The amino acid sequence of the major parvalbumin of the whiting (Gadus merlangus).

1. The amino acid sequence of the major parvalbumin of the Whiting has been determined; the polypeptide chain is made of 108 residues, the terminal amino acid group is acetylated, there is no disulfide bridges, the structure of the two calcium binding sites is preserved and the distribution along the polypeptide chain of the hydrophobic residues implicated in the compact hydrophobic core of the protein is also maintained. 2. The comparison of this amino acid sequence with other parvalbumins indicates that it belongs to the beta type and that within the Gadidae family two types of parvalbumins also occur.     

Parvalbumins from coelacanth muscle. II. Amino acid sequence of the two less acidic components.

The primary structure of the two less acidic parvalbumins (pI = 5.44 and pI = 4.95) from coelacanth muscle (Latimeria chalumnae) has been determined. They differ only by the presence or absence of a N-terminal blocking group. By the use of the automatic degradation, 69 amino acids could be placed unambiguously in the N-terminal part and 24 amino acids following the single arginine 75. Tryptic peptides were used to establish the sequence and the position of the remaining residues. The two parvalbumins examined belong to the alpha-lineage, and the rate of their molecular evolution is comparable to that found in other vertebrates.     

Parvalbumins from the lungfish (Protopterus dolloi).

Five parvalbumins have been isolated from the white muscles of the lungfish. They can be divided into two sub families showing typical amino acid compositions, C-terminal amino acid residues, peptide maps and immuno-reactivity. The red muscles including the cardiac muscle also contain parvalbumins in amounts roughly inversely related to the concentration of myoglobin in the muscle. Parvalbumins have also been detected in the brain and kidney.     

The sarcoplasmic proteins of white muscle of an antarctic hemoglobin-free fish, Champsocephalus gunnari.

1. The protein composition of the sarcoplasm of Champsocephalus gunnari white muscle has been examined by ultracentrifugation and starch-gel electrophoresis. 2. The extracts have been fractionated by several methods in order to compare them more closely to similar extracts of other fish species and to isolate creatine kinase and the parvalbumins IV and V. 3. The creatine kinase does not appear to differ from other fish creatine kinases. Both parvalbumins are also very similar to other parvalbumins except that they are more easily oxidized than all the parvalbumins described so far.     

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 parvalbumins. 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.     

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 conformers revealed by steady-state and time-resolved fluorescence spectroscopy

The binding of calcium to whiting (one tryptophan residue) and pike (one tyrosine residue) parvalbumins has been studied by means of kinetic and steady-state fluorescence techniques. The decay curves of the tryptophan and tyrosine fluorescence of the parvalbumins are best fitted by a sum of two exponents for any metal state of the proteins. The data can be interpreted as a nonexponential decay of the fluorescence of a single-type chromophore or in terms of equilibria between compact and relaxed conformers of the parvalbumins in each metal state. Fluorescence quenching by I-ions and effects of H2O/D2O substitution confirm the second interpretation. The constants of the equilibria have been evaluated.     

Immunological cross-reactions among gadidae parvalbumins

Antisera raised against apparently homogeneous whiting parvalbumin III have been found to recognize two non cross-reacting molecular species of parvalbumins. Aliquots of these antisera have been separately absorbed with two distinct parvalbumins from a near-related fish species, namely haddock parvalbumins II and III, and also with the homologous antigen. The immunochemical reactivities of absorbed and non-absorbed antisera toward parvalbumins from nine Gadidae species have been systematically explored by immunoelectrophoresis. The observed cross-reactions lead to distinguish two groups among Gadidae parvalbumins. So far this discrimination can be correlated with differences in amino-acid compositions, peptide maps and sequences which are known to characterize several protein members from each of the two groups. Using the same anti-whiting antisera, a tenuous common antigenic reactivity is shown between Gadidae and some Cyprinidae parvalbumins.     

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.     

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.     

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)     

Immunological cross-reactions among cyprinidae parvalbumins

The parvalbumin pattern in white muscle from seven Cyprinidae has been determined by starch gel electrophoresis analysis. The immunochemical discrimination of the various components using several monospecific antisera against a number of distinct Cyprinidae parvalbumins has been qualitatively evaluated by immunoelectrophoresis. The results show that immunologically different groups of parvalbumins can be distinguished within these patterns. One of these groups includes at least one component which is present in each of the seven species investigated. A similar component has been found in a species of Siluridae which, like the Cyprinidae, belongs to the well defined systematic superorder of the Ostariophysi.     

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.     

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.     

Conformational studies on muscular parvalbumins cooperative binding of calcium (II) to parvalbumins.

1H NMR and ORD were used to characterize the respective variations of tertiary structure and secondary structure of parvalbumins with calcium content ((Pa(O), without calcium and PaCa2 calcium saturated) and temperature. It has been observed that the tertiary structure can be lost without significant variation of the helical content. Cooperative binding of calcium to Pa(O) has been shown by NMR spectroscopy under low ionic strength conditions and at neutral pH. The present study shows that the calcium binding affinity of parvalbumin is dependent on the tertiary structure. Calcium binding and calcium release functions of parvalbumins in the muscle may be controlled by their tertiary structure.     

Crystal structure determination and refinement of pike 4.10 parvalbumin (minor component from Esox lucius)

The crystal and molecular structure of the minor component of pike parvalbumins has been determined at 1.93 A resolution by molecular replacement (1 A = 0.1 nm). The crystals are orthorhombic, space group P2(1)2(1)2 with a = 59.62 A, b = 59.83 A and c = 26.35 A. A location of the secondary cation binding site is proposed for this parvalbumin of the beta phylogenetic series.     

Heat capacity and entropy changes of the two major isotypes of bullfrog (Rana catesbeiana) parvalbumins induced by calcium binding

The possible structural changes of the two major isotypes (PA1 and PA2) of parvalbumins from bullfrog (Rana catesbeiana) skeletal muscle caused by Ca2+ binding have been analyzed by microcalorimetric titrations. Titrations of the parvalbumins with Ca2+ have been made in both the absence and presence of Mg2+ at pH 7.0 and at 5, 15, and 25 degrees C. The reactions of the parvalbumins with Ca2+ are exothermic in both the presence and absence of Mg2+ and at every temperature. But the contributions of enthalpy and entropy changes are variable; Mg2+-Ca2+ exchange on PA1 at 25 degrees C is driven almost entirely by a favorable enthalpy change, whereas Ca2+ binding to PA2 at 5 degrees C is driven for the most part by a favorable entropy change. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of the parvalbumins on Ca2+ binding and Mg2+-Ca2+ exchange have been estimated by the empirical method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2236-2240]. Although PA1 (beta) and PA2 (alpha) belong to genetically different lineages, the parvalbumins indicate very similar conformational changes to each other on both Ca2+ binding and Mg2+-Ca2+ exchange. On Mg2+-Ca2+ exchange, the vibrational as well as hydrophobic entropy is slightly increased in a parallel manner. In contrast, on Ca2+ binding, the hydrophobic entropy increases but the vibrational entropy decreases. The increase in the hydrophobic entropy indicates the sequestering of nonpolar groups from the surface to the interior of molecules, while the changes in the vibrational entropy suggest that the overall structures are tightened on Ca2+ binding but loosened on Mg2+-Ca2+ exchange.     

Solution and high‐pressure NMR studies of the structure,dynamics, and stability of the cross‐reactive allergenic cod parvalbumin Gad m 1

Beta‐parvalbumins from different fish species have been identified as the main elicitors of IgE‐mediated reactions in fish‐allergic individuals. Here, we report for the first time the NMR determination of the structure and dynamics of the major Atlantic cod (Gadus morhua) allergen Gad m 1 and compare them with other known parvalbumins. Although the Gad m 1 structure and accessibility of putative IgE epitopes are similar to parvalbumins in mackerel and carp, the charge distribution at the putative epitopes is different. The determination of the Gad m 1 structure contributes to a better understanding of cross‐reactivity among fish parvalbumins. In addition, the high‐pressure NMR and temperature variation experiments revealed the important contribution of the AB motif and other regions to the protein folding. This structural information could assist the future identification of hot spots for targeted mutations to develop hypoallergenic Ca²⁺‐free forms for potential use in immunotherapy. Proteins 2014; 82:3032–3042. © 2014 Wiley Periodicals, Inc.     

Remodeling of the AB site of rat parvalbumin and oncomodulin into a canonical EF-hand.

Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF-hand motifs, but the first site (AB) cannot bind Ca2+. Here we aimed to recreate the putative ancestral proteins [D19-28E]PV and [D19-28E]OM by replacing the 10-residue-long nonfunctional loop in the AB site by a 12-residue canonical loop. To create an optical conformational probe we also expressed the homologs with a F102W replacement. Unexpectedly, in none of the proteins did the mutation reactivate the AB site. The AB-remodeled parvalbumins bind two Ca2+ ions with strong positive cooperativity (nH = 2) and moderate affinity ([Ca2+]0.5 = 2 microM), compared with [Ca2+]0.5 = 37 nM and nH = 1 for the wild-type protein. Increasing Mg2+ concentrations changed nH from 2 to 0.65, but without modification of the [Ca2+]0. 5-value. CD revealed that the Ca2+ and Mg2+ forms of the remodeled parvalbumins lost one-third of their alpha helix content compared with the Ca2+ form of wild-type parvalbumin. However, the microenvironment of single Trp residues in the hydrophobic cores, monitored using intrinsic fluorescence and difference optical density, is the same. The metal-free remodeled parvalbumins possess unfolded conformations. The AB-remodeled oncomodulins also bind two Ca2+ with [Ca2+]0.5 = 43 microM and nH = 1.45. Mg2+ does not affect Ca2+ binding. Again the Ca2+ forms display two-thirds of the alpha-helical content in the wild-type, while their core is still strongly hydrophobic as monitored by Trp and Tyr fluorescence. The metal-free oncomodulins are partially unfolded and seem not to possess a hydrophobic core. Our data indicate that AB-remodeled parvalbumin has the potential to regulate cell functions, whereas it is unlikely that [D19-28E]OM can play a regulatory role in vivo. The predicted evolution of the AB site from a canonical to an abortive EF-hand may have been dictated by the need for stronger interaction with Mg2+ and Ca2+, and a high conformational stability of the metal-free forms.