Antifreeze proteins in the primary urine of larvae of the beetle <i>Dendroides canadensis</i> (Latreille)

Nickell, Philip K.; Sass, Sandra J.; Verleye, Dawn M.; Blumenthal, Edward M.; Duman, John G.

doi:10.1242/jeb.082461

Cited by 19 publications

(14 citation statements)

References 57 publications

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“…Freezing and melting points of the resulting samples were measured using a nanoliter osmometer (Nickell et al, 2013) to determine whether the samples displayed TH (melting point and freezing point differ), indicative of the presence of AFP and/or AFGL. Subsamples that exhibited TH were treated with proteomics-grade trypsin (porcine pancreas, Sigma, St Louis, MO, USA) according to the manufacturer's instructions.…”

Section: Screening and Isolation For Afgl And Nmr Spectroscopymentioning

confidence: 99%

Wood frog adaptations to overwintering in Alaska: New limits to freezing tolerance

Larson

Middle

et al. 2014

Journal of Experimental Biology

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We investigated the ecological physiology and behavior of free-living wood frogs [Lithobates (Rana) sylvaticus] overwintering in Interior Alaska by tracking animals into natural hibernacula, recording microclimate, and determining frog survival in spring. We measured cryoprotectant (glucose) concentrations and identified the presence of antifreeze glycolipids in tissues from subsamples of naturally freezing frogs. We also recorded the behavior of wood frogs preparing to freeze in artificial hibernacula, and tissue glucose concentrations in captive wood frogs frozen in the laboratory to −2.5°C. Wood frogs in natural hibernacula remained frozen for 193±11 consecutive days and experienced average (October-May) temperatures of −6.3°C and average minimum temperatures of -14.6±2.8°C (range −8.9 to −18.1°C) with 100% survival (N=18). Mean glucose concentrations were 13-fold higher in muscle, 10-fold higher in heart and 3.3-fold higher in liver in naturally freezing compared with laboratory frozen frogs. Antifreeze glycolipid was present in extracts from muscle and internal organs, but not skin, of frozen frogs. Wood frogs in Interior Alaska survive freezing to extreme limits and durations compared with those described in animals collected in southern Canada or the Midwestern United States. We hypothesize that this enhancement of freeze tolerance in Alaskan wood frogs is due to higher cryoprotectant levels that are produced by repeated freezing and thawing cycles experienced under natural conditions during early autumn.

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Section: Screening and Isolation For Afgl And Nmr Spectroscopymentioning

confidence: 99%

Wood frog adaptations to overwintering in Alaska: New limits to freezing tolerance

Larson

Middle

et al. 2014

Journal of Experimental Biology

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“…The overwintering larvae produce a family of some 30 AFP isomers (DAFPs) that are differentially expressed in various tissues and body fluids including hemolymph, gut, urine, and epidermal cells that function to prevent inoculative freezing and promote supercooling (26,27). The DAFPs consist of 12-and 13-mer repeating units containing highly conserved threonine and cysteine residues (28,29) that form a right-handed β-solenoid structure and a relatively flat ice-binding site on one side of the repeating β-strands (30).…”

mentioning

confidence: 99%

Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature

Wen

Wang

Duman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding insects in winter, and yet, paradoxically are found in some freeze-tolerant insects. Here, we report a previously unidentified role for AFPs in effectively inhibiting trehalose precipitation in the hemolymph (or blood) of overwintering beetle larvae. We determine the trehalose level (29.6 ± 0.6 mg/mL) in the larval hemolymph of a beetle, Dendroides canadensis, and demonstrate that the hemolymph AFPs are crucial for inhibiting trehalose crystallization, whereas the presence of trehalose also enhances the antifreeze activity of AFPs. To dissect the molecular mechanism, we examine the molecular recognition between AFP and trehalose crystal interfaces using molecular dynamics simulations. The theory corroborates the experiments and shows preferential strong binding of the AFP to the fast growing surfaces of the sugar crystal. This newly uncovered role for AFPs may help explain the long-speculated role of AFPs in freeze-tolerant species. We propose that the presence of high levels of molecules important for survival but prone to precipitation in poikilotherms (their body temperature can vary considerably) needs a companion mechanism to prevent the precipitation and here present, to our knowledge, the first example. Such a combination of trehalose and AFPs also provides a novel approach for cold protection and for trehalose crystallization inhibition in industrial applications.T rehalose is a multifunctional nonreducing disaccharide, occurring naturally in all kingdoms (1-4). In addition to being an energy and carbon source, this sugar protects cells and proteins against injuries in extreme environments (1, 2, 4), prevents osteoporosis (5), alleviates certain diseases (6), and acts as a signal molecule in plants (7). Due to its bioprotective properties, trehalose is a potentially useful protectant of cells and proteins in numerous applications (2,8). Its practical use, however, can be impaired by the fact that this sugar is prone to crystallization, in particular, when its aqueous solution is under fluctuating low temperatures. For example, the crystallization of trehalose dihydrate during the freeze-drying processes or from solutions at low temperatures would significantly jeopardize its ability to protect biomolecules (8-10). In comparison with other common sugars including sucrose, trehalose has a high propensity to crystallize at low temperature, forming trehalose dihydrate crystals. This propensity is due to: (i) the solubility of trehalose in water decreases dramatically or exponentially as temperature decreases (few data at...

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“…Some species also remove incidental INPs from hemolymph (Neven et al, 1986), but this is not always possible as these may be essential proteins. However, AFPs can bind to and inhibit ice nucleators and/or embryo crystals (Parody-Morreale et al, 1988;Olsen and Duman, 1997a,b;Nickell et al, 2013), thereby extending supercooling. Terrestrial organisms can also be inoculated by external ice across the body surface, but AFPs can also inhibit this (Olsen et al, 1998).…”

Section: Freeze Avoidancementioning

confidence: 99%

“…D. canadensis produces 30 known AFP isomers, and these exhibit tissue-specific expression Andorfer and Duman, 2000;Nickell et al, 2013), suggesting that their functions vary somewhat. The 12 initially described DAFP isomers were separated into three groups based on sequence variations (Andorfer and Duman, 2000).…”

Section: Dendroides Canadensis Beetlesmentioning

confidence: 99%

“…Combinations of certain groups I, II and III are produced in the single cell layer of epidermal cells underlying the cuticle, and are essential in inhibiting inoculative freezing (Olsen et al, 1998). Numerous isoforms are produced by the Malpighian tubule epithelium and secreted into the primary urine to inhibit ice-nucleating activity of various crystals that form in insect urine over the winter (Nickell et al, 2013). Recently, DAFPs were shown to control the size and shape of α-D-mannopyranoside crystals by binding directly to the crystals , suggesting that Malpighian tubule DAFPs could function to limit the size of non-ice crystals in urine.…”

Section: Dendroides Canadensis Beetlesmentioning

confidence: 99%

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Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function

Duman¹

2015

Journal of Experimental Biology

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136

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Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) icenucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those commonly experienced by the organism. Low thermal hysteresis IBPs are found in freeze-tolerant organisms (those able to survive extracellular freezing), and function to inhibit recrystallization -a potentially damaging process whereby larger ice crystals grow at the expense of smaller ones -and in some cases, prevent lethal propagation of extracellular ice into the cytoplasm. Ice-nucleator proteins inhibit supercooling and induce freezing in the extracellular fluid at high subzero temperatures in many freeze-tolerant species, thereby allowing them to control the location and temperature of ice nucleation, and the rate of ice growth. Numerous nuances to these functions have evolved. Antifreeze glycolipids with significant thermal hysteresis activity were recently identified in insects, frogs and plants.

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Antifreeze proteins in the primary urine of larvae of the beetle Dendroides canadensis (Latreille)

Cited by 19 publications

References 57 publications

Wood frog adaptations to overwintering in Alaska: New limits to freezing tolerance

Wood frog adaptations to overwintering in Alaska: New limits to freezing tolerance

Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature

Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function

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