Identification and characterization of parvalbumin-like protein in Trichophyton violaceum

Parvalbumins play crucial physiological roles in neuromuscular systems of vertebrates, such as cell-cycle, development of neurons, contraction of muscles, and regulation of intracellular calcium. To perform these neuromuscular functions, parvalbumin may be in associated with other proteins including calbindin, carbonic anhydrase, and cytochrome oxidase. Humans may show an IgE-specific hypersensitivity to parvalbumins after consumption of some distinct fish species. While this protein is abundant in fish muscles, literature review of publications related to fish parvalbumins, do not point to the presence of parvalbumins in eukaryotic microbes. In this study, we propose that distantly related parvalbumins may be found in some non-fish species. Bioinformatics studies such as multiple sequence alignment (MSA), phylogenetic analysis as well as molecular-based experiments indicate that, at least two parvalbumins sequences (UniProt IDs: A0A178F775 and A0A178F7E4) with EF-hand domains and Ca²⁺-binding sites could be identified in Trichophyton violaceum, a pathogenic fungal species.It was determined that both genes consisted of a single exon and encoded for parvalbumin proteins possessing conserved amino acid motifs. Antigenicity prediction revealed antigenic sites located in both sides of the Ca²⁺-binding site of the first EF-hand domain. Our phylogenetic analysis revealed that one of parvalbumins (UniProt ID: 0A178F775) can be evolved to other parvalbumins in T. violaceum (UniProt ID: A0A178F7E4) and fish species through evolutionary phenomenon. To confirm our in-silico findings, we designed three primer pairs to detect one of the T. violaceum parvalbumins (UniProt ID: A0A178F7E4) by polymerase chain reaction (PCR); one primer pair showed a strong and specific band in agarose gel electrophoresis. To evaluate the specificity of the method, the primers were tested on extracted DNA from Trichophyton rubrum and T. mentagrophytes. The results demonstrated that the evaluated parvalbumin gene (UniProt ID: A0A178F7E4) was T. violaceum-specific and this pathogenic fungus can be differentiated from T. rubrum and T. mentagrophytes through identification of parvalbumin genes. Further studies are necessary to unravel the biochemical and physiological functions of parvalbumins in T. violaceum.     

Polymorphism of parvalbumins and tissue distribution: characterization of component I, isolated from red muscles of Cyprinus carpio L.

The component I isolated from carp red muscle has been characterized as a true parvalbumin, fairly different from carp parvalbumins described so far. The protein is antigenically related to the parvalbumin III from pike, which belongs to the so called parvalbumin lineage alpha. Immunological investigations on the location of the various carp parvalbumins reveal genuine variation in the pattern of these proteins according to organ and type of muscular tissue.     

Amino acid sequences of lower vertebrate parvalbumins and their evolution: parvalbumins of boa, turtle, and salamander

One major parvalbumin each was isolated from the skeletal muscle of two reptiles, a boa snake, Boa constrictor, and a map turtle, Graptemys geographica, while two parvalbumins were isolated from an amphibian, the salamander Amphiuma means. The amino acid sequences of all four parvalbumins were determined from the sequences of their tryptic peptides, which were ordered partially by homology to other parvalbumins. Phylogenetic study of these and 16 other parvalbumin sequences revealed that the turtle parvalbumin belongs to beta lineage, while the salamander sequences belong, one each, to the alpha and beta lineages defined by Goodman and Pechere (1977). Boa parvalbumin, however, while belonging to the beta lineage, clusters within the fish in all reasonably parsimonious trees. The most parsimonious trees show many parallel or back mutations in the evolution of many parvalbumin residues, although the residues responsible for Ca2+ binding are very well conserved. These most parsimonious trees show an actinopterygian rather than a crossoptyrigian origin of the tetrapods in both the alpha and beta groups. One of two electric eel parvalbumins is evolving more than 10 times faster than its paralogous partner, suggesting it may be on its way to becoming a pseudogene. It is concluded that varying rates of amino acid replacement, much homoplasy, considerable gene duplication, plus complicated lineages make the set of parvalbumin sequences unsuitable for systematic study of the origin of the tetrapods and other higher-taxa divergence, although it may be suitable within a genus or family.      

Parvalbumin isoforms in chicken muscle and thymus. Amino acid sequence analysis of muscle parvalbumin by tandem mass spectrometry

Parvalbumins are high-affinity Ca(2+)-binding proteins characterized by an EF-hand structure. Muscles of lower vertebrates contain up to five isoparvalbumins whereas higher vertebrates were believed to contain only one isoform per species. Recently Brewer et al. [Brewer, J.M., Wunderlich, J.K., & Ragland, W. (1990) Biochimie 72, 653-660] purified and sequenced a protein that they named avian thymic hormone, from chicken thymus. This protein, promoting immunological maturation of bone marrow cells in culture, was identified as a parvalbumin. The amino acid composition of this thymic parvalbumin was, however, considerably different from those of chicken muscle parvalbumin [Strehler, E.E., Eppenberger, H.M., & Heizman, C.W. (1977) FEBS Lett. 75, 127-133], suggesting the existence of two tissue-specific parvalbumins in chicken. We purified parvalbumin from chicken muscle, determined its complete amino acid sequence by tandem mass spectrometry, and showed that this protein is rather homologous to muscle parvalbumins from other species but different in 45 positions from the thymic parvalbumin. We discuss the possibility that a parvalbumin gene family might exist in higher vertebrates, expressed in a tissue-specific and developmentally regulated manner.     

Identification and quantification in single muscle fibers of four isoforms of parvalbumin in the iliofibularis muscle of Xenopus laevis

The major parvalbumins present in the iliofibularis muscle of Xenopus laevis were identified and the total parvalbumin content of different types of single fibers of this muscle was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS). The criteria used in the identification of proteins as parvalbumins were: a relative molecular mass (Mr) between 10,000 and 14,000, an isoelectric point (pI) between 4.0 and 5.0, and a Ca2+-dependent mobility when run on a polyacrylamide gel in the absence of SDS. Four proteins were thus identified as parvalbumins: PA1, Mr 14,000, pI 4.90; PA2, Mr 11,000, pI 4.90; PA3, Mr 11,000, pI 4.95; and PA4, Mr 11,000, pI 4.25. An ultraviolet absorbance spectrum characteristic of parvalbumins was recorded for a purified preparation of these four proteins. Because the apparent Mr of rabbit parvalbumin in the gel system used was 14,000, whereas the true value is 12,100, it is not excluded that the Mr of component PA1 of 14,000 is an overestimation. The total parvalbumin content of muscles and single muscle fibers was determined using the supernatant obtained after centrifugation of tissue homogenates. Analysis of the protein pattern after electrophoresis in the presence of SDS of this fraction indicated that the Mr 14,000 and 11,000 protein bands contained virtually only parvalbumin. Quantification of the total parvalbumin content of relatively fast (type 1) and slow (type 2) contracting and relaxing single muscle fibers, using laser densitometric analysis of minigels, yielded mean values (mg protein/g wet wt., +/- S.D.) of 5.2 +/- 0.8 for nine type 1 fibers, and 1.9 +/- 1.0 for five type 2 fibers. Both fiber types contained about 2.5-times as much of the Mr 14,000 isoform relative to the combined Mr 11,000 isoforms.     

Isolation and characterization of a protein having lipoic acid as prosthetic group from Ascaris muscle microsomes

A protein which had lipoic acid as a prosthetic group was found in $\text{Ascaris}$ muscle microsomes. The protein was solubilized with 0.1% trypsin, and purified by heat-treatment and column chromatographys, and showed a single band on sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, having a molecular weight of about 14,000. The protein was heat stable and capable of oxidizing reduced microsomal lipoamide dehydrogenase in $\text{Ascaris}$ muscle.     

Comparison of the amino acid sequences of tissue-specific parvalbumins from chicken muscle and thymus and possible evolutionary significance

Chicken leg muscle parvalbumin was digested with cyanogen bromide or trypsin or trypsin after citraconylation. Peptides isolated by reverse phase HPLC at pH 7.0 were subjected to acid hydrolysis and amino acid analysis and, in some cases, sequencing. The chicken muscle parvalbumin amino acid sequence has ca. 80% sequence identity with alpha-type parvalbumins from mammalian (rabbit, human and rat) muscle. By contrast, the chicken thymus parvalbumin ("avian thymic hormone") sequence is very similar to reptile (turtle, salamander and frog) muscle beta-type parvalbumins. We hypothesize that the evolutionary appearance of the warm-blooded reptiles was accompanied by recruitment of the beta parvalbumin isozyme for promotion of lymphocyte maturation.     

Parvalbumin in Cat Brain: Isolation, Characterization, and Localization

Because of the increasing evidence that Ca2+-binding proteins have important regulating functions in nerve cells and because of the indications that there are species differences in the structures of these proteins, parvalbumin was purified from cat brain and muscle. Brain and muscle parvalbumins were found to be indistinguishable from each other in their biochemical and immunological properties. However, cat parvalbumin differs from all other mammalian parvalbumins by its apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 10-11K (compared to rat parvalbumin, 12K), and a lower pI of 4.6 (rat parvalbumin, 4.9), in the tryptic peptide maps, and in the immunological properties, indicating a distinct primary structure. With the purified parvalbumin as antigen, polyclonal antibodies were raised in rabbits and these were subsequently used for immunohistochemical localizations of parvalbumin in the cat brain. In the visual cortices of adult cats immunoreactive neurons were present throughout layers II and IV. In cerebellar cortex, Purkinje, basket, and stellate cells were immunoreactive. Comparison with staining patterns obtained with antiserum against rat parvalbumin revealed some cross-reactivity but confirmed the existence of species differences in the antigenic structure of rat and cat parvalbumin.     

A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens,Parvalbumins, and Identification of a Major IgE-Binding Epitope

Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients’ sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. ¹H/¹⁵N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.     

Crystallization and preliminary X-ray data for parvalbumin IIIf of Opsanus tau

The parvalbumin III_f of the superfast swimbladder muscle of Opsanus tau has been crystallized by a vapor diffusion technique using (NH₄)₂SO₄ as a precipitant agent. The crystals belong to space group P2₁, with a unit cell $\text{a = 27.31}\text{A}\text{?}\text{, b = 58.75}\text{A}\text{?}\text{, c = 30.83}\text{A}\text{?}$ and β = 98 ° 30′, and the asymmetric unit contains one molecule. These crystals are suitable for a high resolution crystallographic investigation.     

Isopycnic-zonal centrifugation of plasma membrane,sarcoplasmic reticular fragments,lysosomes, and cytoplasmic proteins from phasic skeletal muscle

Homogenates of the posterior latissimus dorsi muscle, a phasic muscle, were fractionated by a one-step zonal centrifugation technique into four major organelle populations and cytoplasmic constituents. These were: (1) Plasma membrane fragments with a modal equilibrium density of 1.10 and containing 5′-nucleotidase, alkaline phosphodiesterase, $\text{p-}\text{nitrophenylphosphatase}$ and acid phosphatase (β-glycerophosphate was used as the substrate). (2) Sarcoplasmic reticular fragments which could be further subdivided into calcium transport vesicles, with a modal equilibrium density of 1.16, that exhibited calcium uptake; K⁺-ATPase; leucyl-β-naphthylamidase; acid phosphodiesterase; acid phosphatase (using cytidine monophosphate as the substrate); and sarcoplasmic reticular lysosomes, with a modal equilibrium density of 1.18, possessing dipeptidyl-aminopeptidase II, cathepsin D, α-glucosidase, $\text{N-}\text{acetyl}\text{-β-}\text{glucosaminidase}$, and NADH oxidase activity. (3) Mitochondria with a modal equilibrium density of 1.21. (4) Catalase-containing vesicles with a modal equilibrium density of 1.22; and cytoplasmic constituents (modal density of 1.25) with phosphorylase, pyruvate kinase, myosin-ATPase, aldolase, and protein and RNA content. The purity of these organelles was equal to or better than previous efforts, with a 30-fold purification achieved for 5′-nucleotidase and alkaline phosphodiesterase. These results lend support to the hypothesis that the sarcoplasmic reticulum of phasic muscle, in addition to its specialized role in excitation-contraction coupling, represents a multifunctional membrane system, and that, similar to the smooth endoplasmic reticulum of other cells, it includes some membrane-bound lysosomal enzymes and NADH oxidase.     

A cyclic AMP dependent protein kinase in Dictyostelium discoideum

A cyclic AMP-dependent protein kinase was found to appear during the time course of development of $\text{Dictyostelium}\text{discoideum}$. No cyclic AMP dependency was observed at any stage of development in crude 110,000 X G soluble extracts. After partial purification, however, extracts from post-aggregation stages contained enzyme that was activated up to 6-fold by cyclic AMP, whereas protein kinase from earlier stages was not affected by cyclic AMP. Likewise, cyclic AMP binding activity increased from the aggregation to the slug stage of development. Approximately one-half of the total cyclic AMP binding activity co-purified with the cyclic AMP dependent protein kinase. The enzyme from $\text{Dictyostelium}$ showed similarities to mammalian protein kinases with respect to its kinetic properties but differed in its behavior on ion-exchange chromatography.     

Identification and characterization of new protein chemoattractants in the frog skin secretome

The vomeronasal organ is a chemosensory organ present in most vertebrates and involved in chemical communication. In the last decade, the deciphering of the signal transduction process of this organ has progressed. However, less is known about the vomeronasal organ ligands and their structure-function relationships. Snakes possess a highly developed vomeronasal system that is used in various behaviors such as mating, predator detection, or prey selection, making this group a suitable model for study of the vomeronasal chemoreception. In this work, we used a proteomics approach to identify and characterize proteins from frog cutaneous mucus proteome involved in prey recognition by snakes of the genus Thamnophis. Herein we report the purification and characterization of two proteins isolated from the frog skin secretome that elicit the vomeronasal organ-mediated predatory behavior of Thamnophis marcianus. These proteins are members of the parvalbumin family, which are calcium-binding proteins generally associated to muscular and nervous tissues. This is the first report that demonstrates parvalbumins are not strictly restricted to intracellular compartments and can also be isolated from exocrine secretions. Purified parvalbumins from frog muscle and mucus revealed identical chemoattractive properties for T. marcianus. Snake bioassay revealed the Ca(2+)/Mg(2+) dependence of the bioactivity of parvalbumins. So parvalbumins appear to be new candidate ligands of the vomeronasal organ.     

Rapid reduction in parvalbumin concentration during chronic stimulation of rabbit fast twitch muscle

Chronic indirect stimulation of fast twitch rabbit muscle induced a rapid reduction in parvalbumin concentration. When compared to the unstimulated contralateral muscle, parvalbumins were reduced to 55% following 6 days of stimulation. Prolonged stimulation further reduced parvalbumins so that they were undetectable after 28 days. The time course of these changes appears to be related with the previously observed changes in the sarcoplasmic reticulum.     

Purification of thiamine-binding protein from Escherichia coli

An affinity column coupled with thiamine pyrophosphate quantitatively absorbs the thiamine-binding activity from a partially purified preparation of $\text{Escherichia}\text{coli}$. The thiamine-binding protein can be eluted from the affinity column in high yield by use of 8 M urea-containing buffer. Approximately 90-fold purification occurs by affinity chromatography yielding a preparation which appears to be homogenous.     

Preparation and characterization of the major isotype of parvalbumin from skeletal muscle of the toad (Bufo bufo japonicus)

The major isotype of parvalbumin has been isolated from the skeletal muscle of the toad, Bufo bufo japonicus. Unlike the skeletal muscle of every frog so far examined (Rana esculenta, Rana temporaria, and Rana catesbeiana), which contains two major isotypes of parvalbumins, toad skeletal muscle has been shown to contain only one isotype, but the content of parvalbumin in toad skeletal muscle was similar to the sum of those of the two isotypes in skeletal muscles of frogs. This feature of toad skeletal muscle is advantageous to clarify the physiological role of parvalbumin. The relative molecular mass of toad parvalbumin was estimated to be 12,200 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to be 4.81 by polyacrylamide gel isoelectric focusing. The amino acid composition indicated that toad parvalbumin corresponds to bullfrog (R. catesbeiana) pI 4.97 parvalbumin, showing that toad parvalbumin is genetically an alpha-parvalbumin. It was also revealed by the amino acid composition that toad parvalbumin is distinctly different from any of the parvalbumins from frogs. The ultraviolet spectrum of toad parvalbumin is consistent with its amino acid composition. The ultraviolet difference spectrum of the Ca2+-loaded form vs. the metal-free form indicates that some Phe residues in the toad parvalbumin molecule are affected by a conformational change associated with Ca2+ binding. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the metal-free and Mg2+-loaded forms of toad parvalbumin migrated twice as fast as the Ca2+-loaded form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

Relationship of the reduction-oxidation state to protein degradation in skeletal and atrial muscle

Changes in proteolysis were correlated with the cell reduction-oxidation state in rat diaphragm and atrium. Protein degradation was measured in the presence of cycloheximide as the linear release of tyrosine into the medium. Intracellular ratios of lactate/pyruvate, total $\text{NADH}\text{NAD}$, and malate/pyruvate were used as indicators of the muscle reduction-oxidation state. Incubation of diaphragms with leucine (0.5–2.0 mm) or its transamination product, sodium α-ketoisocaproate (0.5 mm), resulted in a lower rate of proteolysis and a higher ratio of lactate/pyruvate and $\text{NADH}\text{NAD}$. These effects of leucine could be abolished by inhibiting its transamination with l-cycloserine. Unlike leucine, neither isoleucine nor valine alone produced any change in these parameters. Incubation of diaphragms with glucose (20 mm) or atria with sodium lactate (2 mm) produced a diminution of tyrosine release from the muscles and a rise in the ratio of total $\text{NADH}\text{NAD}$. Similarly, in incubated diaphragms of fasted rats, the anabolic effects of insulin, epinephrine and isoproterenol on protein degradation were associated with a higher malate/pyruvate ratio. In catabolic states, such as fasting, cortisol treatment of fasted, adrenalectomized rats or traumatization, enhanced muscle proteolysis was observed. Fresh-frozen diaphragms from these rats had both lower lactate/pyruvate and malate/pyruvate ratios than did muscles from control animals. These data show that diminution of proteolysis in diaphragm is accompanied by an increase of the $\text{NAD(P)H}\text{NAD(P)}$ ratios. In contrast to these findings, chymostatin and leupeptin, which inhibit directly muscle proteinases, caused a decrease of the lactate/pyruvate and malate/pyruvate ratios. These results suggest that protein degradation in diaphragm and atrium is linked to the cellular redox state.     

Functional characterization of parvalbumin from the Arctic cod (Boreogadus saida): similarity in calcium affinity among parvalbumins from polar teleosts

Calcium dissociation constants (KD) were measured as a function of temperature for parvalbumin, a small acidic protein expressed abundantly in fast-twitch muscle, from the Arctic cod (Boreogadus saida) and compared to values previously determined for Antarctic and temperate zone teleosts. Estimates of KD were derived independently from fluorometric titrations and calorimetry. In addition, the primary structure of B. saida parvalbumin was determined. Calcium KDs for parvalbumin from B. saida were fundamentally similar to those for parvalbumins from Antarctic species (6.68+/-0.59 nM and 7.77+/-0.72 nM at 5 degrees C, respectively), but significantly different from temperate zone species (1.35+/-0.28 nM at 5 degrees C). However, estimates of KD for B. saida parvalbumin at 5 degrees C closely matched values for temperate zone fish at 25 degrees C (6.54+/-0.56 nM), recapitulating the prior observation that calcium affinity of parvalbumin is conserved at the native temperature of teleost fish. Full sequence of B. saida parvalbumin was generated using reverse-phase HPLC and RACE-PCR. The Arctic parvalbumin showed 83% homology to a carp parvalbumin. None of the 16 total substitutions between the two parvalbumins resided in the cation binding sites of the protein, indicating that the structural locus of the thermal sensitivity of function lies outside the active regions.     

Parvalbumins distribution and physical state inside the muscle cell

Single skinned muscle fibres (frog) have been submitted to double Ouchterlony immunodiffusion assays with antibodies directed against the two species of frog parvalbumin. The antigenic material which diffuses out of each fibre contains the two parvalbumins. Their presence in each cell is thus demonstrated. The amount of parvalbumins having diffused out of the fibre has been quantified. It corresponds to the parvalbumin content of the cell. This implies that these proteins are freely soluble in the muscle sarcoplasm.