Efficiency of beetle (Dendroides canadensis) recombinant antifreeze protein for buffalo semen freezability and fertility.

Overwintering larvae of the beetle Dendroides canadensis produce potent antifreeze proteins to inhibit inoculative freezing and promote supercooling. We hypothesized that addition of Dendroides canadensis recombinant antifreeze proteins (DAFPs) in the extender will improve the quality and fertility of cryopreserved Nili-Ravi buffalo (Bubalus bubalis) sperm. The study was divided into two parts: (1) Evaluation of the effect of DAFPs on the quality of frozen-thawed buffalo bull sperm and (2) Examination of the fertilizing ability of frozen-thawed buffalo bull sperm. Semen was collected from three bulls using an artificial vagina (42 °C). Qualifying ejaculates from each bull were divided into four aliquots and diluted (at 37 °C, 50 × 10(6) sperm/mL) in tris-citric acid extender containing DAFP (at 0.1, 1.0, and 10 µg/mL), and the sperm were evaluated for important characteristics relative to a control without DAFP. D canadensis recombinant antifreeze proteins at any of the three concentrations did not affect sperm progressive motility or plasma membrane integrity (PMI), either before or after the semen was cooled to 4 °C in 2 hours. However, after 24 hours of cryostorage at -196 °C, followed by thawing at 37 °C for 30 seconds, sperm progressive motility and PMI were higher (P < 0.05) in extender containing DAFP at 10 µg/mL compared with control. The in vitro-fertilizing ability of cryopreserved (-196 °C) sperm supplemented with DAFP (10 µg/mL) was slightly higher (P = 0.098) compared with control, as assessed through in vitro cleavage rate of in vitro matured buffalo oocytes. Also, the in vivo fertility rate was evaluated by inseminating 100 buffaloes (50 inseminations per extender) 12 hours after standing heat. The fertility rate of cryopreserved buffalo bull sperm in terms of positive pregnancy at 90 days after insemination was clinically higher but remained statistically nonsignificant in extender containing DAFP at 10 µg/mL (52.0%) compared with control (43.8%). In conclusion, supplementation of 10 µg/mL of DAFP in the extender improved the motility and PMI of Nili-Ravi buffalo sperm after freeze-thawing, and yielded numerically higher, although statistically nonsignificant, in vitro cleavage, and in vivo fertility rate.

Functional evaluation of candidate ice structuring proteins using cell-free expression systems.

Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications.

A molecular characterization of the choroid plexus and stress-induced gene regulation.

The role of the choroid plexus (CP) in brain homeostasis is being increasingly recognized and recent studies suggest that the CP has a more important role in physiological and pathological brain functions than currently appreciated. To obtain additional insight on the CP function, we performed a proteomics and transcriptomics characterization employing a combination of high resolution tandem mass spectrometry and gene expression analyses in normal rodent brain. Using multiple protein fractionation approaches, we identified 1400 CP proteins in adult CP. Microarray-based comparison of CP gene expression with the kidney, cortex and hippocampus showed significant overlap between the CP and the kidney. CP gene profiles were validated by in situ hybridization analysis of several target genes including klotho, CLIC 6, OATP 14 and Ezrin. Immunohistochemical analyses were performed for CP and enpendyma detection of several target proteins including cytokeratin, Rab7, klotho, tissue inhibitor of metalloprotease 1 (TIMP1), MMP9 and glial fibrillary acidic protein (GFAP). The molecular functions associated with various proteins of the CP proteome indicate that it is a blood-cerebrospinal fluid (CSF) barrier that exhibits high levels of metabolic activity. We also analyzed the gene expression changes induced by stress, an exacerbating factor for many illnesses, particularly mood disorders. Chronic stress altered the expression of several genes, downregulating 5HT2C, glucocorticoid receptor and the cilia genes IFT88 and smoothened while upregulating 5HT2A, BDNF, TNFα and IL-1b. The data presented here attach additional significance to the emerging importance of CP function in brain health and CNS disease states.

Deep supercooling, vitrification and limited survival to -100{degrees}C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae.

Larvae of the freeze-avoiding beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) in Alaska have mean supercooling points in winter of -35 to -42 degrees C, with the lowest supercooling point recorded for an individual of -58 degrees C. We previously noted that some larvae did not freeze when cooled to -80 degrees C, and we speculated that these larvae vitrified. Here we present evidence through differential scanning calorimetry that C. c. puniceus larvae transition into a glass-like state at temperatures<-58 degrees C and can avoid freezing to at least -150 degrees C. This novel finding adds vitrification to the list of insect overwintering strategies. While overwintering beneath the bark of fallen trees, C. c. puniceus larvae may experience low ambient temperatures of around -40 degrees C (and lower) when microhabitat is un-insulated because of low snow cover. Decreasing temperatures in winter are correlated with loss of body water from summer high levels near 2.0 to winter lows near 0.4 mg mg(-1) dry mass and concomitant increases in glycerol concentrations (4-6 mol l(-1)) and thermal hysteresis. Finally, we provide direct evidence that Cucujus from Wiseman, Alaska, survive temperatures to -100 degrees C.

Simultaneous freeze tolerance and avoidance in individual fungus gnats, Exechia nugatoria.

Freeze tolerance and freeze avoidance are typically described as mutually exclusive strategies for overwintering in animals. Here we show an insect species that combines both strategies. Individual fungus gnats, collected in Fairbanks, Alaska, display two freezing events when experimentally cooled and different rates of survival after each event (mean +/- SEM: -31.5 +/- 0.2 degrees C, 70% survival and -50.7 +/- 0.4 degrees C, 0% survival). To determine which body compartments froze at each event, we dissected the abdomen from the head/thorax and cooled each part separately. There was a significant difference between temperature levels of abdominal freezing (-30.1 +/- 1.1 degrees C) and head/thorax freezing (-48.7 +/- 1.3 degrees C). We suggest that freezing is initially restricted to one body compartment by regional dehydration in the head/thorax that prevents inoculative freezing between the freeze-tolerant abdomen (71.0 +/- 0.8% water) and the supercooled, freeze-sensitive head/thorax (46.6 +/- 0.8% water).

Membrane fusion correlates with surface charge in exocytic vesicles.

Stimulation of gastric parietal cells results in exocytic recruitment of the proton pump (H(+),K(+)-ATPase) from a pool of intracellular membranes (tubulovesicles) to the apical plasma membrane. We have previously reconstituted a step in this process, the homotypic fusion of tubulovesicles, and shown that they also fuse with liposomes in a protein-dependent manner [Duman, J. G., Singh, G., Lee, G. Y., Machen, T. E., and Forte, J. G. (2002) Traffic 3, 203-17]. Further, the lipid composition of the liposomes affects their ability to undergo fusion with tubulovesicles. In the present study, we investigated the lipid requirements for tubulovesicular membrane fusion using a fluorescent probe relaxation assay as well as transfer of protein between tubulovesicles and liposomes of defined composition. Initially, we tested the ability of tubulovesicles to undergo fusion with a panel of synthetic phosphatidylcholine-based liposomes containing a variety of common membrane lipids of various shapes and charges. We found that anionic lipids such as phosphatidylserine, phosphatidic acid, and phosphoinositides were best able to enhance tubulovesicle-liposome fusion and that they did it in a dose-dependent, apparently saturable manner. Next, we altered the lipid compositions of actual tubulovesicles and observed that addition of anionic lipids was able to enhance tubulovesicle-tubulovesicle fusion in vitro; thus, we hypothesized that the charge imparted by the lipids, per se, was responsible for the enhancement of membrane fusion. Accordingly, addition of negative charges to one of two pools of tubulovesicles in a fusion assay using anionic detergents increased membrane fusion; whereas, addition of positively charged cationic detergent decreased membrane fusion and could be used to back-titrate the anionic effects. Surprisingly, when both pools of fusing membranes were loaded with anionic detergents, fusion was markedly increased. The ability of anionic charges to enhance fusion was diminished as the ionic strength of the fusion medium was increased, suggesting that the mechanism of fusion enhancement depends on the surface charge of the membranes. Finally, the fusion reaction was highly dependent on temperature, and anionic charge appears to lower the activation energy of the fusion reaction. Taken together, these data suggest that (1) tubulovesicular fusion is enhanced by an increase in membrane surface negative charge associated with a lower activation energy and (2) neutralization or reversal of the surface charge prevents tubulovesicular fusion.

Antifreeze proteins in Alaskan insects and spiders.

Prior to this study, antifreeze proteins (AFPs) had not been identified in terrestrial arthropods from the Arctic or anywhere in Alaska. The hemolymph of 75 species of insects and six spiders from interior and arctic Alaska were screened for thermal hysteresis (a difference between the freezing and melting points), characteristic of the presence of AFPs. Eighteen species of insects and three spiders were shown to have AFPs. Ten of the insects with AFPs were beetles including the first species from the families Chrysomelidae, Pythidae, Silphidae and Carabidae. In addition, the first Neuropteran to have AFPs was identified, the lacewing Hemerobius simulans together with the second and third Diptera (the first Tipulids) and the second and third Hemiptera, the stinkbug Elasmostethus interstinctus (the first Pentatomid), and the water strider Limnoporus dissortis (the first Gerrid). Prior to this study, 33 species of insects and three spiders had been reported to have AFPs. Most AFP-producing terrestrial arthropods are freeze avoiding, and the AFPs function to prevent freezing. However, some of the AFP- producing insects identified in this study are known to be freeze tolerant (able to survive freezing) to very low temperatures (-40 to -70 degrees C).

Site-specific forms of antifreeze protein in the beetle Dendroides canadensis.

It was known previously that overwintering larvae of the beetle Dendroides canadensis produce antifreeze proteins (DAFPs) consisting of a family of 12 similar proteins, and based on sequence variations the DAFPs may be separated into three groups. DAFPs were known to be present in hemolymph, midgut fluid and in/on epidermal cells located immediately under the cuticle. However, only DAFPs-1, 2, and 4 were known to be present in the hemolymph, leaving the location of the others unknown. In this study, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry of hemolymph confirmed the presence of DAFPs-1, 2, and 4 (Group I), plus a protein consistent with the mass of DAFP-6 (Group I). Also, a review of older data revealed the co-purification of DAFP-6 along with DAFP-4 in hemolymph. However, none of the other DAFPs (Groups II and III) were present in hemolymph. In contrast, mass spectrometry of midgut fluid demonstrated the absence of DAFPs-1, 2, 4, or 6, however, proteins consistent with the masses of all, or a subset of, Groups II and/or III were present. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that transcripts of all 12 DAFPs were present in the fat body. However, consistent with the MALDI-TOF data, only Groups II (8, 9, 10, 11) and III (3, 5, 7, 12) transcripts were found in midgut epithelia. RT-PCR of epidermal tissue identified dafps- 4, 6, 8 and 11 (and sometimes 1 and/or 2) as the major transcripts. These data suggest that various DAFPs may have evolved to function best in certain sites.

The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate.

Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.

Antifreeze and ice nucleator proteins in terrestrial arthropods.

Terrestrial arthropods survive subzero temperatures by becoming either freeze tolerant (survive body fluid freezing) or freeze avoiding (prevent body fluid freezing). Protein ice nucleators (PINs), which limit supercooling and induce freezing, and antifreeze proteins (AFPs), which function to prevent freezing, can have roles in both freeze tolerance and avoidance. Many freeze-tolerant insects produce hemolymph PINs, which induce freezing at high subzero temperatures thereby inhibiting lethal intracellular freezing. Some freeze-tolerant species have AFPs that function as cryoprotectants to prevent freeze damage. Although the mechanism of this cryoprotection is not known, it may involve recrystallization inhibition and perhaps stabilization of the cell membrane. Freeze-avoiding species must prevent inoculative freezing initiated by external ice across the cuticle and extend supercooling abilities. Some insects remove PINs in the winter to promote supercooling, whereas others have selected against surfaces with ice-nucleating abilities on an evolutionary time scale. However, many freeze-avoiding species do have proteins with ice-nucleating activity, and these proteins must be masked in winter. In the beetle Dendroides canadensis, AFPs in the hemolymph and gut inhibit ice nucleators. Also, hemolymph AFPs and those associated with the layer of epidermal cells under the cuticle inhibit inoculative freezing. Two different insect AFPs have been characterized. One type from the beetles D. canadensis and Tenebrio molitor consists of 12- and 13-mer repeating units with disulfide bridges occurring at least every six residues. The spruce budworm AFP lacks regular repeat units. Both have much higher activities than any known AFPs.

Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization.

Clathrin from H-K-ATPase-rich membranes derived from the tubulovesicular compartment of rabbit and hog gastric acid secretory (parietal) cells was characterized biochemically, and the subcellular localization of membrane-associated clathrin in parietal cells was characterized by immunofluorescence, electron microscopy, and immunoelectron microscopy. Clathrin from H-K- ATPase-rich membranes was determined to be comprised of conventional clathrin heavy chain and a predominance of clathrin light chain A. Clathrin and adaptors could be induced to polymerize quantitatively in vitro, forming 120-nm-diameter basketlike structures. In digitonin-permeabilized resting parietal cells, the intracellular distribution of immunofluorescently labeled clathrin was suggestive of labeling of the tubulovesicular compartment. Clathrin was also unexpectedly localized to canalicular (apical) membranes, as were alpha-adaptin and dynamin, suggesting that this membrane domain of resting parietal cells is endocytotically active. At the ultrastructural level, clathrin was immunolocalized to canalicular and tubulovesicular membranes. H-K-ATPase was immunolocalized to the same membrane domains as clathrin but did not appear to be enriched at the specific subdomains that were enriched in clathrin. Finally, in immunofluorescently labeled primary cultures of parietal cells, in contrast to the H-K-ATPase, intracellular clathrin was found not to translocate to the apical membrane on secretagogue stimulation. Taken together, these biochemical and morphological data provide a framework for characterizing the role of clathrin in the regulation of membrane trafficking from tubulovesicles and at the canalicular membrane in parietal cells.

An osmotin-like cryoprotective protein from the bittersweet nightshade Solanum dulcamara.

Cold acclimation in plants is a polygenic phenomenon involving increased expression of several genes. The gene products participate either directly or indirectly towards increasing cold tolerance. Evidence of proteins having a direct effect on cold tolerance is emerging but limited. With isolated protoplasts from warm-grown kale (Brassica oleracea) as a model system, we tested protein fractions from winter bittersweet nightshade, Solanum dulcamara, stems for the presence of proteins that have a cryoprotective effect. Purification of one such fraction resulted in isolation of a 25 kDa protein. N-terminal Edman degradation amino acid sequence analysis showed that it has high homology to osmotin and osmotin-like proteins. When added to warm-grown protoplasts, it increased the cryosurvival of frozen-thawed protoplasts by 24% over untreated or BSA-treated controls at -8 degrees C. A cDNA library which was made in November from stems and leaves of S. dulcamara was successfully screened for the corresponding cDNA clone. The deduced amino acid sequence indicated that the protein consists of 206 amino acid residues including a N-terminal signal sequence and a putative C-terminal propeptide. The mature protein, without the N-terminal signal sequence, was expressed in Escherichia coli. The partially purified protein in the supernatant fraction of the culture medium had cryoprotective activity.

Isolation and characterization of cDNA clones encoding antifreeze proteins of the pyrochroid beetle Dendroides canadensis.

Temporal differences in the expression of Dendroides canadensis antifreeze protein (DAFP) are indicated from seasonal comparison of dafp-1 transcript level, thermal hysteresis activity and temperature changes. DAFP-1 transcript abundance correlates with the thermal hysteresis activity level in late fall/early winter and appears to follow overall seasonal temperature changes with peak transcript levels occurring in December. A cDNA library created from December larvae yielded clones encoding a set of novel putative DAFPs. Some of the cDNA clones isolated display significant divergence at the primary amino acid level, yet, maintain conservation of key residues that are presumably important for structure and function of antifreeze proteins in this cold-hardy organism. Seasonal analysis of two dafps (dafp-1 and dafp-7) revealed differences on the transcriptional level, suggesting that DAFPs may serve somewhat different functions.

Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase.

Stimulation of the gastric parietal cell results in a massive redistribution of H+-K+-ATPase from cytoplasmic tubulovesicles to the apical plasma membrane. Previous studies have implicated the small GTPase rab11 in this process. Using matrix-assisted laser desorption mass spectrometry, we confirmed that rab11 is associated with H+-K+-ATPase-enriched gastric microsomes. A stoichiometry of one rab11 per six copies of H+-K+-ATPase was estimated. Furthermore, rab11 exists in at least three forms on rabbit gastric microsomes: the two most prominent resemble rab11a, whereas the third resembles rab11b. Using an adenoviral expression system, we expressed the dominant negative mutant rab11a N124I in primary cultures of rabbit parietal cells under the control of the tetracycline transactivator protein (tTA). The mutant was well expressed with a distribution similar to that of the H+-K+-ATPase. Stimulation of these cultures with histamine and IBMX was assessed by measuring the aminopyrine (AP) uptake relative to resting cells (AP index). In experiments on six culture preparations, stimulated uninfected cells gave an AP index of 10.0 +/- 2.9, whereas parallel cultures expressing rab11a N124I were poorly responsive to stimulation, with a mean AP index of 3.2 +/- 0. 9. Control cultures expressing tTA alone or tTA plus actin responded equally well to stimulation, giving AP index values of 9.0 +/- 3.1 and 9.6 +/- 0.9, respectively. Thus inhibition by rab11a N124I is not simply due to adenoviral infection. The AP uptake data were confirmed by immunocytochemistry. In uninfected cells, H+-K+-ATPase demonstrated a broad cytoplasmic distribution, but it was cleared from the cytoplasm and associated with apically derived membranes on stimulation. In cells expressing rab11a N124I, H+-K+-ATPase maintained its resting localization on stimulation. Furthermore, this effect could be alleviated by culturing infected cells in the presence of tetracycline, which prevents expression of the mutant rab11. We therefore conclude that rab11a is the prominent GTPase associated with gastric microsomes and that it plays a role in parietal cell activation.

Cytological transformations associated with parietal cell stimulation: critical steps in the activation cascade.

Cultured rabbit parietal cells were used to evaluate morphological responses to activators and inhibitors of HCl secretion. Immunofluorescence was used to localize the proton pump protein, H, K-ATPase, and the apical membrane-cytoskeletal linker protein, ezrin; fluorescent-labeled phalloidin was used as a marker of F-actin. Treatment of healthy control parietal cells with secretagogues resulted in exaggerated swelling of apical membrane vacuoles, presumably with the accumulation of HCl and water. Thus stimulation-associated swelling of apical vacuoles was blocked by inhibitors that work at various steps in the secretion-activation cascade. When secretion was blocked by agents that prevent the translocation of H,K-ATPase-rich tubulovesicles to apical membrane vacuoles (such as H2-receptor antagonists and protein kinase A inhibitors), the general resting morphology was maintained. ME-3407 (a functional analogue of wortmannin) was unique in preventing H, K-ATPase redistribution and effecting the delocalization of ezrin from apical membrane vacuoles. When secretion was blocked by agents that inhibit the H+ pump or induce H+ backflux, the translocation of H,K-ATPase to apical membrane vacuoles occurred but the large vacuolar swelling associated with HCl and H2O accumulation was greatly diminished. These data support the membrane recycling/recruitment hypothesis of HCl secretion in which H, K-ATPase-rich tubulovesicles are recruited from a cytoplasmic domain to the apical surface, and they are inconsistent with models proposing that the tubulovesicles, regardless of shape, are contiguous with the apical plasma membrane. These studies also demonstrate the utility of the parietal cell culture model in distinguishing a general site of action for various inhibitors and antisecretory agents.

Secondary structure of antifreeze proteins from overwintering larvae of the beetle Dendroides canadensis.

Antifreeze proteins from overwintering larvae of the beetle Dendroides canadensis are among the most active antifreeze proteins known. The Dendroides AFPs (DAFPs) consist of 6 or 7, 12- or 13-mer repeat units with a consensus sequence of -C-T-X3-S-X5-X6-C-X8-X9-A-X11-T-X13-. Nearly all of the Cys residues are in internal disulfide bridges between positions 1 and 7 within the repeats. The study presented here identified the secondary structure of the DAFPs using infrared and circular dichroism (CD) spectroscopies. The eight disulfide bridges impose significant constraints on potential secondary structural features (i.e., a number of three-residue gamma-turns) which may lead to unusual infrared and CD spectra that require special interpretation. At 25 degreesC the DAFPs contain approximately 46% beta-sheet, 39% turn, 2% helix, and 13% random structure. In the presence of ice there is a slight increase in helix and beta-sheet structures and a decrease in both turn and especially random structures. This change in the presence of ice may reflect a certain amount of flexibility in the DAFP structure. These structural changes may permit an improved lattice match between the DAFPs and ice, a requisite for the noncolligative freezing-point-depressing activity of the DAFPs.

O-Mannosylation of Pichia pastoris cellular and recombinant proteins.

O-linked saccharides were released from a major cell wall glycoprotein and from cellular mannan-protein complexes obtained from Pichia pastoris cells. Analysis by a variety of chromatographic methods and exoglycosidase digestions revealed the presence of mannose and (alpha1-2)-linked dimer, trimer and tetramer saccharides of mannose. The recombinant kringle 1-4 domain of human plasminogen expressed in P. pastoris was subjected to hydrazinolysis of both O- and N-linked saccharides. Only a very small quantity of N-linked oligosaccharides was present on the Asn289 consensus site. The major products were O-linked (alpha1-2)-linked mannans, containing dimeric, trimeric, tetrameric and pentameric oligosaccharides with the major amount of the total saccharides being distributed approximately equally between the dimer and trimer components. These results show that short O-linked saccharides of mannose containing (alpha1-2) glycosidic linkages are present in P. pastoris cells and expressed proteins.

Enhancement of insect antifreeze protein activity by solutes of low molecular mass.

Antifreeze proteins (AFPs) lower the non-equilibrium freezing point of water (in the presence of ice) below the melting point, thereby producing a difference between the freezing and melting points that has been termed thermal hysteresis. In general, the magnitude of the thermal hysteresis depends upon the specific activity and concentration of the AFP. This study describes several low-molecular-mass solutes that enhance the thermal hysteresis activity of an AFP from overwintering larvae of the beetle Dendroides canadensis. The most active of these is citrate, which increases the thermal hysteresis nearly sixfold from 1.2 degrees C in its absence to 6.8 degrees C. Solutes which increase activity approximately fourfold are succinate, malate, aspartate, glutamate and ammonium sulfate. Glycerol, sorbitol, alanine and ammonium bicarbonate increased thermal hysteresis approximately threefold. Interestingly, 0.5 mol l-1 sodium sulfate eliminated activity. Solute concentrations between 0.25 and 1 mol l-1 were generally required to elicit optimal thermal hysteresis activity. Glycerol is the only one of these enhancing solutes that is known to be present at these concentrations in overwintering D. canadensis, and therefore the physiological significance of most of these enhancers is unknown. The mechanism(s) of this enhancement is also unknown. The AFP used in this study (DAFP-4) is nearly identical to previously described D. canadensis AFPs. The mature protein consists of 71 amino acid residues arranged in six 12- or 13-mer repeats with a consensus sequence consisting of Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X1 3, where X3 and X11 tend to be charged residues, X5 tends to be Thr or Ser, X6 to be Asn or Asp, X9 to be Asn or Lys and X13 to be Ala in the 13-mers. DAFP-4 is shorter by one repeat than previously described D. canadensis AFPs.

Mapping of disulfide bridges in antifreeze proteins from overwintering larvae of the beetle Dendroides canadensis.

Antifreeze proteins (AFPs) have been identified in certain high-latitude marine fish, insects and other terrestrial arthropods, and plants. Despite considerable structural variation, the mechanisms of their noncolligative antifreeze activity are probably quite similar. AFPs hydrogen bond onto the surface of potential seed ice crystals at preferred growth sites, thereby preventing growth of the crystals. AFPs from overwintering larvae of the beetle Dendroidescanadensis are among the most active AFPs. These 8.7-kDa proteins consist of seven 12- or 13-mer repeating units. Their most striking feature is the location of cysteines every six residues throughout their length. Consequently, identification of the disulfide linkages of these cysteines is essential to understanding the structure of these AFPs. This study demonstrated that all of the 16 Cys residues in the Dendroides AFPs are disulfide bridged. All of the seven 12- or 13-mer repeats have internal disulfide bridges, and in all but the first repeat the Cys residues at positions 1 and 7 of the repeats are linked. In repeat 1 the Cys at position 1 is linked to the Cys at position 10, rather than the Cys at position 7 as in the other repeats, and the Cys at position 7 of the first repeat is linked to a Cys at position 4 of the second repeat. The disulfide bridges probably function to position the hydrophilic side chains of serine and threonine residues so that they hydrogen bond with ice.

Molecular characterization and sequencing of antifreeze proteins from larvae of the beetle Dendroides canadensis.

The deduced amino acid sequences of antifreeze proteins (AFPs) from larvae of the beetle Dendroides canadensis were determined from both complementary DNAs (cDNAs) and from peptide sequencing. These consisted of proteins with a 25-residue signal peptide and mature proteins 83 (Dendroides antifreeze protein; DAFP-1) or 84 (DAFP-2) amino acids in length which differed at only two positions. Peptide sequencing yielded sequences which overlapped exactly with those of the deduced cDNA sequences of DAFP-1 and DAFP-2, while the partial sequence of another AFP (DAFP-3) matched 21 of 28 residues. Seven 12- or 13-mer repeating units are present in these antifreeze proteins with a consensus sequence consisting of: Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X1 3, where X3 and X11 tend toward charged residues, X5 tends toward threonine or serine, X6 toward asparagine or aspartate, X9 toward asparagine or lysine, and X13 toward alanine in the 13-mers. The most interesting feature of these proteins is that throughout the length of the mature antifreeze proteins every sixth residue is a cysteine. These sequences are not similar to any of the known fish AFPs, but they are similar to AFPs from the beetle Tenebrio molitor.