Structural and physicochemical characteristics of novel basic proteins isolated from duck egg white

Novel basic proteins, duck basic protein small 1 (dBPS(1)) and 2 (dBPS(2)), were isolated from duck egg white by cation-exchange and gel filtration chromatography. Protein sequence analyses indicated that they possessed 39 amino acid residues with three disulfide bonds. The amino acid sequence of dBPS(1) showed 45% identity with dBPS(2). The amino acid sequence of dBPS(2) was the same as cygnin, a small protein from black swan, and strongly homologous with meleagrin from turkey and chicken. Phylogenic relationships implied that dBPS(1) and dBPS(2) share a common ancestry with cygnin and meleagrin. Based on MALDI-TOF mass spectra, the molecular masses of dBPS(1) and dBPS(2) were 4,373, and the 4,486 Da. pI of dBPS(1) and dBPS(2) elucidated by isoelectric focusing were 9.35 and 9.44. FT-IR spectra classified these proteins as (beta) proteins. Both dBPS(1) and dBPS(2), possessed high heat stability, Td 101.2 and 98.3 degrees C. Indirect ELISA results showed that the dBPS(1)/dBPS(2)-related proteins were distributed in the oviduct and gallbladder.     

Covalent structure of a low-molecular-mass protein, meleagrin, present in a turkey (Meleagris gallopavo) ovomucoid preparation

A low-molecular-mass protein, tentatively named meleagrin, was isolated from a commercial preparation of turkey (Meleagris gallopavo) ovomucoid. This 40-amino acid protein contains 3 disulfide bonds and high concentrations of aromatic residues (2 tryptophans and 3 tyrosines). It lacks threonine, methionine, phenylalanine, and arginine residues. The complete amino acid sequence was determined to be the following: less than Glu-Val-Leu-Lys-Tyr-Cys-Pro-Lys-Ile-Gly-Tyr-Cys-Ser-Ser-Lys-Cys-Ser-Lys- Ala- Glu-Val-Trp-Ala-Tyr-Ser-Pro-Asp-Cys-Lys-Val-His-Cys-Cys-Val-Pro-Ala-Asn- Gln-Lys - Trp. One of the three disulfide bonds exists between Cys12 and Cys28, and the two others links Cys32-Cys33 with Cys6 and Cys16. The amino acid sequence of meleagrin shows a strong homology to a similar basic protein, cygnin (Simpson, G.R. & Morgan, F.J. [1983] Int. J. Pep. Protein Res. 22, 476-481), of a rather distantly related aves, black swan (Cygnus atratus), suggesting some vital role of this protein in avian eggs. Similarity to a part (exon 9) of transferrins was also recognized.     

The chicken egg white proteome

Using 1-D PAGE and LC-MS/MS and MS(3) we identified 78 chicken egg white proteins, 54 of which were identified in egg white for the first time. All proteins were quantitated by calculating their exponentially modified protein abundance index (emPAI). Some previously known egg white components not characterized by amino acid sequences before, such as alpha-2-macroglobulin, were associated to a sequence for the first time. The predicted sequence was confirmed by MS-sequenced peptides covering 42% of the entire sequence. alpha-2-Macroglobulin occurred in egg white at the same concentration as ovostatin with which it showed 35% identity. For other proteins, which were previously only characterized by partial sequences, such as beta-ovomucin or ovalbumin X, we identified and confirmed predicted complete sequences with a high coverage by MS-sequenced peptides. New proteins included a 7 kDa protein consisting of a single secretoglobin sequence (ovosecretoglobin), a 7 kDa protein with similarity to black swan cygnin and turkey meleagrin (gallin) and proteins involved in binding, modification, and possibly detoxification, of bacterial lipopolysaccaride. The list of egg white proteins provided is by far the most comprehensive at present and is intended to serve as a starting point for the isolation and functional characterization of interesting new proteins.     

Isolation and complete amino acid sequence of a basic low molecular weight protein from black swan egg white

The following complete amino acid sequence of a low molecular weight basic protein (Mr 4,454) from black swan egg white has been determined: less than Glu-Val-Arg-Lys-Tyr-Cys-Pro-Lys-Val-Gly-Tyr-Cys-Ser-Ser-Lys-Cys-Ser-Lys-Ala-Asp-Val-Trp-Ser-Leu-Ser-Ser-Asp-Cys-Lys-Phe-Tyr-Cys-Cys-Leu-Pro-Pro-Gly-Trp-Lys. There is significant homology between this protein, provisionally designated cygnin, and the NH2-terminal region of the second domain of chicken ovotransferrin. The disulfide bonds have not been assigned; however, the arrangement of half-cystines in cygnin is sufficiently different from that of the known transferrins to suggest that cygnin is derived from another gene.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Abolition of anion-activation of mitochondrial F1-ATPase by the partial ADP-induced hysteretic inhibition

An M_r 21 000 polypeptide, designated APPG, has been purified by reverse-phase, high-performance liquid chromatography (RP-HPLC), from acid extracts of porcine anterior pituitary glands. This acidic protein possesses an isoelectric point of 4.9. Amino acid analysis shows that it is not a glycoprotein and estimates it to contain about 173 amino acids. NH₂-terminal sequence analysis allowed the determination of the first 50 residues unambiguously. A computer data bank search using a mutation data matrix and comparison with 269 012 protein segments indicated that this is a novel polypeptide sequence. However, this search revealed suggestive sequence homologies to a number of peptides of known sequence, including duck proinsulin (30%), Rous sarcoma virus transforming protein TVFV60 (24%) and pig secretin (26%).     

Phosphorylation in vivo of four basic proteins of rat brain myelin

When rat brain myelin was examined by sodium dodecyl sulphate/polyacrylamideslab-gel electrophoresis followed by fluorography of the stained gel, it was found that a host of proteins of rat brain myelin were labelled 2, 4 and 24h after the intracerebral injection of H₃³²PO₄. Among those labelled were proteins migrating to the positions of myelin-associated glycoprotein, Wolfgram proteins, proteolipid protein, DM-20 and basic proteins. The four basic proteins with mol.wts. 21000, 18000 (large basic protein), 17000 and 14000 (small basic protein) were shown to be phosphorylated after electrophoresis in both acid-urea- and sodium dodecyl sulphate-containing gel systems followed by fluorography. The four basic proteins imparted bluish-green colour, after staining with Amido Black, which is characteristic of myelin basic proteins. The four basic proteins were purified to homogeneity. Fluorography of the purified basic proteins after re-electrophoresis revealed the presence of phosphorylated high-molecular-weight `polymers' associated with each basic protein. The amino acid compositions of the phosphorylated large basic protein and small basic proteins are compatible with the amino acid sequences. Proteins with mol.wts. 21000 and 17000 gave the expected amino acid composition of myelin basic proteins. Radiolabelled phosphoserine and phosphothreonine were identified after partial acid hydrolysis of the four purified basic proteins. The [³²P]phosphate–protein bond in the basic protein was stable at an acidic pH but was readily hydrolysed at alkaline pH, as would be expected of phosphoester bonds involving both serine and threonine residues. Double-immunodiffusion analysis demonstrated that the four phosphorylated proteins showed complete homology when diffused against antiserum to a mixture of small and large basic proteins. Since the four basic proteins of rat brain myelin were phosphorylated both in vivo and in vitro it is postulated that the same protein kinase is responsible for their phosphorylation in both conditions.     

Conformation and aggregation of bovine myelin proteins

CD and PMR spectra were obtained on three major protein fractions of bovine CNS myelin: the basic A-1 protein, the Folch-Lees proteolipid apoprotein (APL), and the Wolfgram proteolipid protein (WPP). Most PMR peaks of the A-1 broadened on going from D₂O to salt solutions or to 100% 2-Chloroethanol (2-CE). CD spectra showed no α-helix in water or salt solutions, but showed 42% in 2-CE. The APL showed no PMR in D₂O, but did show aromatic amino acid peaks in 1.5% SDS. CD spectra showed 37% α-helix in both cases. The PMR of the WPP in 1.5% SDS showed aromatic amino acids, and the CD showed <20% α-helix. All three proteins showed sharp PMR spectra in trifluoroacetic acid with α-CH chemical shifts characteristic of random coils. It was concluded that the A-1 and the APL aggregate.     

Diverse dextranase genes from <Emphasis Type="Italic">Paenibacillus</Emphasis> species

Genes encoding dextranolytic enzymes were isolated from Paenibacillus strains Dex40-8 and Dex50-2. Single, similar but non-identical dex1 genes were isolated from each strain, and a more divergent dex2 gene was isolated from strain Dex50-2. The protein deduced from the Dex40-8 dex1 gene sequence had 716 amino acids, with a predicted M_r of 80.8 kDa. The proteins deduced from the Dex50-2 dex1 and dex2 gene sequences had 905 and 596 amino acids, with predicted M_r of 100.1 kDa and 68.3 kDa, respectively. The deduced amino acid sequences of all three dextranolytic proteins had similarity to family 66 glycosyl hydrolases and were predicted to possess cleavable N-terminal signal peptides. Homology searches suggest that the Dex40-8 and Dex50-2 Dex1 proteins have one and two copies, respectively, of a carbohydrate-binding module similar to CBM_4_9 (pfam02018.11). The Dex50-2 Dex2 deduced amino acid sequence had highest sequence similarity to thermotolerant dextranases from thermophilic Paenibacillus strains, while the Dex40-8 and Dex50-2 Dex1 deduced protein sequences formed a distinct sequence clade among the family 66 proteins. Examination of seven Paenibacillus strains, using a polymerase chain reaction-based assay, indicated that multiple family 66 genes are common within this genus. The three recombinant proteins expressed in Escherichia coli possessed dextranolytic activity and were able to convert ethanol-insoluble blue dextran into an ethanol-soluble product, indicating they are endodextranases (EC 3.2.1.11). The reaction catalysed by each enzyme had a distinct temperature and pH dependence.     

Genetic variation of the VP1 gene of the virulent duck hepatitis A virus type 1 (DHAV-1) isolates in Shandong province of China

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1 (DHAV-1) isolates, the virulence, cross neutralization assays and the complete sequence of the virion protein 1 (VP1) gene of nine virulent DHAV-1 strains, which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007–2008, were tested. The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses (ELD₅₀s) and the median lethal doses (LD₅₀s), respectively. The results showed that the ELD₅₀s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10⁶/mL to 1.44 × 10⁷/mL, while the LD₅₀s were 2.39 × 10⁵/mL to 6.15 × 10⁶/mL. Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates, respectively. Compared with other virulent, moderate virulent, attenuated vaccine and mild strains, the VP1 genes of the 9 strains shared 89.8%–99.7% similarity at the nucleotide level and 92.4%–99.6% at amino acid level with other DHAV-1 strains. There were three hypervariable regions at the C-terminus (aa 158–160, 180–193 and 205–219) and other variable points in VP1 protein, but which didn’t cause virulence of DHAV-1 change.     

Determination of the Amino Acid Sequence of a New Phospholipase A<Subscript>2</Subscript> (MIDCA1) Isolated from <Emphasis Type="Italic">Micrurus dumerilii carinicauda</Emphasis> Venom

A new Phospholipase A₂ (PLA₂) from Micrurus dumerilii carinicauda venom was isolated and its primary structure determined. This new PLA₂ showed a low enzymatic activity when compared with other PLA₂s and it is moderately basic with an isoelectric point of 8.0. Its amino acid sequence showed the presence of 120 amino acid residues and its sequence was: NLIQFLNMIQCTTPGREPLVAFANYGCYCGRGGSGTPVDELDRCCQVHDNCYDTAKKVFGCSPYFTMYSYDCSEGKLTCKDNNTKCKAAVCNCDRTAALCFAKAPYNDKNYKIDLTKRCQ. The structural model of MIDCA1, when compared with other strong neurotoxic PLA₂s, such as Naja naja, showed significant differences in the β-wing and neurotoxic sites, despite the high level of amino acid sequence similarity. These observations indicate a dissociation between the biological and catalytic activity of this new PLA₂, supporting the view that other regions of the protein are involved in the biological effects.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

The γ1 heavy-chain disease FOR protein is present in two molecular forms

Heavy chain disease proteins (FOR) were isolated from human plasma. These proteins were also detected immunochemically in the urine of the patient. The proteins were disulphide-linked Fc-like dimers with molar mass 64.2 kg/mol and sedimentation rate S _20,w ⁰ = = 0.356 ps (3.56 S). Similar amounts of aspartic acid and pyroglutamic acid were found at the N-terminus. After cyanogen bromide cleavage of the FOR proteins, three peptides were isolated and their amino acid composition and partial amino acid sequence was determined. We suggest that two Fc-like proteins of similar sizes are present in the plasma: (1) the first with N-terminal aspartic acid corresponding to position 221 of γ1 EU chain and (2) the second with N-terminal pyroglutamic acid. The first protein and small amounts of related low-molar mass fragments found also in the plasma could be degradation products of the second protein. Evidence is given on structural differences between the FOR proteins and the corresponding portion of the γ1 EU chain.     

Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein

We previously generated a monoclonal antibody (mAb), G2, by immunizing mice with Residues 174–247 of the chicken prion protein (ChPrP^C). In this study, we found that G2 possessed an extremely unusual characteristic for a mAb; in particular, it could react with at least three proteins other than ChPrP^C, the original antigenic protein. We immunoscreened a complementary DNA library from chicken brain DNA and found three proteins (SEPT3, ATP6V1C1, and C6H10orf76) that reacts with G2. There were no regions of amino acid sequence similarity between ChPrP^C and SEPT3, ATP6V1C1, or C6H10orf76. We selected ATP6V1C1 as a representative of the three proteins and identified the epitope within ATP6V1C1 that reacts with G2. The amino acid sequence of the G2 epitope within ATP6V1C1 (Pep8) was not related to the G2 epitope within ChPrP^C (Pep18mer). However, enzyme-linked immunosorbent assay, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) experiments indicated that these two peptides have similar binding affinity for G2. The apparent K_D values of Pep18mer and Pep8 obtained from SPR experiments were 2.9 × 10⁻⁸ and 1.6 × 10⁻⁸ M, respectively. Antibody inhibition test using each peptide indicated that the binding sites of the two different peptides overlapped each other. We observed that these two peptides substantially differed in several binding characteristics. Based on the SPR experiments, the association and dissociation rate constants of Pep18mer were higher than those of Pep8. A clear difference was also observed in ITC experiments. These differences may be explained by G2 adopting different binding conformations and undergoing different binding pathways.     

Identification of a hybrid-specific expressed gene encoding novel RNA-binding protein in wheat seedling leaves using differential display of mRNA

A hybrid-specific expressed cDNA fragment, designated as AG5, has been identified in wheat seedling leaves using differential mRNA display. AG5 contains an open reading frame (ORF) encoding 183 amino acid residues. Comparison with amino acid sequences in GenBank revealed that the AG5 protein is homologous to a group of Gly-rich proteins with consensus sequence-type RNA-binding domains (CS-RBD). Structural analysis showed that AG5 protein contains five motifs, including a consensus sequence-type RNA-binding domain near its N-terminus, arginine/aspartic acid repeats and a Gly-rich region in its center, a Cys-X₂-Cys-X₄-His-X₄-Cys (CCHC) zinc finger motif in the Gly-rich region, and TrySer₂ArgAsp₂Arg repeats towards its C-terminus. Of all previously described RNA-binding proteins, only RZ-1 from tobacco has a similar structure to the AG5 protein, but RZ-1 lacks a TrySer2ArgAsp2Arg repeat motif, indicating that the two proteins may belong to a family of closely related proteins in plants. The possible role of AG5 and its relation to wheat heterosis are discussed. Received: 21 November 1999 / Accepted: 20 March 2000     

Partial structural analysis of a highly basic low molecular weight protein from rat testis

Automated Edman degradation of a testis-specific basic protein isolated from the rat gave the following NH₂-terminal sequence of amino acids:

Cleavage of the native protein with cyanogen bromide produced two fragments which were purified by gel filtration. Amino acid analysis of the smaller fragment revealed it to be the NH₂-terminal undecapeptide resulting from cleavage at Met₁₁. The partial sequence analysis of the intact protein coupled with compositional analyses of these cyanogen bromide peptides indicate that the basic testis protein contains 24 basic amino acids and a single methionine in a sequence of 54 amino acids.     

Specific interaction of central nervous system myelin basic protein with lipids. Specific regions of the protein sequence protected from the proteolytic action of trypsin

The tryptic hydrolysis of the basic protein of central nervous system myelin (A₁ basic protein) and of A₁ basic-lipid complexes was studied. The tryptic digestion was monitored by “finger printing”, column chromatography and amino acid analysis of the resulting pure peptides.Specific regions of the protein sequence were found to be protected from the hydrolytic action of the trypsin only after the protein was recombined with specific lipids. The degree of protection was in the order: cerebroside sulphate > acidic lipid fraction of myelin > phosphatidylsrine = total lipid extract of myelin. The protected Lys-X, Arg-X bonds were all situated in the region amino acid 20 to amino acid 113 of the intact protein. This region contains the (proline)₃ bend in the protein which is stabilized by interaction with lipids and also the encephalitogenic site for monkey and rabbit.From the results reported in this publication we would like to suggest a specific interaction between a region of the A₁ basic protein molecule and cerebroside sulphate. Differences in A₁ basic protein-lipid interaction in different animals arising from differences in lipid composition and fatty acid composition of the different lipid species combined with minor changes in the protein sequence could explain the species variability of the encephalitogenic sites of the A₁ basic protein.     

Duck skeletal muscle acylphosphatase: Primary structure

The complete amino acid sequence of duck skeletal muscle acylphosphatase is presented. The sequence was studied by the manual Edman degradation of the complete series of tryptic peptides and the amino acid composition of peptic peptides. The NH₂-terminus is acetylated, and the polypeptide consists of 102 amino acid residues. The sequence is compared with other known acylphosphatases from the skeletal muscle of several vertebrate species.     

A stress protein is induced in the deep-sea barophilic hyperthermophile Thermococcus barophilus when grown under atmospheric pressure

The whole-cell protein inventory of the deep-sea barophilic hyperthermophile Thermococcus barophilus was examined by one-dimensional SDS gradient gel electrophoresis when grown under different pressure conditions at 85°C (T _opt). One protein (P60) with a molecular mass of approximately 60 kDa was prominent at low pressures (0.3 MPa hydrostatic pressure and 0.1 MPa atmospheric pressure) but not at deep-sea pressures (10, 30, and 40 MPa). About 17 amino acids were sequenced from the N-terminal end of the protein. Sequence homology analysis in the GenBank database showed that P60 most closely resembled heat-shock proteins in some sulfur-metabolizing Archaea. A high degree of amino acid identity (81%–93%) to thermosome subunits in Thermococcales strains was found. Another protein (P35) with molecular mass of approximately 35.5 kDa was induced at 40 MPa hydrostatic pressure but not under low-pressure conditions. No amino acid sequence homology was found for this protein when the 40 amino acids from the N-terminal end were compared with homologous regions of proteins from databases. A PTk diagram was generated for T. barophilus. The results suggest that P _habitat is about 35 MPa, which corresponds to the in situ pressure where the strain was obtained. Received: May 14, 1999 / Accepted: July 30, 1999