A natural variant of type I antifreeze protein with four ice-binding repeats is a particularly potent antifreeze.

A 4.3-kDa variant of Type I antifreeze protein (AFP9) was purified from winter flounder serum by size exclusion chromatography and reversed-phase HPLC. By the criteria of mass, amino acid composition, and N-terminal sequences of tryptic peptides, this variant is the posttranslationally modified product of the previously characterized AFP gene 21a. It has 52 amino acids and contains four 11-amino acid repeats, one more than the major serum AFP components. The larger protein is completely alpha-helical at 0 degree C, with a melting temperature of 18 degrees C. It is considerably more active as an antifreeze than the three-repeat winter flounder AFP and the four-repeat yellowtail flounder AFP, both on a molar and a mg/mL basis. Several structural features of the four-repeat winter flounder AFP, including its larger size, additional ice-binding residues, and differences in ice-binding motifs might contribute to its greater activity. Its abundance in flounder serum, together with its potency as an antifreeze, suggest that AFP9 makes a significant contribution to the overall freezing point depression of the host.     

Structural variations in the alanine-rich antifreeze proteins of the pleuronectinae

The sequence and activity of antifreeze proteins from two right eye flounder species were compared to assess the influence of structural variations on antifreeze capacity. The cDNA encoding the major serum antifreeze protein in the yellowtail flounder (Limanda ferruginea) was cloned from liver tissue. Its DNA sequence shows that the precursor to the antifreeze is a 97-residue preproportion. Edman degradation identified the N-terminus of the 48-amino-acid mature serum antifreeze protein and confirmed the sequence of the first 36 residues. A comparison with the previously determined winter flounder antifreeze protein and mRNA sequences shows strong homology through the 5' and 3' untranslated regions and in the peptide region. The mature protein section has the greatest sequence variation. Specifically, the yellowtail antifreeze protein, in contrast to that of the winter flounder, contains a fourth 11-amino-acid repeat and lacks several of the hydrophilic residues that have been postulated to aid in the binding of the protein to ice crystals. Intramolecular salt bridges are present in the antifreeze proteins from both species but in different registries with respect to the 11-amino-acid repeats. On a mass basis the yellowtail flounder antifreeze, though longer than that of the winter flounder, is only 80% as effective at depressing the freezing temperature of aqueous solutions. This lower activity might be due to the reduced number of hydrophilic ice-binding residues per molecule.     

Inhibition of recrystallization in ice by chimeric proteins containing antifreeze domains

Using synthetic DNA, we assembled a gene encoding a protein identical in sequence to one of the antifreeze proteins produced by the fish Pseudopleuronectes americanus (winter flounder). To address the relationship between structure and function, we also assembled genes encoding proteins varying in sequence and length. The synthetic genes were cloned into a bacterial expression vector to generate translational fusions to the 3' end of a truncated staphylococcal protein A gene; the chimeric proteins encoded by these fusions, varying only in their antifreeze domains, were isolated from Escherichia coli. The antifreeze domains conferred the ability to inhibit ice recrystallization, which is characteristic of naturally occurring antifreeze proteins, on the chimeric proteins. The chimeric proteins varied in their effectiveness of inhibiting ice recrystallization according to the number of 11-amino acid repeats present in the antifreeze moiety. A protein with only two repeats lacked activity, while the inhibitory activity increased progressively for proteins containing three, four, and five repeats. Some activity was lost upon removal of either the salt bridge or the carboxyl-terminal arginine, but surprisingly, not when both features were absent together.     

Hyperactive antifreeze protein in flounder species. The sole freeze protectant in American plaice

The recent discovery of a large hyperactive antifreeze protein in the blood plasma of winter flounder has helped explain why this fish does not freeze in icy seawater. The previously known, smaller and much less active type I antifreeze proteins cannot by themselves protect the flounder down to the freezing point of seawater. The relationship between the large and small antifreezes has yet to be established, but they do share alanine-richness (> 60%) and extensive alpha-helicity. Here we have examined two other righteye flounder species for the presence of the hyperactive antifreeze, which may have escaped prior detection because of its lability. Such a protein is indeed present in the yellowtail flounder judging by its size, amino acid composition and N-terminal sequence, along with the previously characterized type I antifreeze proteins. An ortholog is also present in American plaice based on the above criteria and its high specific antifreeze activity. This protein was purified and shown to be almost fully alpha-helical, highly asymmetrical, and susceptible to denaturation at room temperature. It is the only detectable antifreeze protein in the blood plasma of the American plaice. Because this species appears to lack the smaller type I antifreeze proteins, the latter may have evolved by descent from the larger antifreeze.     

A complex family of highly heterogeneous and internally repetitive hyperactive antifreeze proteins from the beetle Tenebrio molitor.

We have previously identified a Thr- and Cys-rich thermal hysteresis (antifreeze) protein (THP) in the beetle Tenebrio molitor that has 10-100 times the freezing point depression activity of fish antifreeze proteins. Because this 8.4 kDa protein is significantly different in its properties from THP preparations previously reported from this insect, a thorough search was undertaken for other antifreeze types. Many active proteins were observed, but all appeared to be isoforms of the THP that differed in their number of 12-amino acid repeats (consensus sequence CTxSxxCxxAxT), amino acid substitutions, and N-linked glycosylation. Mass spectral analysis has matched most of these isoforms with cDNA sequences of 17 different clones from a larval fat body library that encode eight different mature THPs containing 84, 96, or 120 amino acids. Genomic Southern blots suggest there may be 30-50 tightly linked copies of the gene, which is a signature consistently seen with unrelated fish antifreeze protein genes, and one that has been associated with the need to rapidly increase gene product in response to climate change. A three-dimensional model is proposed for the fully disulfide-bonded structure of T. molitor THP, which can accommodate addition or deletion of 12-amino acid repeats. The structure is a beta-helix that places most of the Thr in a regular array on one side of the protein to form a putative ice-binding surface.     

Chicken apolipoprotein A-I: cDNA sequence,tissue expression and evolution

Using an antibody against chicken apolipoprotein (apo) A-I, we identified multiple cDNA clones for the protein in two intestinal cDNA libraries in λgtll. The complete nucleotide sequence of chicken apoA-I cDNA was determined. The sequence predicts a mature protein of 240 amino acids, a 6-amino acid propeptide and an 18-amino acid signal peptide. Using a ³²P-cDNA probe, we detected the presence of apoA-I mRNA in 21 day old chicken intestine, liver, kidney, spleen, breast muscle and brain. The primary sequence of apoA-I contains numerous tandem repeats of 11 and 22 residues in a manner similar to the mammalian proteins. Our analysis of apoA-I sequences from human, rabbit, dog, rat, and chicken indicates that the rate of amino acid substitution is considerably faster in the rat lineage than in other mammalian lineages.     

Evidence that translational control mechanisms operate to optimize antifreeze protein production in the winter flounder

In fall and winter, the liver of the winter flounder produces large amounts of alanine-rich (60 mol %) antifreeze proteins for export to the circulation. We have examined the tRNA in the liver to see if the seasonal production of antifreeze protein is accompanied by changes in tRNAAla isoacceptors. Total tRNA from the liver of winter fish showed an approximate 40% increase in alanine acceptor capacity over tRNA from summer fish. In contrast, the acceptor capacities for other amino acids showed no seasonal difference. When labeled alanyl-tRNAs were separated by reverse phase chromatography-5 chromatography, a large proportion of the increase in alanine acceptor capacity was in one of three main peaks. Measurements of the optimum temperatures for various flounder amino-acyl-tRNA synthetases suggest that alanyl-tRNA synthetase functions best between 0 and 5 degrees C, which is the sea water temperature when antifreeze protein synthesis occurs, while prolyl- and valyl-tRNA synthetases are most active between 20 and 30 degrees C. These differences in temperature optima and the seasonal variation in tRNAAla levels and isoaccepting species may both serve to optimize antifreeze protein production by increasing the translational efficiency of its mRNA.     

Wound-induced proteinase inhibitors from tomato leaves. I. The cDNA-deduced primary structure of pre-inhibitor I and its post-translational processing

A cDNA containing the coding region for the complete amino acid sequence of wound-induced proteinase Inhibitor I from tomato leaves was constructed in the plasmid pUC9 and characterized. The open reading frame codes for a protein of 111 amino acids. This deduced amino acid sequence revealed the presence of a 42-amino acid N-terminal sequence that is not found in the native protein. This sequence appears to contain a 23-amino acid segment typical of a signal sequence followed by a 19-amino acid sequence containing 9 charged amino acids. The 42-amino acid sequence is apparently lost during maturation to the native Inhibitor I and represents 38% of the translated protein. The Inhibitor I amino acid sequence contains 71% identity with potato tuber Inhibitor I sequence and 35% identity with an inhibitor from the leech.     

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.     

Use of a novel strategy for the preparation and characterization of an antipeptide antibody capable of recognizing members of the annexin family

Annexins are structurally-related proteins which bind phospholipids in a Ca2+-dependent manner. We have used a novel coupling strategy to prepare an antiserum directed against a 17-amino acid synthetic peptide that resembles the sequence of a highly-conserved portion of these proteins. This antipeptide serum specifically recognizes 5 of 6 human annexins on Western blots, despite differences between the protein and peptide sequences of 3 or 4 amino acids. The antiserum does not recognize endonexin II, whose sequence differs from that of the peptide by 6 amino acids. The availability of multiple proteins with known amino acid sequence has allowed analysis of structural requirements for recognition by this antibody. In some situations, use of such an antibody may allow the identification of a protein as a member of a family.     

Hyperactive antifreeze protein from winter flounder is a very long rod-like dimer of alpha-helices

The winter flounder (Pseudopleuronectes americanus) produces short, monomeric alpha-helical antifreeze proteins (type I AFP), which adsorb to and inhibit the growth of ice crystals. These proteins alone are not sufficiently active to protect this fish against freezing at -1.9 degrees C, the freezing point of seawater. We have recently isolated a hyperactive antifreeze protein from the plasma of the flounder with activity 10-100-fold higher than type I AFP. It is comparable in activity to the AFPs produced by insects, and is capable of conferring freeze resistance to the flounder. This novel AFP has a molecular mass of 16,683 Da and a remarkable amino acid composition that is >60% alanine. CD spectra indicate that the protein is almost entirely alpha-helical at 4 degrees C but partially denatures at 20 degrees C, resulting in a species with a moderately reduced helix content that is stable at up to 50 degrees C. This transformation correlates with irreversible loss of activity. Analytical ultracentrifugation (sedimentation velocity and equilibrium) indicates that the predominant species in solution is dimeric (molecular weight, 32,275). Size-exclusion chromatography reveals a 2-fold higher apparent molecular weight suggesting that this molecule has an unusually large Stokes radius. The axial ratio of the dimer calculated from the sedimentation velocity data is 18:1, confirming that this protein has an extraordinarily long, rod-like structure, consistent with a novel dimeric alpha-helical arrangement. The structural model that best fits these data is one in which the approximately 195 amino acids of each monomer form one approximately 290-A long alpha-helix and associate via a unique dimerization motif that is distinct from that of the leucine zipper and any other coiled-coil.     

A single WAP domain-containing protein from Litopenaeus vannamei hemocytes

A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.     

Differential amplification of antifreeze protein genes in the pleuronectinae

Summary The organization of antifreeze protein (AFP) genes in the yellowtail flounder was investigated by Southern blotting and the characterization of clones from a genomic library. This flounder, like the closely related winter flounder, has a set of 10–12 linked but irregularly spaced AFP genes. However, it lacks the tandemly amplified set of 20 such genes that are present in the winter flounder. DNA sequence analysis of a tandemly repeated gene from winter flounder showed that it can code for one of the two most abundant AFP components in the serum. Consistent with this higher AFP gene dosage, the peak serum AFP level in midwinter was 9 mg/ml in the winter flounder and only 4 mg/ml in the yellowtail flounder. A recent amplification of the AFP gene in the winter flounder lineage might be responsible for the higher serum AFP levels in this fish. This increase in gene dosage might have helped the winter flounder colonize the ice-laden, shallow-water niche that it currently occupies along the east coast of North America. Genomic Southern blotting of two other righteye flounders, the smooth flounder and the American plaice, illustrates another example of a differential amplification of AFP genes that correlates with a species' exposure to ice.     

Rat vitamin D binding protein. Determination of the full-length primary structure from cloned cDNA

Vitamin D binding protein (DBP) is an abundant serum glycoprotein secreted by the liver which transports vitamin D sterols, binds to actin, and is found on the surface of B-lymphocytes and subpopulations of T-lymphocytes. In the current study, a cDNA to rat DBP mRNA was cloned from a bacteriophage lambda gt 11 rat liver expression library. This DBP cDNA clone was identified by immunoblotting and its identity was confirmed by immunoprecipitation of a 54-kDa protein after hybrid-assisted translation. Northern analysis and primer extension mapping of rat liver mRNA indicated that the full-length DBP mRNA contains 1700 bases. By DNA sequence analysis this 1655-base pair clone contains a single open reading frame encoding the 476-amino acid containing full-length DBP and includes its 16-amino acid signal sequence. Analysis of the sequence reveals about 40% nucleotide and 23% amino acid homology to both albumin and alpha-fetoprotein. The encoded DBP contains a characteristic placement of cysteine residues, identical to that in albumin, suggesting a similar secondary folding structure. Albumin and alpha-fetoprotein are composed of three internally homologous domains. DBP mRNA terminates 122 amino acids before the larger albumin mRNA in the third internal domain, but retains the characteristic homology among the first two domains and the truncated portion of the third domain. These data support the conclusion that DBP is a member of a multigene family which includes albumin and alpha-fetoprotein.     

Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease inhibitor superfamily

A cDNA library in lambda-phage lambda gt11 containing DNA inserts prepared from human liver mRNA was screened with monoclonal antibodies to human protein C inhibitor. Six positive clones were isolated from 6 X 10(6) phages and plaque purified. The cDNA in the phage containing the largest insert, which hybridized to a DNA probe prepared on the basis of the amino-terminal amino acid sequence of the mature inhibitor, was sequenced. This cDNA insert contained 2106 base pairs coding for a 5'-noncoding region, a 19-amino acid signal peptide, a 387-amino acid mature protein, a stop codon, and a long 3'-noncoding region of 839 base pairs. Based on the amino acid sequence of the carboxyl-terminal peptide released by cleavage of protein C inhibitor by activated protein C as well as by thrombin, the reactive site peptide bond of protein C inhibitor is Arg354-Ser355. Five potential carbohydrate-binding sites were found in the mature protein. The high homology of the amino acid sequence of protein C inhibitor to the other known inhibitors clearly demonstrates that protein C inhibitor is a member of the superfamily of serine protease inhibitors including alpha 1-antichymotrypsin, alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. Based on the difference matrices for these proteins, we present possible phylogenetic trees for these proteins.     

Cloning, Golgi localization, and enzyme activity of the full-length heparin/heparan sulfate-glucuronic acid C5-epimerase

While studying the cellular localization and activity of enzymes involved in heparan sulfate biosynthesis, we discovered that the published sequence for the glucuronic acid C5-epimerase responsible for the interconversion of d-glucuronic acid and l-iduronic acid residues encodes a truncated protein. Genome analysis and 5'-rapid amplification of cDNA ends was used to clone the full-length cDNA from a mouse mastocytoma cell line. The extended cDNA encodes for an additional 174 amino acids at the amino terminus of the protein. The murine sequence is 95% identical to the human epimerase identified from genomic sequences and fits with the general size and structure of the gene from Drosophila melanogaster and Caenorhabditis elegans. Full-length epimerase is predicted to have a type II transmembrane topology with a 17-amino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An assay with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike other enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myc tag or green fluorescent protein to the highly conserved COOH-terminal portion of the protein inhibits its activity. The amino-terminally truncated epimerase does not localize to any cellular compartment, whereas the full-length enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur.     

Targeted expression of redesigned and codon optimised synthetic gene leads to recrystallisation inhibition and reduced electrolyte leakage in spring wheat at sub-zero temperatures

Antifreeze proteins (AFPs) adsorb to ice crystals and inhibit their growth, leading to non-colligative freezing point depression. Crops like spring wheat, that are highly susceptible to frost damage, can potentially be made frost tolerant by expressing AFPs in the cytoplasm and apoplast where ice recrystallisation leads to cellular damage. The protein sequence for HPLC-6 α-helical antifreeze protein from winter flounder was rationally redesigned after removing the prosequences in the native protein. Wheat nuclear gene preferred amino acid codons were used to synthesize a recombinant antifreeze gene, rAFPI. Antifreeze protein was targeted to the apoplast using a Murine leader peptide sequence from the mAb24 light chain or retained in the endoplasmic reticulum using C-terminus KDEL sequence. The coding sequences were placed downstream of the rice Actin promoter and Actin-1 intron and upstream of the nopaline synthase terminator in the plant expression vectors. Transgenic wheat lines were generated through micro projectile bombardment of immature embryos of spring wheat cultivar Seri 82. Levels of antifreeze protein in the transgenic lines without any targeting peptide were low (0.06–0.07%). The apoplast-targeted protein reached a level of 1.61% of total soluble protein, 90% of which was present in the apoplast. ER-retained protein accumulated in the cells at levels up to 0.65% of total soluble proteins. Transgenic wheat line T-8 with apoplast-targeted antifreeze protein exhibited the highest levels of antifreeze activity and provided significant freezing protection even at temperatures as low as −7°C.     

Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum

Habu (Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habu serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca2+ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with serotriflin. It was bound to triflin and serotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood.