-helical type I antifreeze protein known. A recombinant fusion protein containing an N-terminal-linked His-tag was produced and purified from Escherichia coli. This protein is α-helical at 0 °C and exhibits significant antifreeze activity. Northern blot and reverse transcription-polymerase chain reaction analyses indicate that sssAFP-2 mRNA has limited tissue distribution and is present in peripheral tissues such as skin and dorsal fin, but is notably absent in the liver. These studies reinforce recent evidence that indicate that the external tissues of cold water marine fishes are major organs for antifreeze protein synthesis and are likely the first line of defense against the threat of freezing. antifreeze protein/polypeptide antifreeze glycoprotein untranslated region base pair(s) open reading frame reverse transcription polymerase chain reaction polyacrylamide gel electrophoresis high performance liquid chromatography winter flounder liver type AFP kilobase(s). Many cold water marine fishes produce antifreeze proteins/polypeptides (AFPs)1or antifreeze glycoproteins (AFGPs) to prevent freezing in icy sea waters (1DeVries A.L. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1984; 304: 575-588Crossref Google Scholar, 2Davies P.L. Hew C.L. Fletcher G.L. Can. J. Zool. 1988; 66: 2611-2617Crossref Google Scholar, 3Davies P.L. Sykes B.D. Curr. Opin. Struct. Biol. 1997; 7: 828-834Crossref PubMed Scopus (186) Google Scholar). Whereas AFGPs from different fish species are similar in structure, the AFPs are structurally diverse. Biochemical characterization has grouped the AFPs into four distinct groups: the type I α-helical AFPs from righteye flounders and shorthorn sculpins; type II lectin-like AFPs from sea raven and herring; type III globular AFPs from eelpouts (4Davies P.L. Hew C.L. FASEB J. 1990; 4: 2460-2468Crossref PubMed Scopus (370) Google Scholar, 5Hew C.L. Yang D.S.C. Eur. J. Biochem. 1992; 203: 33-42Crossref PubMed Scopus (171) Google Scholar, 6Shears M.A. Kao M.H. Scott G.K. Davies P.L. Fletcher G.L. Mol. Mar. Biol. Biotechnol. 1993; 2: 104-111Google Scholar); and more recently, a type IV helix bundle AFP has been reported from longhorn sculpin, Myoxocephalus octodecimspinosis (7Deng G. Andrews D.W. Laursen R.A. FEBS Lett. 1997; 402: 17-20Crossref PubMed Scopus (120) Google Scholar).Most, if not all of the antifreeze proteins were isolated and characterized from the sera and synthesized in the liver. Recently, our laboratories have demonstrated the presence of new isoforms of type I AFPs in the winter flounder, Pleuronectes americanus, that are synthesized in the peripheral tissues such as the skin and gills (8Gong Z. Ewart K.V. Hu Z. Fletcher G.L. Hew C.L. J. Biol. Chem. 1996; 271: 4106-4112Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). These skin-type AFPs are encoded by a distinct set of AFP genes that lack the signal peptide, which is indicative of an intracellular location. The presence of both 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 freezing protection, their structure/function, and evolutionary relationships. These findings have prompted us to reexamine the presence of skin-type AFPs in other fishes. Shorthorn sculpin, like the winter flounder, has been found to produce type I α-helical AFPs (9Hew C.L. Fletcher G.L. Ananthanarayanan V.S. Can. J. Biochem. 1980; 58: 377-383Crossref PubMed Scopus (43) Google Scholar, 10Fletcher G.L. Addison R.F. Slaughter D. Hew C.L. Arctic. 1982; 35: 302-306Crossref Google Scholar, 11Hew C.L. Joshi S. Wang N.C. Kao M.H. Ananthanarayanan V.S. Eur. J. Biochem. 1985; 151: 167-172Crossref PubMed Scopus (50) Google Scholar), and antifreeze peptides have been isolated from the skin of European shorthorn sculpin (12Schneppenheim R. Theede H. Polar Biol. 1982; 1: 115-123Google Scholar). The latter peptides, however, were not further characterized. Therefore, the present investigation attempts to clarify the occurrence, structure/function relationship, and tissue distribution of AFPs in the shorthorn sculpin.DISCUSSIONIn the present study, we have demonstrated that the shorthorn sculpin, similar to the winter flounder, possesses skin-type AFPs along with serum (liver-type) AFPs as an intergral part of its defense strategy against freezing. The cDNA sequence of sssAFP-2 indicates that it lacks the pre- and prosequences, implying that it is intracellular and functionally analogous to the skin-type AFPs found in flounder. We suggest that an updated nomenclature of these proteins to denote the fish species and the nature of the AFP isoform is warranted,i.e. liver-type (l) versus the skin-type (s). Thus, the compliment of AFPs of the shorthorn sculpin includes the serum-type AFPs, ss-3 (renamed sslAFP-3 to denote ShorthornSculpin Liver-type AFP-3) and ss-8 (renamed as sslAFP-8), and the new skin-type AFP, sssAFP-2, identified here. Furthermore, evidence is provided that demonstrates sssAFP-2 possesses antifreeze activity comparable with winter flounder HPLC-6 (renamed wflAFP-6 for Winter FlounderLiver-type AFP-6). However, the amino acid composition of sssAFP-2 does not match that of either FPDP I or II, the two antifreeze peptides previously isolated from the skin of European shorthorn sculpin (12Schneppenheim R. Theede H. Polar Biol. 1982; 1: 115-123Google Scholar). In fact, FPDP I and II seem more closely related to sslAFP-3 and sslAFP-8, raising the possibility that these preparations may have been contaminated by serum-derived proteins. Furthermore, GenBankTM searches using the s3–2 cDNA sequence or sssAFP-2 primary sequence did not indicate any identity with any known sequences.Northern analysis, RT-PCR, and primer extension studies indicate that sssAFP-2 is produced in a wide variety of tissues, most notably the skin, dorsal fin, brain, and gill filament, but not in the liver. The relatively abundant sssAFP-2 mRNA level in brain is interesting, and its functional significance remains to be determined. It is also interesting to note that skin-type AFP genes are not expressed in the liver of sculpin, but are expressed in the liver of flounder (8Gong Z. Ewart K.V. Hu Z. Fletcher G.L. Hew C.L. J. Biol. Chem. 1996; 271: 4106-4112Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Furthermore, in contrast to the expression of wfsAFPs, which only show moderate seasonal fluctuation (17Gong Z. King M.J. Fletcher G.L. Hew C.L. Biochem. Biophys. Res. Commun. 1995; 206: 387-392Crossref PubMed Scopus (18) Google Scholar), the sculpin skin-type AFP mRNA levels show significant seasonal variation, which may reflect the different habitats of these two species.Type I AFPs are alanine-rich, partially amphipathic α-helical peptides. It has been proposed that type I AFPs present hydrophilic polar residues for interaction with the ice-crystal lattice through hydrogen bonding while presenting a hydrophobic surface to incoming water molecules, thereby preventing further crystal growth (18Raymond J.A. DeVries A.L. Proc. Natl. Acad. Sci., U. S. A. 1977; 74: 2589-2593Crossref PubMed Scopus (660) Google Scholar, 19DeVries A.L. Lin Y. Biochim. Biophys. Acta. 1977; 495: 388-392Crossref PubMed Scopus (138) Google Scholar, 20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar). As well, recent work has suggested that nonpolar interactions, such as van der Waals forces and hydrophobic effects, may have greater relevance in ice-binding than previously assumed (21Chao H. Houston Jr., M.E. Hodges R.S. Kay C.M. Sykes B.D. Loewen M.C. Davies P.L. Sönnichsen F.D. Biochemistry. 1997; 36: 14652-14660Crossref PubMed Scopus (201) Google Scholar, 22Cheng A. Merz K.M. Biophys. J. 1997; 73: 2851-2873Abstract Full Text PDF PubMed Scopus (93) Google Scholar). The majority of type I AFPs that have been studied in the past possess 11-residue repeats that consist of Thr-X 2-Asn/Asp-X 7 whereX can be any residue but is usually alanine (20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar, 23Wen D. Laursen R.A. J. Biol. Chem. 1992; 267: 14102-14108Abstract Full Text PDF PubMed Google Scholar, 24Wen D. Laursen R.A. Biophys. J. 1992; 63: 1659-1662Abstract Full Text PDF PubMed Scopus (144) Google Scholar). The Thr and Asn/Asp residues are located on the same face of the helix in a periodic nature which presumably leads to hydrogen-bonding with ice in a lattice-matching manner. One notable exception is the shorthorn sculpin serum AFP, sslAFP-8, which does not possess the typical 11-residue repeats (11Hew C.L. Joshi S. Wang N.C. Kao M.H. Ananthanarayanan V.S. Eur. J. Biochem. 1985; 151: 167-172Crossref PubMed Scopus (50) Google Scholar, 25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). In sssAFP-2, there are many putative 11-residue repeats (indicated by boxed residues in Fig. 1); however, none of these putative repeats match the established Thr-X 2-Asn/Asp-X 7 motif. The alanine content of sssAFP-2 is approximately 70%, the secondary structure of sssAFP-2 is predicted to be entirely helical (26Chou P.Y. Fasman G.D. Adv. Enzymol. 1978; 47: 45-148PubMed Google Scholar) and was confirmed by CD spectroscopy (Fig. 6). Furthermore, a helical wheel presentation of sssAFP-2 suggests a partially amphipathic molecule (see Fig. 7). Thus, sssAFP-2 meets the criteria necessary to be classified as a type I AFP, and at 92 residues, it is the largest known naturally occurring type I AFP identified thus far.The predominant 11-residue repeat within sssAFP-2 is Pr-X 2-Pr-X 7 (see Fig. 1), where Pr represents another polar residue and X is predominantly alanine. Three of the repeats highlighted in Fig. 1 begin with lysine. Although threonine is not present, the correct spacing of polar residues within the 11-residue repeat itself is maintained. Proper positioning of the polar residues in a lattice-matching manner is believed to be critical for AFP binding to ice (19DeVries A.L. Lin Y. Biochim. Biophys. Acta. 1977; 495: 388-392Crossref PubMed Scopus (138) Google Scholar, 27Knight C.A. Cheng C.C. DeVries A.L. Biophys. J. 1991; 59: 409-418Abstract Full Text PDF PubMed Scopus (457) Google Scholar). This belief is supported by crystal and NMR solution structures determined for wflAFP-6 (20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar, 28Gronwald W. Chao H. Reddy D.V. Davies P.L. Sykes B.D. Sönnichsen F.D. Biochemistry. 1996; 35: 16698-16704Crossref PubMed Scopus (52) Google Scholar), and molecular dynamics simulation techniques (22Cheng A. Merz K.M. Biophys. J. 1997; 73: 2851-2873Abstract Full Text PDF PubMed Scopus (93) Google Scholar). Furthermore, the possibility of an 11-residue repeat beginning with lysine has been noted before (20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar).Recently, a combination of molecular dynamics and modeling of the interaction of sslAFP-8 with the ice lattice has produced another possible mechanism of interaction (25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). This approach involves two binding models: accommodation of binding surface residues within ice cages, and the inclusion of key lysine side chains into the ice lattice through their tetrahedral end groups (25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). A comparison of the secondary structure of sssAFP-2 with sslAFP-8 (Fig. 7) shows that sssAFP-2 is similar to sslAFP-8 in that many lysine side chains project in a similar manner to one face of the helix. Furthermore, a comparison of the sequences for the two proteins reveals that many lysine residues within sssAFP-2 possess the correct spacing within the protein as defined by Wierzbicki et. al. (25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). Nonetheless, more structural work will be required to resolve and define any similarities or differences in the binding mechanisms of the sculpin AFPs.It appears that the defense against the dangers of freezing in ice-laden, sub-zero sea water of the shorthorn sculpin is identical to that of the winter flounder. Both species secrete AFP into the blood serum that serves to depress the freezing temperature of their extracellular fluids, and both produce skin-type AFPs that lack signal peptides, suggesting that they function as intracellular protectants. Precisely how these skin-type AFPs help to protect fish from cold and freezing is subject to some debate (for review, see Ref. 29Fletcher G.L. Goddard S.V. Davies P.L. Gong Z. Ewart K.V. Hew C.L. Portner H.O. Playle R. Cold Ocean Physiology. Cambridge University Press, Cambridge, United Kingdom1998: 239-265Google Scholar). Valerioet. al. (30Valerio P.F. Kao M.H. Fletcher G.L. J. Exp. Biol. 1992; 164: 135-151Crossref Scopus (14) Google Scholar) have demonstrated that winter flounder skin is an effective barrier to ice propagation and that the effectiveness of this barrier can be increased by the addition of antifreeze proteins to the extracellular space. This suggests that despite the lack of a signal peptide, the skin-type AFPs may use an alternative pathway for secretion of the cells into the intercellular space, thereby acting to block ice propagation.Recently, Murray et al. 2H. M. Murray, C. L. Hew, K. R. Kao, and G. L. Fletcher, manuscript in preparation. have identified the gill epithelial cells of the winter flounder as a major site of skin-type AFP production utilizing in situ hybridization and immuno-cytochemical techniques. Because of their importance in gas exchange, gill epithelia are the thinnest of all external epithelial tissues in the fish, and the most likely site to come into intimate contact with potentially lethal ice crystals. Thus, it is possible that the skin-type AFP simply serves to lower the freezing temperature of the intracellular fluids and thus ensure that this essential layer of cells cannot freeze. Many cold water marine fishes produce antifreeze proteins/polypeptides (AFPs)1or antifreeze glycoproteins (AFGPs) to prevent freezing in icy sea waters (1DeVries A.L. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1984; 304: 575-588Crossref Google Scholar, 2Davies P.L. Hew C.L. Fletcher G.L. Can. J. Zool. 1988; 66: 2611-2617Crossref Google Scholar, 3Davies P.L. Sykes B.D. Curr. Opin. Struct. Biol. 1997; 7: 828-834Crossref PubMed Scopus (186) Google Scholar). Whereas AFGPs from different fish species are similar in structure, the AFPs are structurally diverse. Biochemical characterization has grouped the AFPs into four distinct groups: the type I α-helical AFPs from righteye flounders and shorthorn sculpins; type II lectin-like AFPs from sea raven and herring; type III globular AFPs from eelpouts (4Davies P.L. Hew C.L. FASEB J. 1990; 4: 2460-2468Crossref PubMed Scopus (370) Google Scholar, 5Hew C.L. Yang D.S.C. Eur. J. Biochem. 1992; 203: 33-42Crossref PubMed Scopus (171) Google Scholar, 6Shears M.A. Kao M.H. Scott G.K. Davies P.L. Fletcher G.L. Mol. Mar. Biol. Biotechnol. 1993; 2: 104-111Google Scholar); and more recently, a type IV helix bundle AFP has been reported from longhorn sculpin, Myoxocephalus octodecimspinosis (7Deng G. Andrews D.W. Laursen R.A. FEBS Lett. 1997; 402: 17-20Crossref PubMed Scopus (120) Google Scholar). Most, if not all of the antifreeze proteins were isolated and characterized from the sera and synthesized in the liver. Recently, our laboratories have demonstrated the presence of new isoforms of type I AFPs in the winter flounder, Pleuronectes americanus, that are synthesized in the peripheral tissues such as the skin and gills (8Gong Z. Ewart K.V. Hu Z. Fletcher G.L. Hew C.L. J. Biol. Chem. 1996; 271: 4106-4112Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). These skin-type AFPs are encoded by a distinct set of AFP genes that lack the signal peptide, which is indicative of an intracellular location. The presence of both 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 freezing protection, their structure/function, and evolutionary relationships. These findings have prompted us to reexamine the presence of skin-type AFPs in other fishes. Shorthorn sculpin, like the winter flounder, has been found to produce type I α-helical AFPs (9Hew C.L. Fletcher G.L. Ananthanarayanan V.S. Can. J. Biochem. 1980; 58: 377-383Crossref PubMed Scopus (43) Google Scholar, 10Fletcher G.L. Addison R.F. Slaughter D. Hew C.L. Arctic. 1982; 35: 302-306Crossref Google Scholar, 11Hew C.L. Joshi S. Wang N.C. Kao M.H. Ananthanarayanan V.S. Eur. J. Biochem. 1985; 151: 167-172Crossref PubMed Scopus (50) Google Scholar), and antifreeze peptides have been isolated from the skin of European shorthorn sculpin (12Schneppenheim R. Theede H. Polar Biol. 1982; 1: 115-123Google Scholar). The latter peptides, however, were not further characterized. Therefore, the present investigation attempts to clarify the occurrence, structure/function relationship, and tissue distribution of AFPs in the shorthorn sculpin. DISCUSSIONIn the present study, we have demonstrated that the shorthorn sculpin, similar to the winter flounder, possesses skin-type AFPs along with serum (liver-type) AFPs as an intergral part of its defense strategy against freezing. The cDNA sequence of sssAFP-2 indicates that it lacks the pre- and prosequences, implying that it is intracellular and functionally analogous to the skin-type AFPs found in flounder. We suggest that an updated nomenclature of these proteins to denote the fish species and the nature of the AFP isoform is warranted,i.e. liver-type (l) versus the skin-type (s). Thus, the compliment of AFPs of the shorthorn sculpin includes the serum-type AFPs, ss-3 (renamed sslAFP-3 to denote ShorthornSculpin Liver-type AFP-3) and ss-8 (renamed as sslAFP-8), and the new skin-type AFP, sssAFP-2, identified here. Furthermore, evidence is provided that demonstrates sssAFP-2 possesses antifreeze activity comparable with winter flounder HPLC-6 (renamed wflAFP-6 for Winter FlounderLiver-type AFP-6). However, the amino acid composition of sssAFP-2 does not match that of either FPDP I or II, the two antifreeze peptides previously isolated from the skin of European shorthorn sculpin (12Schneppenheim R. Theede H. Polar Biol. 1982; 1: 115-123Google Scholar). In fact, FPDP I and II seem more closely related to sslAFP-3 and sslAFP-8, raising the possibility that these preparations may have been contaminated by serum-derived proteins. Furthermore, GenBankTM searches using the s3–2 cDNA sequence or sssAFP-2 primary sequence did not indicate any identity with any known sequences.Northern analysis, RT-PCR, and primer extension studies indicate that sssAFP-2 is produced in a wide variety of tissues, most notably the skin, dorsal fin, brain, and gill filament, but not in the liver. The relatively abundant sssAFP-2 mRNA level in brain is interesting, and its functional significance remains to be determined. It is also interesting to note that skin-type AFP genes are not expressed in the liver of sculpin, but are expressed in the liver of flounder (8Gong Z. Ewart K.V. Hu Z. Fletcher G.L. Hew C.L. J. Biol. Chem. 1996; 271: 4106-4112Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Furthermore, in contrast to the expression of wfsAFPs, which only show moderate seasonal fluctuation (17Gong Z. King M.J. Fletcher G.L. Hew C.L. Biochem. Biophys. Res. Commun. 1995; 206: 387-392Crossref PubMed Scopus (18) Google Scholar), the sculpin skin-type AFP mRNA levels show significant seasonal variation, which may reflect the different habitats of these two species.Type I AFPs are alanine-rich, partially amphipathic α-helical peptides. It has been proposed that type I AFPs present hydrophilic polar residues for interaction with the ice-crystal lattice through hydrogen bonding while presenting a hydrophobic surface to incoming water molecules, thereby preventing further crystal growth (18Raymond J.A. DeVries A.L. Proc. Natl. Acad. Sci., U. S. A. 1977; 74: 2589-2593Crossref PubMed Scopus (660) Google Scholar, 19DeVries A.L. Lin Y. Biochim. Biophys. Acta. 1977; 495: 388-392Crossref PubMed Scopus (138) Google Scholar, 20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar). As well, recent work has suggested that nonpolar interactions, such as van der Waals forces and hydrophobic effects, may have greater relevance in ice-binding than previously assumed (21Chao H. Houston Jr., M.E. Hodges R.S. Kay C.M. Sykes B.D. Loewen M.C. Davies P.L. Sönnichsen F.D. Biochemistry. 1997; 36: 14652-14660Crossref PubMed Scopus (201) Google Scholar, 22Cheng A. Merz K.M. Biophys. J. 1997; 73: 2851-2873Abstract Full Text PDF PubMed Scopus (93) Google Scholar). The majority of type I AFPs that have been studied in the past possess 11-residue repeats that consist of Thr-X 2-Asn/Asp-X 7 whereX can be any residue but is usually alanine (20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar, 23Wen D. Laursen R.A. J. Biol. Chem. 1992; 267: 14102-14108Abstract Full Text PDF PubMed Google Scholar, 24Wen D. Laursen R.A. Biophys. J. 1992; 63: 1659-1662Abstract Full Text PDF PubMed Scopus (144) Google Scholar). The Thr and Asn/Asp residues are located on the same face of the helix in a periodic nature which presumably leads to hydrogen-bonding with ice in a lattice-matching manner. One notable exception is the shorthorn sculpin serum AFP, sslAFP-8, which does not possess the typical 11-residue repeats (11Hew C.L. Joshi S. Wang N.C. Kao M.H. Ananthanarayanan V.S. Eur. J. Biochem. 1985; 151: 167-172Crossref PubMed Scopus (50) Google Scholar, 25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). In sssAFP-2, there are many putative 11-residue repeats (indicated by boxed residues in Fig. 1); however, none of these putative repeats match the established Thr-X 2-Asn/Asp-X 7 motif. The alanine content of sssAFP-2 is approximately 70%, the secondary structure of sssAFP-2 is predicted to be entirely helical (26Chou P.Y. Fasman G.D. Adv. Enzymol. 1978; 47: 45-148PubMed Google Scholar) and was confirmed by CD spectroscopy (Fig. 6). Furthermore, a helical wheel presentation of sssAFP-2 suggests a partially amphipathic molecule (see Fig. 7). Thus, sssAFP-2 meets the criteria necessary to be classified as a type I AFP, and at 92 residues, it is the largest known naturally occurring type I AFP identified thus far.The predominant 11-residue repeat within sssAFP-2 is Pr-X 2-Pr-X 7 (see Fig. 1), where Pr represents another polar residue and X is predominantly alanine. Three of the repeats highlighted in Fig. 1 begin with lysine. Although threonine is not present, the correct spacing of polar residues within the 11-residue repeat itself is maintained. Proper positioning of the polar residues in a lattice-matching manner is believed to be critical for AFP binding to ice (19DeVries A.L. Lin Y. Biochim. Biophys. Acta. 1977; 495: 388-392Crossref PubMed Scopus (138) Google Scholar, 27Knight C.A. Cheng C.C. DeVries A.L. Biophys. J. 1991; 59: 409-418Abstract Full Text PDF PubMed Scopus (457) Google Scholar). This belief is supported by crystal and NMR solution structures determined for wflAFP-6 (20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar, 28Gronwald W. Chao H. Reddy D.V. Davies P.L. Sykes B.D. Sönnichsen F.D. Biochemistry. 1996; 35: 16698-16704Crossref PubMed Scopus (52) Google Scholar), and molecular dynamics simulation techniques (22Cheng A. Merz K.M. Biophys. J. 1997; 73: 2851-2873Abstract Full Text PDF PubMed Scopus (93) Google Scholar). Furthermore, the possibility of an 11-residue repeat beginning with lysine has been noted before (20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar).Recently, a combination of molecular dynamics and modeling of the interaction of sslAFP-8 with the ice lattice has produced another possible mechanism of interaction (25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). This approach involves two binding models: accommodation of binding surface residues within ice cages, and the inclusion of key lysine side chains into the ice lattice through their tetrahedral end groups (25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). A comparison of the secondary structure of sssAFP-2 with sslAFP-8 (Fig. 7) shows that sssAFP-2 is similar to sslAFP-8 in that many lysine side chains project in a similar manner to one face of the helix. Furthermore, a comparison of the sequences for the two proteins reveals that many lysine residues within sssAFP-2 possess the correct spacing within the protein as defined by Wierzbicki et. al. (25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). Nonetheless, more structural work will be required to resolve and define any similarities or differences in the binding mechanisms of the sculpin AFPs.It appears that the defense against the dangers of freezing in ice-laden, sub-zero sea water of the shorthorn sculpin is identical to that of the winter flounder. Both species secrete AFP into the blood serum that serves to depress the freezing temperature of their extracellular fluids, and both produce skin-type AFPs that lack signal peptides, suggesting that they function as intracellular protectants. Precisely how these skin-type AFPs help to protect fish from cold and freezing is subject to some debate (for review, see Ref. 29Fletcher G.L. Goddard S.V. Davies P.L. Gong Z. Ewart K.V. Hew C.L. Portner H.O. Playle R. Cold Ocean Physiology. Cambridge University Press, Cambridge, United Kingdom1998: 239-265Google Scholar). Valerioet. al. (30Valerio P.F. Kao M.H. Fletcher G.L. J. Exp. Biol. 1992; 164: 135-151Crossref Scopus (14) Google Scholar) have demonstrated that winter flounder skin is an effective barrier to ice propagation and that the effectiveness of this barrier can be increased by the addition of antifreeze proteins to the extracellular space. This suggests that despite the lack of a signal peptide, the skin-type AFPs may use an alternative pathway for secretion of the cells into the intercellular space, thereby acting to block ice propagation.Recently, Murray et al. 2H. M. Murray, C. L. Hew, K. R. Kao, and G. L. Fletcher, manuscript in preparation. have identified the gill epithelial cells of the winter flounder as a major site of skin-type AFP production utilizing in situ hybridization and immuno-cytochemical techniques. Because of their importance in gas exchange, gill epithelia are the thinnest of all external epithelial tissues in the fish, and the most likely site to come into intimate contact with potentially lethal ice crystals. Thus, it is possible that the skin-type AFP simply serves to lower the freezing temperature of the intracellular fluids and thus ensure that this essential layer of cells cannot freeze. In the present study, we have demonstrated that the shorthorn sculpin, similar to the winter flounder, possesses skin-type AFPs along with serum (liver-type) AFPs as an intergral part of its defense strategy against freezing. The cDNA sequence of sssAFP-2 indicates that it lacks the pre- and prosequences, implying that it is intracellular and functionally analogous to the skin-type AFPs found in flounder. We suggest that an updated nomenclature of these proteins to denote the fish species and the nature of the AFP isoform is warranted,i.e. liver-type (l) versus the skin-type (s). Thus, the compliment of AFPs of the shorthorn sculpin includes the serum-type AFPs, ss-3 (renamed sslAFP-3 to denote ShorthornSculpin Liver-type AFP-3) and ss-8 (renamed as sslAFP-8), and the new skin-type AFP, sssAFP-2, identified here. Furthermore, evidence is provided that demonstrates sssAFP-2 possesses antifreeze activity comparable with winter flounder HPLC-6 (renamed wflAFP-6 for Winter FlounderLiver-type AFP-6). However, the amino acid composition of sssAFP-2 does not match that of either FPDP I or II, the two antifreeze peptides previously isolated from the skin of European shorthorn sculpin (12Schneppenheim R. Theede H. Polar Biol. 1982; 1: 115-123Google Scholar). In fact, FPDP I and II seem more closely related to sslAFP-3 and sslAFP-8, raising the possibility that these preparations may have been contaminated by serum-derived proteins. Furthermore, GenBankTM searches using the s3–2 cDNA sequence or sssAFP-2 primary sequence did not indicate any identity with any known sequences. Northern analysis, RT-PCR, and primer extension studies indicate that sssAFP-2 is produced in a wide variety of tissues, most notably the skin, dorsal fin, brain, and gill filament, but not in the liver. The relatively abundant sssAFP-2 mRNA level in brain is interesting, and its functional significance remains to be determined. It is also interesting to note that skin-type AFP genes are not expressed in the liver of sculpin, but are expressed in the liver of flounder (8Gong Z. Ewart K.V. Hu Z. Fletcher G.L. Hew C.L. J. Biol. Chem. 1996; 271: 4106-4112Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Furthermore, in contrast to the expression of wfsAFPs, which only show moderate seasonal fluctuation (17Gong Z. King M.J. Fletcher G.L. Hew C.L. Biochem. Biophys. Res. Commun. 1995; 206: 387-392Crossref PubMed Scopus (18) Google Scholar), the sculpin skin-type AFP mRNA levels show significant seasonal variation, which may reflect the different habitats of these two species. Type I AFPs are alanine-rich, partially amphipathic α-helical peptides. It has been proposed that type I AFPs present hydrophilic polar residues for interaction with the ice-crystal lattice through hydrogen bonding while presenting a hydrophobic surface to incoming water molecules, thereby preventing further crystal growth (18Raymond J.A. DeVries A.L. Proc. Natl. Acad. Sci., U. S. A. 1977; 74: 2589-2593Crossref PubMed Scopus (660) Google Scholar, 19DeVries A.L. Lin Y. Biochim. Biophys. Acta. 1977; 495: 388-392Crossref PubMed Scopus (138) Google Scholar, 20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar). As well, recent work has suggested that nonpolar interactions, such as van der Waals forces and hydrophobic effects, may have greater relevance in ice-binding than previously assumed (21Chao H. Houston Jr., M.E. Hodges R.S. Kay C.M. Sykes B.D. Loewen M.C. Davies P.L. Sönnichsen F.D. Biochemistry. 1997; 36: 14652-14660Crossref PubMed Scopus (201) Google Scholar, 22Cheng A. Merz K.M. Biophys. J. 1997; 73: 2851-2873Abstract Full Text PDF PubMed Scopus (93) Google Scholar). The majority of type I AFPs that have been studied in the past possess 11-residue repeats that consist of Thr-X 2-Asn/Asp-X 7 whereX can be any residue but is usually alanine (20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar, 23Wen D. Laursen R.A. J. Biol. Chem. 1992; 267: 14102-14108Abstract Full Text PDF PubMed Google Scholar, 24Wen D. Laursen R.A. Biophys. J. 1992; 63: 1659-1662Abstract Full Text PDF PubMed Scopus (144) Google Scholar). The Thr and Asn/Asp residues are located on the same face of the helix in a periodic nature which presumably leads to hydrogen-bonding with ice in a lattice-matching manner. One notable exception is the shorthorn sculpin serum AFP, sslAFP-8, which does not possess the typical 11-residue repeats (11Hew C.L. Joshi S. Wang N.C. Kao M.H. Ananthanarayanan V.S. Eur. J. Biochem. 1985; 151: 167-172Crossref PubMed Scopus (50) Google Scholar, 25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). In sssAFP-2, there are many putative 11-residue repeats (indicated by boxed residues in Fig. 1); however, none of these putative repeats match the established Thr-X 2-Asn/Asp-X 7 motif. The alanine content of sssAFP-2 is approximately 70%, the secondary structure of sssAFP-2 is predicted to be entirely helical (26Chou P.Y. Fasman G.D. Adv. Enzymol. 1978; 47: 45-148PubMed Google Scholar) and was confirmed by CD spectroscopy (Fig. 6). Furthermore, a helical wheel presentation of sssAFP-2 suggests a partially amphipathic molecule (see Fig. 7). Thus, sssAFP-2 meets the criteria necessary to be classified as a type I AFP, and at 92 residues, it is the largest known naturally occurring type I AFP identified thus far. The predominant 11-residue repeat within sssAFP-2 is Pr-X 2-Pr-X 7 (see Fig. 1), where Pr represents another polar residue and X is predominantly alanine. Three of the repeats highlighted in Fig. 1 begin with lysine. Although threonine is not present, the correct spacing of polar residues within the 11-residue repeat itself is maintained. Proper positioning of the polar residues in a lattice-matching manner is believed to be critical for AFP binding to ice (19DeVries A.L. Lin Y. Biochim. Biophys. Acta. 1977; 495: 388-392Crossref PubMed Scopus (138) Google Scholar, 27Knight C.A. Cheng C.C. DeVries A.L. Biophys. J. 1991; 59: 409-418Abstract Full Text PDF PubMed Scopus (457) Google Scholar). This belief is supported by crystal and NMR solution structures determined for wflAFP-6 (20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar, 28Gronwald W. Chao H. Reddy D.V. Davies P.L. Sykes B.D. Sönnichsen F.D. Biochemistry. 1996; 35: 16698-16704Crossref PubMed Scopus (52) Google Scholar), and molecular dynamics simulation techniques (22Cheng A. Merz K.M. Biophys. J. 1997; 73: 2851-2873Abstract Full Text PDF PubMed Scopus (93) Google Scholar). Furthermore, the possibility of an 11-residue repeat beginning with lysine has been noted before (20Sicheri F. Yang D.S.C. Nature. 1995; 375: 427-431Crossref PubMed Scopus (348) Google Scholar). Recently, a combination of molecular dynamics and modeling of the interaction of sslAFP-8 with the ice lattice has produced another possible mechanism of interaction (25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). This approach involves two binding models: accommodation of binding surface residues within ice cages, and the inclusion of key lysine side chains into the ice lattice through their tetrahedral end groups (25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). A comparison of the secondary structure of sssAFP-2 with sslAFP-8 (Fig. 7) shows that sssAFP-2 is similar to sslAFP-8 in that many lysine side chains project in a similar manner to one face of the helix. Furthermore, a comparison of the sequences for the two proteins reveals that many lysine residues within sssAFP-2 possess the correct spacing within the protein as defined by Wierzbicki et. al. (25Wierzbicki A. Taylor M.S. Knight C.A. Madura J.D. Harrington J.P. Sikes C.S. Biophys. J. 1997; 71: 8-18Abstract Full Text PDF Scopus (69) Google Scholar). Nonetheless, more structural work will be required to resolve and define any similarities or differences in the binding mechanisms of the sculpin AFPs. It appears that the defense against the dangers of freezing in ice-laden, sub-zero sea water of the shorthorn sculpin is identical to that of the winter flounder. Both species secrete AFP into the blood serum that serves to depress the freezing temperature of their extracellular fluids, and both produce skin-type AFPs that lack signal peptides, suggesting that they function as intracellular protectants. Precisely how these skin-type AFPs help to protect fish from cold and freezing is subject to some debate (for review, see Ref. 29Fletcher G.L. Goddard S.V. Davies P.L. Gong Z. Ewart K.V. Hew C.L. Portner H.O. Playle R. Cold Ocean Physiology. Cambridge University Press, Cambridge, United Kingdom1998: 239-265Google Scholar). Valerioet. al. (30Valerio P.F. Kao M.H. Fletcher G.L. J. Exp. Biol. 1992; 164: 135-151Crossref Scopus (14) Google Scholar) have demonstrated that winter flounder skin is an effective barrier to ice propagation and that the effectiveness of this barrier can be increased by the addition of antifreeze proteins to the extracellular space. This suggests that despite the lack of a signal peptide, the skin-type AFPs may use an alternative pathway for secretion of the cells into the intercellular space, thereby acting to block ice propagation. Recently, Murray et al. 2H. M. Murray, C. L. Hew, K. R. Kao, and G. L. Fletcher, manuscript in preparation. have identified the gill epithelial cells of the winter flounder as a major site of skin-type AFP production utilizing in situ hybridization and immuno-cytochemical techniques. Because of their importance in gas exchange, gill epithelia are the thinnest of all external epithelial tissues in the fish, and the most likely site to come into intimate contact with potentially lethal ice crystals. Thus, it is possible that the skin-type AFP simply serves to lower the freezing temperature of the intracellular fluids and thus ensure that this essential layer of cells cannot freeze. We thank Lingya Liao for technical assistance and Linda Mark for the preparation of the manuscript." @default.
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• W2014580500 title "Skin-type Antifreeze Protein from the Shorthorn Sculpin,Myoxocephalus scorpius" @default.
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