In vitro polymerization of mussel polyphenolic proteins catalyzed by mushroom tyrosinase

The in vitro enzymatic polymerization of the polyphenolic protein purified from the mussels Aulacomya ater, Mytilus edulis chilensis and Choromytilus chorus was studied. Mushroom tyrosinase was used to oxidize the dopa residues present in these proteins, and polymerization was monitored by acid-urea polyacrylamide gel electrophoresis. The protein from A. ater polymerized at a faster rate than the other two. Amino acid analysis of the crosslinked protein showed a notable decrease in the content of dopa, but no significant change of other amino acids. This suggests that crosslink formation may be limited to the oxidized dopa derivatives of the protein molecules.     

Identification of a novel eosinophil chemotactic cytokine (ECF-L) as a chitinase family protein

A novel eosinophil chemotactic cytokine (ECF-L) was purified from the culture supernatant of splenocytes of mice by a combination of anion-exchange chromatography, Procion red-agarose affinity chromatography, size exclusion high performance liquid chromatography (HPLC), and reverse phase HPLC. The NH(2)-terminal amino acid sequence was determined by direct protein sequencing. An ECF-L cDNA clone of 1,506 nucleotides was isolated from a cDNA library, and the nucleotide sequence predicted a mature protein of 397 amino acids. A recombinant ECF-L showed a level of eosinophil chemotactic activity comparable with that of natural ECF-L, and the activity was inhibited by a monoclonal antibody to ECF-L. ECF-L also attracted T lymphocytes and bone marrow polymorphonuclear leukocytes in vitro, whereas it caused selective extravasation of eosinophils in vivo. ECF-L mRNA was highly expressed in spleen, bone marrow, lung, and heart. A comprehensive GenBank data base search revealed that ECF-L is a chitinase family protein. ECF-L retains those amino acids highly conserved among chitinase family proteins, but Asp and Glu residues essential for the proton donation in hydrolysis were replaced by Asn and Gln, respectively. Although ECF-L contains a consensus CXC sequence near the NH(2) terminus akin to chemokine family proteins, the rest of ECF-L shows poor homology with chemokines.     

The F-actin capping proteins of Physarum polycephalum: cap42(a) is very similar, if not identical, to fragmin and is structurally and functionally very homologous to gelsolin; cap42(b) is Physarum actin.

We have carried out a primary structure analysis of the F-actin capping proteins of Physarum polycephalum. Cap42(b) was completely sequenced and was found to be identical with Physarum actin. Approximately 88% of the sequence of cap42(a) was determined. Cap42(a) and fragmin were found to be identical by amino acid composition, isoelectric point, mol. wt, elution time on reversed-phase chromatography and amino acid sequence of their tryptic peptides. The available sequence of cap42(a) is greater than 36% homologous with the NH2-terminal 42-kd domain of human gelsolin. A highly homologous region of 16 amino acids is also shared between cap42(a), gelsolin and the Acanthamoeba profilins. Cap42(a) binds two actin molecules in a similar way to gelsolin suggesting a mechanism of F-actin modulation that has been conserved during evolution.     

Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8

A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named alpha and beta, which were separated by C(2)-C(18) reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 alpha and PLNC8 beta, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (alpha) and 4,000 Da (beta). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature alpha and beta peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.     

The purification and amino acid sequence of toxin CM-13b from Naja haje annulifera (Egyptian cobra) venom.

Toxin CM-13b was purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. The toxin comprises 65 amino acid residues and is cross-linked by five disulphide bridges. The complete amino acid sequence of toxin CM-13b was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides purified by DEAE-cellulose chromatography and chromatography or electrophoresis on paper. The amino acid sequences of the intact toxin and its constituent peptides were determined by the Edman-Begg procedure, either through the use of the automatic sequenator or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The primary structure of toxin CM-13b shows a high degree of homology with that of protein S4C11 from Naja melanoleuca venom[1], but their toxicities are very different.     

Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2

We report here a partial primary structure for human complement protein H. Tryptic peptides comprising 27% of the H molecule were isolated by conventional techniques and were sequenced (333 amino acid residues). Several mixed-sequence oligonucleotide probes were constructed, based on the peptide sequence data, and were used to screen a human liver cDNA library. The largest recombinant plasmid (pH1050), which hybridized with two probes, was further characterized. The cDNA insert of this plasmid contained coding sequence (672 bp) for 224 amino acids of H. The 3' end of this clone had a polyadenylated tail preceded by a polyadenylation recognition site (ATTAAA) and a 3'-untranslated region (229 bp). Four regions of internal homology, each about 60 amino acids in length, were observed in the derived protein sequence from this cDNA clone, and a further seven from the tryptic peptide sequences. The consensus sequence for each of the repetitive units of H was four cysteines, two prolines, three glycines, one tryptophan, and two tyrosines/phenylalanines. Based on the mole percent values for each of these amino acids, it is likely that H is composed of about 20 repetitive units of this nature. Furthermore, the repetitive unit of H shows pronounced homology with the Ba fragment of B, the C4b binding protein, and beta 2-glycoprotein I. Therefore, it seems that at least portions of these proteins have evolved from a common ancestral DNA element.     

The major myosin-binding domain of skeletal muscle MyBP-C (C protein) resides in the COOH-terminal, immunoglobulin C2 motif

A common feature shared by myosin-binding proteins from a wide variety of species is the presence of a variable number of related internal motifs homologous to either the Ig C2 or the fibronectin (Fn) type III repeats. Despite interest in the potential function of these motifs, no group has clearly demonstrated a function for these sequences in muscle, either intra- or extracellularly. We have completed the nucleotide sequence of the fast type isoform of MyBP-C (C protein) from chicken skeletal muscle. The deduced amino acid sequence reveals seven Ig C2 sets and three Fn type III motifs in MyBP-C. alpha-chymotryptic digestion of purified MyBP-C gives rise to four peptides. NH2-terminal sequencing of these peptides allowed us to map the position of each along the primary structure of the protein. The 28-kD peptide contains the NH2-terminal sequence of MyBP-C, including the first C2 repeat. It is followed by two internal peptides, one of 5 kD containing exclusively spacer sequences between the first and second C2 motifs, and a 95-kD fragment containing five C2 domains and three fibronectin type III motifs. The C-terminal sequence of MyBP-C is present in a 14- kD peptide which contains only the last C2 repeat. We examined the binding properties of these fragments to reconstituted (synthetic) myosin filaments. Only the COOH-terminal 14-kD peptide is capable of binding myosin with high affinity. The NH2-terminal 28-kD fragment has no myosin-binding, while the long internal 100-kD peptide shows very weak binding to myosin. We have expressed and purified the 14-kD peptide in Escherichia coli. The recombinant protein exhibits saturable binding to myosin with an affinity comparable to that of the 14-kD fragment obtained by proteolytic digestion (1/2 max binding at approximately 0.5 microM). These results indicate that the binding to myosin filaments is mainly restricted to the last 102 amino acids of MyBP-C. The remainder of the molecule (1,032 amino acids) could interact with titin, MyBP-H (H protein) or thin filament components. A comparison of the highly conserved Ig C2 domains present at the COOH- terminus of five MyBPs thus far sequenced (human slow and fast MyBP-C, human and chicken MyBP-H, and chicken MyBP-C) was used to identify residues unique to these myosin-binding Ig C2 repeats.     

Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein.

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings.

Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA.     

Viral and cellular fos proteins are complexed with a 39,000-dalton cellular protein.

The structure of viral and cellular fos gene products and their association with a 39,000-dalton cellular protein (p39) were investigated by using antisera raised against synthetic peptides. The first peptide, termed M, corresponded to amino acids 127 to 152 of the v-fos sequence, a region which is identical in c-fos. The second peptide, termed V, corresponded to the nine C-terminal amino acids of v-fos; this region is not present in c-fos. Rabbit antisera were purified by affinity chromatography against their respective peptides before being used for immunoprecipitation. M peptide antisera precipitated p55v-fos and p55c-fos, whereas V peptide antisera precipitated only p55v-fos. This observation confirms the prediction from nucleotide sequence analysis that these proteins are distinct at their C termini. p39 was precipitated in association with p55v-fos and p55c-fos by M and V peptide antisera. However, V peptide antisera did not precipitate p39 from cells expressing p55c-fos, even though the presence of p39 in such cells was demonstrated with M peptide antisera. Denaturation of cell lysates completely abolished the precipitation of p39, whereas the precipitation of p55v-fos was unaffected. Taken together, the data demonstrate that p39 exists in a complex with p55.     

Peptide repeats in a mussel glue protein: theme and variations

The adhesive protein from Mytilus edulis contains 75-80 closely related, repeated peptide sequences in its primary structure. These peptides can be resolved following digestion with trypsin by reversed-phase high-pressure liquid chromatography. The most frequently repeated sequence is the decapeptide Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Dopa-Lys (peptide E). Variations of this occur in peptides B with Hyp-3 and Dopa-5, C with Dopa-5, and D with Hyp-3, respectively. Lesser amounts of hexapeptides (A and B') that are lacking residues 4-7 also occur. Peptide A has the sequence Ala-Lys-Pro-Thr-Dopa-Lys, whereas B' contains Tyr instead of Dopa. 4-Hydroxyproline occurs at positions 3 and 7 and occasionally at position 6 of the decapeptide; 3-hydroxyproline occurs only at position 6. Adhesiveness of the protein may be related to the repetition of Dopa residues, the catecholic moiety of which has strong hydrogen-bonding and metal-liganding capabilities.     

Isolation, cloning, and primary structure of a cationic 16-kDa outer membrane protein of Salmonella typhimurium

By using acid-urea polyacrylamide gel electrophoresis, two cationic proteins were found in the isolated outer membranes of Salmonella typhimurium SH5014. Also, all the other enterobacterial strains studied (five additional strains of S. typhimurium, one strain of Salmonella minnesota, and three strains of Escherichia coli K12) had those proteins. The most abundant (OMB2) was purified in preparative acid-urea polyacrylamide gel electrophoresis and reversed-phase high pressure liquid chromatography (HPLC). It had a molecular mass of 16 kDa, a pI above 10.0, and was rich in arginine and lysine. 72% of the total amino acid sequence was determined by sequencing several HPLC-purified proteolytic fragments and 55 amino acids from the NH2 terminus. Furthermore, we isolated by molecular cloning the corresponding gene, named it ompH, and determined its nucleotide sequence. By combining protein and nucleotide sequence data, we determined the primary structure of the entire OmpH protein. It consists of 141 amino acids, possesses regions very rich in basic amino acids, and has a molecular mass of 15,862 kDa.     

Nucleotide and deduced amino acid sequences of a GTP-binding protein family with molecular weights of 25,000 from bovine brain

We have purified a novel GTP-binding protein (G protein) with a Mr of about 24,000 to homogeneity from bovine brain membranes (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904). In the present studies, we have isolated and sequenced the cDNA of this G protein from a bovine brain cDNA library using oligonucleotide probes designed from the partial amino acid sequences. The cDNA of the G protein has an open reading frame encoding a protein of 220 amino acids with a calculated Mr of 24,954. This G protein is designated as the smg-25A protein (smg p25A). The amino acid sequence deduced from the smg-25A cDNA contains the consensus sequences of GTP-binding and GTPase domains. smg p25A shares about 28 and 44% amino acid homology with the ras and ypt1 proteins, respectively. In addition to this cDNA, we have isolated two other homologous cDNAs encoding G proteins of 219 and 227 amino acids with calculated Mr values of 24,766 and 25,975, respectively. These G proteins are designated as the smg-25B and smg-25C proteins (smg p25B and smg p25C), respectively. The amino acid sequences deduced from the three smg-25 cDNAs are highly homologous with one another in the overall sequences except for C-terminal 32 amino acids. Moreover, three smg p25s have a consensus C-terminal sequence, Cys-X-Cys, which is different from the known C-terminal consensus sequences of the ras and ypt1 proteins, Cys-X-X-X and Cys-Cys, respectively. These results together with the biochemical properties of smg p25A described previously indicate that three smg p25s constitute a novel G protein family.     

Complete amino acid sequence of human T-cell leukemia virus structural protein p15

The complete amino acid sequence of human T-cell leukemia virus (HTLV) structural protein p15 has been determined. The intact protein and peptides generated by enzymatic digestion and acid cleavage were purified by reversed-phase liquid chromatography and subjected to semi-automated Edman degradation. HTLV p15 is a basic linear polypeptide composed of 85 amino acids with Mr 9458. The primary structure indicates that HTLV p15 is homologous to the nucleic acid binding proteins of other type-C retroviruses and especially related to bovine leukemia virus p12.     

Extensive homology between the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen

We have determined the complete primary structure for the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen; which have 229 and 227 amino acids, respectively. The amino acid sequences are 63% identical and conservatively substituted in 28 positions. A striking feature of these peptides is that the first half of each sequence is homologous with the second half, 37% in alpha 1(IV) and 36% in alpha 2(IV). These results suggest that the carboxyl-terminal peptides of type IV collagen are closely related in their structure and evolution. Presumably, they were first derived by internal duplication of a common ancestral DNA sequence which later, by gene duplication, gave rise to the two different but homologous carboxyl-terminal peptides of type IV collagen.     

The primary structure of the Chloroflexus aurantiacus reaction-center polypeptides

The complete nucleotide sequence of two Chloroflexus aurantiacus reaction-center genes has been obtained. The amino acid sequence deduced from the first gene showed 40% similarity to the L subunit of the Rhodobacter sphaeroides reaction center. This L subunit was 310 amino acids long and had an approximate molecular mass of 35 kDa. The second gene began 17 bases downstream from the first gene. The amino acid sequence deduced from it (307 amino acids; 34950 Da) was 42% similar to the M subunit of the Rhodobacter sphaeroides reaction center. 20% of the deduced primary structure were confirmed through automated Edman degradation of cyanogen bromide peptide fragments or N-chlorosuccinimide peptide fragments isolated from the purified reaction-center complex or from the individual subunits. The peptides were isolated by preparative gel electrophoresis combined with molecular sieve chromatography in the presence of a mixture of formic acid, acetonitrile, 2-propanol and water. This method appeared to be applicable to the isolation of other hydrophobic proteins and their peptides.     

Sequence similarities of protein kinase peptide substrates and inhibitors: comparison of their primary structures with immunoglobulin repeats

Forty original sequences of peptide substrates and inhibitors of protein kinases and phosphatases were aligned in a chain matrix without artificial gaps. Fifteen protein kinase peptide substrates and inhibitors (PKSI peptides) contained a common dipeptide ArgArg and also additional important tetra-, tri- and dipeptide homologies. Three further peptide substrates were significantly similar to these peptides but lacked the ArgArg dipeptide. Sequence comparison of individual PKSI peptides revealed probabilistically restricted consensus sequence—PKSI motif—comprising 8 homologous and 13 non-randomly distributed amino acids without considering mutation analysis. This template motif was compared with the consensus sequences of 12 different immunoglobulin domains. In 11 of 12 these domains, the starts of homologous segments were found at nearly the same domain related sites, beginning with serine. A single-triplet mutation, of any of the first two triplet bases that encode equally localized amino acids in each of the two sequence sets (PKSI and Ig) revealed additional homologies with the other set. A primary derived motif version composed of 9 homologous and seven non-randomly distributed amino acids was consequently established by its feedback projection into the original sequence sets. This procedure yielded a second preliminary motif version (revised motif) formed by a sequence of 9 homologous amino acids and two non-randomly distributed amino acids. In addition, three shorter oligopeptide motifs called important stereotypes were derived, based on repeated homology between Ig chains and the revised motif. The most extensive similarities in terms of these stereotypes occurred in the CH2 and CH4 domains of Ig peptides, and inhibitors of cAMP dependent protein kinase and protein kinase A. Further comparisons based on a reference sequence set arranged with the aid of feedback projection revealed a lower similarity between variable Ig chains reflected in a decreased number of homologous amino acids. Two final motif versions, FMC and FMV, were found in two different subsets of constant and variable Ig chains, respectively. FMC was composed of seven homologous and one non-randomly distributed amino acids forming the dispersed structure STLR(C)LVSD, whereas 6 homologous and one questionable amino acid constituted FMV. Only CH4 and CH1 domain segments contained all five high-incidence amino acids, which represented a higher level of similarity than homologous amino acids of all preliminary and final motifs. Four such amino acids were present also in three PKSI peptides. All similarities described here occur in domain segments positionally overlapping with the CDR1 region of variable chains. The results are discussed in terms of immunoglobulin evolution, the position of Fc receptor binding sites and degeneration or mutability of the triplets of motif-constituting amino acids.     

Peptide mimicking sialyl-Lewis(a) with anti-inflammatory activity

Peptides mimicking carbohydrate structure sialyl-Lewis a (SA-Le(a)) have been selected from a diverse dodecapeptide library using monoclonal antibody (MAb) NS19-9. Families of peptides with a consensus sequence consisting of three to nine amino acids and peptides that do not show a conserved core amino acid region were identified. Peptide DLWDWVVGKPAG was selected based on the consensus sequence DXXDXXVG shared with other peptides and strong binding in Western blot. Peptide competes with antibody binding to its native carbohydrate antigen, SA-Le(a), at 50% inhibitory concentration (IC(50)), 700 microM, implying that it represents a structural mimic of the carbohydrate epitope recognized by MAb. Statistically significant reduction of neutrophil recruitment into the intraperitoneal cavity was observed upon administration of this peptide in a murine acute inflammation model in vivo. Results suggest that the peptide mimic of SA-Le(a) carbohydrate might bind to E-selectin and block its interaction with another ligand, sialyl-Lewis X (SA-LeX), expressed on neutrophils.