Purification and primary sequences of the major arginine-containing antifreeze glycopeptides from the fish Eleginus gracilis

An Arg-containing antifreeze glycoprotein from the polar fish Eleginus gracilis was isolated, and the major components were purified to homogeneity. The general protocol for purification was chromatography of serum on DEAE-cellulose, followed by chromatography on a cation exchanger. DEAE-cellulose chromatography resulted in two fractions, A and B. Fraction A contained most of the antifreeze glycoprotein found in E. gracilis (approximately 80% by weight) and consisted of 13 distinct components. Unlike antifreeze glycoproteins from other previously studied polar fish, Fraction A contained both low and high molecular weight antifreeze glycoprotein components. The two major components of Fraction A were sequenced and compared with the sequence of antifreeze glycoproteins 7 and 8 from both Boreogadus saida and Pagothenia borchgrevinki. The antifreeze glycoproteins from E. gracilis were shown to have a similar composition to those previously studied, except for an additional Ala-Arg dipeptide at the carbon terminal in the major components of Fraction A and the position of Pro in the low molecular weight components. The activity of E. gracilis antifreeze glycoproteins is the subject of a companion article (Burcham, T. S., Osuga, D. T., Yeh, Y., and Feeney, R. E. (1986) J. Biol. Chem. 261, 6390-6397).     

Type I antifreeze proteins expressed in snailfish skin are identical to their plasma counterparts

Type I antifreeze proteins (AFPs) are usually small, Ala-rich alpha-helical polypeptides found in right-eyed flounders and certain species of sculpin. These proteins are divided into two distinct subclasses, liver type and skin type, which are encoded by separate gene families. Blood plasma from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish contain type I AFPs that are significantly larger than all previously described type I AFPs. In this study, full-length cDNA clones that encode snailfish type I AFPs expressed in skin tissues were generated using a combination of library screening and PCR-based methods. The skin clones, which lack both signal and pro-sequences, produce proteins that are identical to circulating plasma AFPs. Although all fish examined consistently express antifreeze mRNA in skin tissue, there is extreme individual variation in liver expression - an unusual phenomenon that has never been reported previously. Furthermore, genomic Southern blot analysis revealed that snailfish AFPs are products of multigene families that consist of up to 10 gene copies per genome. The 113-residue snailfish AFPs do not contain any obvious amino acid repeats or continuous hydrophobic face which typify the structure of most other type I AFPs. These structural differences might have implications for their ice-crystal binding properties. These results are the first to demonstrate a dual liver/skin role of identical type I AFP expression which may represent an evolutionary intermediate prior to divergence into distinct gene families.     

Antifreeze proteins differentially affect model membranes during freezing

Over the past decade antifreeze proteins from polar fish have been shown either to stabilize or disrupt membrane structure during low temperature and freezing stress. However, there has been no systematic study on how membrane composition affects the interaction of antifreeze proteins with membranes under stress conditions. Therefore, it is not possible at present to predict which antifreeze proteins will protect, and which will damage a particular membrane during chilling or freezing. Here, we analyze the effects of freezing on spinach thylakoid membranes and on model membranes of varying lipid composition in the presence of antifreeze protein type I (AFP I) and specific fractions of antifreeze glycoproteins (AFGP). We find that the addition of galactolipids to phospholipid model membranes changes the effect each protein has on the membrane during freezing. However, the greatest differences observed in this study are between the different types of antifreeze proteins. We find that AFP type I and the largest molecular weight fractions of AFGP induce concentration dependent leakage from, and are fusogenic to the liposomes. This is the first report that an antifreeze protein induces membrane fusion. In contrast, the smallest fraction of AFGP offers a limited degree of protection during freezing and does not induce membrane fusion at concentrations up to 10 mg/ml.     

Antifreeze glycoproteins from an Antarctic fish. Quasi-elastic light scattering studies of the hydrodynamic conformations of antifreeze glycoproteins.

A quasi-elastic light-scattering technique was used to study the hydrodynamic conformations of antifreeze glycoproteins from an Antarctic fish. Antifreeze glycoprotein is composed of repeating units of Ala-Ala-Thr, with each threonine O-linked to a disaccharide, and it exists as several polymers of different numbers of this repeating unit. Molecular weights of the two major active polymers are 10,500 and 17,500 by such methods as centrifugation and osmotic pressure, but smaller than 20 by freezing-point depression. Translational diffusion coefficients at 20 degrees were 8.35 times 10-7 cm2 s-1 and 6.15 times 10-7 cm2 s-1 for the M-r-10,500 and 17,500 polymers, respectively. Measurements at -0.2 degrees in the presence of ice crystals did not indicate any conformational changes that might be related to the lowering of the freezing temperature. Lowering the temperature of these glycoprotein solutions close to temperatures of freezing caused a decrease in the effective hydrodynamic radius of both active and inactive glycoprotein components.     

Biosynthesis of the Sindbis Virus Carbohydrates

The sequence in which sugars are added to the Sindbis virus glycoproteins was studied. Infected cells contain three glycosylated virus-specific proteins: the two virion glycoproteins and the immediate precursor to the smaller virion glycoprotein. Larger Sindbis-specific proteins are not glycosylated. The cell-associated forms of both of the virion glycoproteins contain glucosamine, mannose, galactose, and fucose. The glycosylated precursor contains only glucosamine, mannose, and some galactose. The conversion of precursor to virion protein involves both the addition of galactose and fucose and the loss of mannose. The apparent extent of glycosylation of each virus-specific protein is not influenced by the host cell.     

Functional importance of short-range binding and long-range solvent interactions in helical antifreeze peptides

Short-range ice binding and long-range solvent perturbation both have been implicated in the activity of antifreeze proteins and antifreeze glycoproteins. We study these two mechanisms for activity of winter flounder antifreeze peptide. Four mutants are characterized by freezing point hysteresis (activity), circular dichroism (secondary structure), Förster resonance energy transfer (end-to-end rigidity), molecular dynamics simulation (structure), and terahertz spectroscopy (long-range solvent perturbation). Our results show that the short-range model is sufficient to explain the activity of our mutants, but the long-range model provides a necessary condition for activity: the most active peptides in our data set all have an extended dynamical hydration shell. It appears that antifreeze proteins and antifreeze glycoproteins have reached different evolutionary solutions to the antifreeze problem, utilizing either a few precisely positioned OH groups or a large quantity of OH groups for ice binding, assisted by long-range solvent perturbation.     

Use of specific monoclonal and polyclonal antibodies to define distinct antigens of the porcine zonae pellucidae

Specific monoclonal and polyclonal antibodies to solubilized porcine and rabbit zonae pellucidae (ZP) and to purified ZP glycoprotein components have been used to define distinct ZP antigens. These studies demonstrate that the individual ZP glycoproteins contain both unique and shared determinants. One monoclonal antibody (R5) has been used to demonstrate that the major porcine ZP glycoprotein, which has multiple charge species ranging in molecular weight from 42,000 to 120,000, is composed of two distinct polypeptide antigens unique to this glycoprotein class. These distinct antigens can be differentiated by immunoblotting after high-resolution two-dimensional polyacrylamide gel electrophoretic separation of trypsin-treated or deglycosylated glycoproteins. The two polypeptides also differ in their staining properties with the silver-based color stain and in their susceptibility to proteolysis. A second monoclonal antibody (PSI) has been used to define a determinant shared by all three major porcine ZP glycoprotein classes. This determinant appears to involve either a carbohydrate moiety or some other molecular feature related to post-translational modification, since the antibody recognizes only the acidic species of each glycoprotein class, and does not recognize the deglycosylated forms of the proteins. This work demonstrates that there are both unique and shared antigenic determinants present in the individual components of the ZP, but that the immunodominant determinants appear to be unique to each glycoprotein.     

A kinetic description of antifreeze glycoprotein activity

The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.     

Antifreeze glycoproteins: influence of polymer length and ice crystal habit on activity

The effect of the ice crystalline habit and the length of the polymer on the ability of the antifreeze glycoproteins (AFGP) from polar fish to depress the freezing temperature of water was investigated. The low-molecular-weight components of the glycoproteins, AFGP- 6–8, are inactive when a solution of such a sample is nucleated at ?6°C. A solution of large AFGP (1–4) is fully functional under the same conditions. The low-molecular-weight components differ from the height-molecular-weight components in that they contain some proline replacing the alanine in the Ala-Ala-Thr · disaccharide polymer unit. In the present experiments, antifreeze activity was examined in the presence of two different forms of ice crystal growth habits, and homodimders of AFGP 6 and 8 were prepared to investigate the function of polymer length and the on antifreeze activity at different degrees of supercooling. The results indicate that the ice crystal growth habit and the introduction of proline into the polymer unit may be responsible for the loss of activity at deep supercooling (?6°C) of AFGP 6–8. The loss in the ability of AFGP to depress the freezing temperature of water at deep supercooling is not solely due to polymer length, as carbodiimide-linked dimers of AFGP 6 do not function under these freezing conditions. A Model of antifreezing action based on Langmuirian adsorption of AFGP on the ice surface and direct competition between water and AFGP molecules for the incorporation sites in the ice crystal lattice is presented.     

Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris

Antifreeze proteins and antifreeze glycoproteins are structurally diverse molecules that share a common property in binding to ice crystals and inhibiting ice crystal growth. Type II fish antifreeze protein of Atlantic herring (Clupea harengus harengus) is unique in its requirement of Ca(2+) for antifreeze activity. In this study, we utilized the secretion vector pGAPZalpha A to express recombinant herring antifreeze protein (WT) and a fusion protein with a C-terminal six-histidine tag (WT-6H) in yeast Pichia pastoris wild-type strain X-33 or protease-deficient strain SMD1168H. Both recombinant proteins were secreted into the culture medium and properly folded and functioned as the native herring antifreeze protein. Furthermore, our studies demonstrated that expression at a lower temperature increased the yield of the recombinant protein dramatically, which might be due to the enhanced protein folding pathway, as well as increased cell viability at lower temperature. These data suggested that P. pastoris is a useful system for the production of soluble and biologically active herring antifreeze protein required for structural and functional studies.     

Structural and functional similarity between fish antifreeze proteins and calcium-dependent lectins.

A cDNA for a type II antifreeze protein was isolated from liver of smelt (Osmerus mordax). The predicted protein sequence is homologous to that from sea raven (Hemitripterus americanus) and both show homology to a family of calcium-dependent lectins. Smelt and sea raven belong to taxonomic orders believed to have diverged prior to Cenozoic glaciation. Thus, type II antifreeze proteins appear to have evolved independently in these fish species from pre-existing calcium-dependent lectins. Sequence alignment of the antifreezes and the lectins suggest that these proteins adopt a similar fold, that the sea raven antifreeze has lost its Ca2+ binding sites, and the smelt antifreeze has retained one site. Experiments show that smelt antifreeze protein activity is responsive to Ca2+ but that of sea raven antifreeze protein is not. These results suggest that the type II fish antifreeze proteins and calcium-dependent lectins share a common ancestry, related folding structures, and functional similarity.     

A comparison of membrane components of normal and transformed BALB/c cells.

Membrane glycolipids, glycoproteins, and surface proteins of normal and transformed BALB/c cell lines have been compared. Several virally and spontaneously transformed cell lines showed differences in membrane components compared to normal A31 cells. These differences consisted of increased amounts of simpler gangliosides, absence of the large external transformation sensitive (LETS) protein, and the appearance of a major new glycoprotein band of about 105 000 molecular weight. In contrast, the spontaneously transformed cell line that caused the fastest growing tumors in vivo and the most rapid animal death (3T12T) did not have these changes. A31 and 3T12T glycolipid profiles appear similar as did glycoproteins and cell surface proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When Pronase-generated glycopeptides were analyzed by Sephadex G-50 chromatography, and enrichment in faster-eluting species was seen in two killing tumor lines (c5T and 3T12T) compared to A31. Regressing tumor lines (MSC, c5) did not show this change. Isolated membrane glycoproteins yield glycopeptides of different sized after Pronase digestion. In addition, several 3T12T glycoproteins yield glycopeptides that are larger than those from the corresponding glycoproteins of A31 cells. It appears that glycopeptide alterations associated with transformation occur in several membrane glycoproteins.     

Calorimetric analysis of antifreeze glycoproteins of the polar fish, Dissostichus mawsoni

Solutions of antifreeze glycoproteins 1 through 5 and 8 were analyzed for activity by differential scanning calorimetry. With a scan rate of 1 degree C min-1, antifreeze glycoproteins 1-5 (20 mg/ml) revealed antifreeze activity with a delay in the freeze exotherm during cooling in the presence of ice. Antifreeze glycoprotein 8 (60 mg/ml), however, did not reveal antifreeze activity. When a 0.1 degree C min-1 scan rate was used, glycoproteins 1-5 again yielded a delay in the freeze onset, but the exotherm consisted of multiple events. At the slower scan glycoprotein 8 revealed an initial freeze followed by multiple exothermic events resembling those of glycoproteins 1-5. Thermograms exhibiting antifreeze activity had an initial shoulder in the exotherm direction upon cooling followed by a delay before the exotherm. The shoulders were correlated with c-axis ice growth observed in visual methods. The glycoprotein antifreezes had a linear increase in activity with decreased ice content.     

Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from Streptomyces griseus, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.     

Metal detoxification and homeostasis in Antarctic Notothenioids. A comparative survey on evolution, expression and functional properties of fish and mammal metallothioneins

Organisms from yeast to mammals contain cysteine-rich, heavy metal binding proteins termed metallothioneins. The putative roles of these proteins are trace metal homeostasis and detoxification of poisonous heavy metals. The highly conserved chemical composition and the structural constraints led to the conclusion that metallothioneins of different origin must display remarkably similar features. The present review aims at surveying the studies carried out on the metallothioneins of Antarctic Notothenioidei, a dominating fish group endowed of a number of striking adaptive characters, including reduced (or absent) hematocrit and presence of antifreeze glycoproteins. Given the unique peculiarities of the Antarctic environment, a comparative study of the features of notothenioid metallothioneins could provide new insights into the role of these proteins in physiology and toxicology. The results summarized here show that the metallothioneins of this fish group display a number of features at the level of evolution, expression pattern, structure and function remarkably different from those of mammal metallothioneins.     

Identification of glycoproteins associated with HIV latently infected cells using quantitative glycoproteomics

HIV infection is not curable due to viral latency. Compelling reports suggest that there is a distinct profile of surface proteins that can be used for targeting latently infected cells. We have recently reported that glycoproteins were differentially secreted from HIV latently infected ACH‐2 cells compared to the parental A3.01 cells. This finding suggests that glyco‐phenotype might be different in these two cell lines. To determine the difference, the ACH‐2 and A3.01 cell lines were subjected to a glycoproteomic analysis. A total number of 940 unique N‐linked glycosite‐containing peptides from 515 glycoproteins were identified. Among the glycoproteins, 365 and 104 were annotated as cell surface and membrane‐associated proteins, respectively. Quantitative LC‐MS/MS analysis revealed a change of 236 glycosite‐containing peptides from 172 glycoproteins between the two cell lines without reactivation. Bioinformatic analysis suggests that cell adhesion, immune response, glycoprotein metabolic process, cell motion, and cell activation were associated with the changed proteins. After reactivation of latency, changes in glycosite‐containing peptides were observed in both cell lines. The changed proteins suggest that cell migration, response to wounding and immune response might be impaired in reactivated latently infected cells. Glycoproteomics merits future application using primary cells to discover reveal mechanisms in HIV pathogenesis.     

Antifreeze protein-induced morphological modification mechanisms linked to ice binding surface

The mechanisms by which the antifreeze protein (AFP) modifies the ice morphology are identified precisely as surface poisoning by the ice binding surface (IBS) of insect AFPs and as bridge-induced surface reconstruction by the IBS of fish AFPs and antifreeze glycoproteins. The primary surfaces of hexagonal ice have predetermined face indices. The "two-dimensional" insect type IBS has regularly spaced binding intervals in two directions. It causes surface poisoning by matching and reinforcing simultaneously intersecting strong bonding directions on the primary ice surfaces. The secondary ice surfaces have variable face indices. The "one-dimensional" and "irregular" IBS variants of fish AFPs and antifreeze glycoproteins are either linearly extended with regular ice binding intervals or have ice binding sites lacking spacing regularity. These variants can bridge transversely lattice periods or shorter oxygen-oxygen distances between parallel adjacent strong bonding directions that do not intersect. Thus, one-dimensional and irregular IBS variants induce supplementary bridges cross-wise on selected secondary surfaces by mimicking strong bonding directions that are not present in the ice structure. These proteins cause surfaces with variable face indices, which in the absence of the AFPs would not grow flat, to appear in the morphology. Whereas for the primary ice surfaces it is only the morphological importance that is determined by the experimental conditions, for the secondary ice surfaces it is the face indices themselves that become adjusted in the process of maximizing the AFP-substrate interaction through attainment of the best structural match. The growth morphology of the AFP-ice system is derived from various factors, including the face indices, surface molecular compositions, relative growth rates, and the mechanisms responsible for that morphology. The theoretical formulation agrees with experiments over a wide range and resolves these, to date, unexplained phenomena.     

Use of carbohydrate probes in conjunction with fluorescence-activated cell sorting to select mutant cell lines of Chlamydomonas with defects in cell surface glycoproteins

Two carbohydrate-binding probes (the lectin concanavalin A and the anti-carbohydrate monoclonal antibody FMG-1) have been utilized in conjunction with fluorescence-activated cell sorting to select cell lines of Chlamydomonas reinhardtii that contain defects in cell surface-exposed glycoproteins. Two very different selection strategies (sorting cells with the lowest binding for the FMG-1 monoclonal antibody or the highest binding of concanavalin A) yield a class of mutant cells that exhibit a total lack of binding of the monoclonal antibody to cell wall and plasma membrane glycoproteins along with an increased affinity for concanavalin A. Detailed characterization of one such mutant cell line, designated L-23, is provided. The subtle glycosylation defect exhibited by this cell line does not alter the ability of the affected glycoproteins to be targeted to the flagellar membrane and does not affect the expression of flagellar surface motility, a phenomenon that appears to involve the major concanavalin A-binding glycoprotein of the flagellar membrane. This approach has general applicability for dissecting the role of carbohydrate epitopes in the targeting and function of any cell surface glycoprotein for which suitable carbohydrate probes are available.     

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.     

Identification of glycoproteins in goblet cells of epidermis and gill of plaice (Pleuronectes platessa L.), flounder (Platichthys flesus (L.)) and rainbow trout (Salmo gairdneri Richardson)

Synopsis A quantitative analysis has been made of the glycoproteins present in the goblet cells of the epidermis, gill filaments and gill lamellae of three species of teleost fish. The glycoproteins have been identified by a combination of techniques, including the use of the enzyme sialidase followed by Alcian Blue staining, at pH 2.6 or I. o, in combination with periodic acid-Schiff. The selected fish were representative of species living in marine, freshwater and estuarine environments.The range of glycoproteins identified in these fish was similar to that found in mammalian tissue in that both neutral and acid glycoproteins were present, the latter included both sialomucins sensitive and resistant to sialidase, and sulphomucin. A single goblet cell contained either neutral or acid glycoproteins alone or in combination. Only the epidermis of the plaice and rainbow trout contained uniform cell populations producing acid glycoproteins, the former sulphomucin and the latter mainly sialomucin. At each site in the flounder and in the gill epithelia of the plaice and rainbow trout, the goblet cell population was mixed, with cells producing each type of glycoprotein. The number of goblet cells producing each type of glycoprotein varied at each tissue site.