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.     

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.     

The evolution of muscular parvalbumins investigated by the maximum parsimony method

Summary Phylogenetic trees requiring the lowest sum of nucleotide replacements and gene duplicative events were constructed from the amino acid sequence data on ten gnathostome parvalbumins (PAR) and two related myofibrillar proteins troponin-C (TNC) and myosin alkali-light-chain (ALC). The origin and differentiation of the structural domains within these proteins were also investigated by the maximum parsimony method and by an alignment statistic for identifying evolutionarily related protein sequences. The results suggest, in agreement with the Weeds-McLachlan model, that tandem duplications in a precursor gene caused a primordial one-domain polypeptide (consisting of two helices with a calcium binding region in between) to double and then quadruple in size. Duplications of the gene coding for this four domain (I–II–III–IV) protein in an early metazoan, pre-gnathostome lineage gave rise to the separate loci for TNC, ALC, and PAR. TNC, which alone retained the Ca-binding function in each of its four domains, evolved much more slowly than either the ALC or PAR lineages. In the PAR lineage the I–II–III–IV structure was degraded, presumably by a partial gene deletion, to the II–III–IV structure during descent to the gnathostome ancestor of parvalbumins. Also during this period the mid region in domain II lost its Ca-binding function and, as it did so, evolved at an accelerated rate over other regions, a pattern indicative of positive selection for a change in function. In turn, from the gnathostome ancestor to the present, the mid regions of domains III and IV, which each retained Ca-bindung function, evolved much more slowly than other regions, a pattern indicative of stabilizing selection for preservation of function. Between the gnathostome and teleost-tetrapod ancestor a gene duplication separated the parvalbumins into an-lineage and a-lineage. During this early vertebrate period PAR genes evolved at the extremely fast rate of 89 nucleotide replacements per 100 codons per 10⁸ years (i.e. 89 NR %), but from the teleost-tetrapod ancestor to the present, both- and-PAR lineages evolved at a much slower rate, about 8 NR %. The use of-parvalbumins as phylogenetic markers was complicated by presumptive evidence that paralogous (i.e. duplication dependent) gene lineages occur within this group. As a final point, in the genealogy of TNC, ALC, and PAR lineages, a non-random pattern of nucleotide replacements was observed between the reconstructed ancestral and descendant mRNA sequences. The pattern was similar to that observed for other protein genealogies and seems to reflect a bias in the genetic code for guanine to adenine and adenine to guanine transitions (especially at the first nucleotide position of the RNA codons) to produce amino acid substitutions which are compatible with the preservation of protein three-dimensional structure.     

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.     

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.     

The amino acid sequence of the parvalbumin from the very fast swimbladder muscle of the toadfish (Opsanus tau)

1. The parvalbumin from the very fast striated muscle of the swimbladder of the toadfish (Opsanus tau) has been purified to homogeneity and its amino acid sequence has been completely elucidated. 2. The polypeptide chain is made of 109 residues, belongs to the beta group of parvalbumins and presents characteristics common to many other parvalbumins, i.e. a blocked N-terminal group, a cysteine and an arginine residue at positions 18 and 75 respectively and a quadruplet of acidic residues in the region 59-62.     

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.     

The phylogenetic relationship of tetrapod, coelacanth, and lungfish revealed by the sequences of forty-four nuclear genes

The origin of tetrapods is a major outstanding issue in vertebrate phylogeny. Each of the three possible principal hypotheses (coelacanth, lungfish, or neither being the sister group of tetrapods) has found support in different sets of data. In an attempt to resolve the controversy, sequences of 44 nuclear genes encoding amino acid residues at 10,404 positions were obtained and analyzed. However, this large set of sequences did not support conclusively one of the three hypotheses. Apparently, the coelacanth, lungfish, and tetrapod lineages diverged within such a short time interval that at this level of analysis, their relationships appear to be an irresolvable trichotomy.     

Amino acid sequence of alpha chain of sarcoplasmic calcium binding protein obtained from shrimp tail muscle

The isotypes of sarcoplasmic Ca2+ binding protein (SCP) were purified from shrimp tail muscle. SCP exists in a dimeric form. One sample of shrimp contained only alpha A chain, whereas another contained alpha B and beta chains, and a heterodimer of alpha B beta which was not analyzed precisely. The amino acid sequences of the two alpha chains were determined. The two alpha chains are composed of 190 and 192 amino acid residues, respectively. The sequences of the two alpha chains differed in only four amino acids out of 192 residues. The sequences indicate that the alpha chain has three Ca2+-binding sites which are common to EF-hand type Ca2+-binding protein. In the absence of added Ca2+ and Mg2+, the amounts of bound Ca2+ in alpha A, alpha B, and beta chains were 3.0, 3.3, and 2.4 mol/22,000 g protein, respectively. Thus, it is suggested that all three isotypes of shrimp SCP have three Ca2+-binding sites which have high affinity to Ca2+. The sequence homology of shrimp SCP with other EF-hand type Ca2+-binding proteins is very low. The protein having the greatest homology with this SCP was cod parvalbumin; the sequence homology is 18%.     

Inference of the Protokaryotypes of Amniotes and Tetrapods and the Evolutionary Processes of Microchromosomes from Comparative Gene Mapping

Comparative genome analysis of non-avian reptiles and amphibians provides important clues about the process of genome evolution in tetrapods. However, there is still only limited information available on the genome structures of these organisms. Consequently, the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes in tetrapods remain poorly understood. We constructed chromosome maps of functional genes for the Chinese soft-shelled turtle (Pelodiscus sinensis), the Siamese crocodile (Crocodylus siamensis), and the Western clawed frog (Xenopus tropicalis) and compared them with genome and/or chromosome maps of other tetrapod species (salamander, lizard, snake, chicken, and human). This is the first report on the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes inferred from comparative genomic analysis of vertebrates, which cover all major non-avian reptilian taxa (Squamata, Crocodilia, Testudines). The eight largest macrochromosomes of the turtle and chicken were equivalent, and 11 linkage groups had also remained intact in the crocodile. Linkage groups of the chicken macrochromosomes were also highly conserved in X. tropicalis, two squamates, and the salamander, but not in human. Chicken microchromosomal linkages were conserved in the squamates, which have fewer microchromosomes than chicken, and also in Xenopus and the salamander, which both lack microchromosomes; in the latter, the chicken microchromosomal segments have been integrated into macrochromosomes. Our present findings open up the possibility that the ancestral amniotes and tetrapods had at least 10 large genetic linkage groups and many microchromosomes, which corresponded to the chicken macro- and microchromosomes, respectively. The turtle and chicken might retain the microchromosomes of the amniote protokaryotype almost intact. The decrease in number and/or disappearance of microchromosomes by repeated chromosomal fusions probably occurred independently in the amphibian, squamate, crocodilian, and mammalian lineages.     

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.     

Sodium and potassium binding to parvalbumins measured by means of intrinsic protein fluorescence

The binding of Na+ and K+ to whiting parvalbumin (pI 4.4) and pike parvalbumins (pI 4.2 and 5.0) results in a shift of the tryptophan fluorescence spectrum towards shorter wavelengths by 2-4 nm for the whiting protein and in a rise of the tyrosine and phenylalanine fluorescence quantum yield for the pike proteins. The effective binding constants of Na+ and K+ to parvalbumins are within the range of 10 M-1 to 100 M-1. Physiological concentrations of Na+ and K+ lower the affinity of whiting parvalbumin for Ca2+ and Mg2+ by almost an order of magnitude.     

A reappraisal of the relationship of phosphate-acceptor protein to parvalbumins.

A phosphate-acceptor protein thought to be related to parvalbumins was described from dogfish muscle (Blum, H.E. $\text{et al.}$, 1974 Proc. Nat. Acad. Sci. USA $\text{71}$, 2198–2202). Further examination of this material indicated that the fraction obtained contained mainly classical parvalbumin, contaminated by less than 5% of a true phosphate-acceptor protein of MW ca 18 000 that accompanies parvalbumin throughout its purification. No such acceptor could be found in hake subjected to identical purification procedures. It is concluded that a phosphate-acceptor protein such as found in dogfish muscle bears no relation to parvalbumins.     

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.     

Amino acid sequence of a myoglobin from lace monitor lizard, Varanus varius, and its evolutionary implications

Myoglobin was purified from a muscle extract of lace monitor lizard, Varanus varius, by Sephadex G-75, followed by DEAE-cellulose column chromatography. The apomyoglobin was cleaved with cyanogen bromide. The largest fragment was further digested with pepsin, trypsin, and alpha-chymotrypsin. From the amino acid sequence of the cyanogen bromide fragments, together with those of tryptic peptides of apomyoglobin, the complete amino acid sequence of lizard myoglobin was deduced. To investigate the tetrapod and amniote origins, many possible phylogenetic trees were constructed using the myoglobin sequences, including those of map turtle and lace monitor lizard. The tree that requires the minimum number of nucleotide substitutions in their genes for the myoglobin sequences to have evolved from a common ancestor was different from the similarly most parsimonious trees for cytochrome c or for alpha-hemoglobin. The trees were different from each other and from the tree that best reflects current biological opinions.     

Rapid determination of parvalbumin amino acid sequence from Rana catesbeiana (pI 4.78) by combination of ESI mass spectrometry, protein sequencing, and amino acid analysis

The complete amino acid sequence of beta-type parvalbumin (PA) from bullfrog Rana catesbeiana (pI 4.78) was determined by tandem mass spectrometry in combination with amino acid analysis and peptide sequencing following Arg-C and V(8) protease digestion. The primary structure of the protein was compared with that of beta-type PA from R. esculenta (pI 4.50), with which it is highly homologous. Compared with R. esculenta beta-type PA4.50, R. catesbeiana beta-type parvalbumin (PA 4.78) differed in 15 out of 108 amino acid residues (14% displacement), PA4.78 had Cys at residue 64 and was acetylated at the amino terminus, but 25 residues of the carboxyl terminus were completely conserved. Several amino acid displacements were found between residues 51 and 80 (30% displacement), although the functionally important sequence of PA was completely conserved. The amino acids residues of putative calcium-binding sites were Asp-51, Asp-53, Ser-55, Phe-57, Glu-59, Glu-62, Asp-90, Asp-92, Asp-94, Lys-96, and Glu-101, which were conserved in all a and b-types of R. catesbeiana as well as other parvalbumins. In addition, Arg-75 and Glu-81, which are thought to form a salt bridge located in the interior of the molecule [Coffee, C.J. et al. (1976) Biochim. Biophys. Acta 453, 67-80], were also conserved in PA4.78.     

Pike eel (Muraenesox cinereus) gonadotropin. Amino acid sequences of both alpha and beta subunits

The amino acid sequences of pike eel gonadotropin alpha and beta subunits have been determined by standard sequencing analytical methods. The alpha subunit is composed of 93 amino acid residues while the beta subunit comprises 113 amino acid residues. All the invariant half-cystine residues are in the same positions as those found in other gonadotropins. It is noteworthy that the first, putative glycosylation site (Asn56) found in the alpha subunit of other gonadotropins was replaced by Asp56 in the alpha subunit of pike eel gonadotropin. Similarity analyses indicate that both subunits are structurally more similar to other known fish gonadotropin subunits than to those of the mammalian gonadotropins.     

Dealing with saturation at the amino acid level: a case study based on anciently duplicated zebrafish genes

The ray-finned fishes (Actinopterygii) seem to have two copies of many tetrapod (Sarcopterygii) genes. The origin of these duplicate fish genes is the subject of some controversy. One explanation for the existence of these extra fish genes could be an increase in the rate of independent gene duplications in fishes. Alternatively, gene duplicates in fish may have been formed in the ancestor of all or most Actinopterygii during a complete genome duplication event. A third possibility is that tetrapods have lost more genes than fish after gene or genome duplication events in the common ancestor of both lineages. These three hypotheses can be tested by phylogenetic reconstruction. Previously, we found that a large number of anciently duplicated genes of zebrafish are sister sequences in evolutionary trees suggesting that they were produced in Actinopterygii after the divergence of Sarcopterygii [Phil. Trans. R. Soc. Lond. B 356 (2001) 119]. On the other hand, several well-supported trees showed one of the two fish genes as the sister sequence to a monophyletic clade that included the second fish gene and genes from frog, chicken, mouse and human. These so-called outgroup topologies suggest that the origin of many fish duplicates predates the divergence of the Sarcopterygii and Actinopterygii and support the hypothesis that tetrapods have lost duplicates that have been retained in fish. Here we show that many of these 'outgroup' tree topologies are erroneous and can be corrected when mutational saturation is taken into account. To this end, a Java-based application has been developed to visualize the amount of saturation in amino acid sequences. The program graphically displays the number of observed frequent and rare amino acid replacements between pairs of sequences against their overall evolutionary distance. Discrimination between frequent and rare amino acid replacements is based on substitution probability matrices (e.g. PAM and BLOSUM). Evolutionary distances between sequences can be computed from the fraction of unsaturated sites only and evolutionary trees inferred by pairwise distance methods. When trees are computed by omitting the saturated fraction of sites, most fish duplicates are sister sequences.     

Paenungulata: a comparison of the hemoglobin sequences from elephant, hyrax, and manatee

Inspection of the amino acid differences among hemoglobin sequences of a wide range of mammalian species suggested that at alpha 19, alpha 110, alpha 111, beta 23, beta 44, and beta 56, synapomorphies group manatee (Trichechus inungius, Sirenia), Indian and African elephant (Elephas maximus and Loxodonta africana, Proboscidea), and rock hyrax (Procavia habessinica, Hyracoidea) into a monophyletic clade. Results obtained by parsimony analysis provide evidence for this grouping--and thus support for the genealogical validity of Simpson's superorder Paenungulata, which contains as the extant orders Proboscidea, Sirenia, and Hyracoidea. All of the 39 most, or nearly most, parsimonious of 10,395 trees constructed from a tandemly combined alpha- and beta- hemoglobin sequence for 103 vertebrate species (of which 79 were mammals from 16 extant orders), depicted Paenungulata as one of the most anciently separated branches of Eutheria. It was found on examining thousands of alternative trees that to not group Proboscidea, Hyracoidea, and Sirenia in a monophyletic clade required at least four additional substitutions.