A 49-residue peptide from adhesin F1 of Streptococcus pyogenes inhibits fibronectin matrix assembly

F1 is an adhesin of Streptococcus pyogenes which binds the N-terminal 70-kDa region of fibronectin with high affinity. The fibronectin binding region of F1 is comprised of a 43-residue upstream domain and a repeat domain comprised of five tandem 37-residue sequences. We investigated the effects of these domains on the assembly of fibronectin matrix by human dermal fibroblasts, MG63 osteosarcoma cells, or fibroblasts derived from fibronectin-null stem cells. Subequimolar or equimolar concentrations of recombinant proteins containing both the upstream and repeat domains or just the repeat domain enhanced binding of fibronectin or its N-terminal 70-kDa fragment to cell layers; higher concentrations of these recombinant proteins inhibited binding. The enhanced binding did not result in greater matrix assembly and was caused by increased ligand binding to substratum. In contrast, recombinant or synthetic protein containing the 43 residues of the upstream domain and the first 6 residues from the repeat domain exhibited monophasic inhibition with an IC(50) of approximately 10 nm. Truncation of the 49-residue sequence at its N or C terminus caused loss of inhibitory activity. The 49-residue upstream sequence blocked incorporation of both endogenous cellular fibronectin and exogenous plasma fibronectin into extracellular matrix and inhibited binding of 70-kDa fragment to fibronectin-null cells in a fibronectin-free system. Inhibition of matrix assembly by the 49-mer had no effect on cell adhesion to substratum, cell growth, formation of focal contacts, or formation of stress fibers. These results indicate that the 49-residue upstream sequence of F1 binds in an inhibitory mode to N-terminal parts of exogenous and endogenous fibronectin which are critical for fibronectin fibrillogenesis.     

Salivary statherin. Dependence on sequence, charge, hydrogen bonding potency, and helical conformation for adsorption to hydroxyapatite and inhibition of mineralization.

The structural domains of salivary statherin that are partly responsible for the protection and recalcification of tooth enamel were examined with respect to charge, sequence, hydrophobicity, hydrogen bonding potential, and conformation. Several fragments of statherin, 1-15 (SN15), 5-15 (SN11), 15-29 (SM15), 29-43 (SC15), 19-43 (SC25), and analogs of the N-terminal 15-residue sequence, where phosphoserines at positions 2 and 3 have been replaced by Ser (SNS15) and Asp (SNA15), respectively, were synthesized. The abilities of these fragments to adsorb at hydroxyapatite (HAP) surfaces and to inhibit its mineralization in supersaturated solutions were determined and compared with those of the whole statherin molecule, reported previously. The conformational preferences of the fragments both in aqueous and nonaqueous solutions were examined by circular dichroism. The highly charged N-terminal SN15 fragment has the greatest adsorption to HAP as compared with statherin and all other fragments. Its mineralization inhibitory activity is significantly greater than those of other fragments and comparable with that of the whole molecule. The dephosphorylated N-terminal fragment SNS15 shows a decreased tendency to adhere to and inhibit the formation of HAP, as compared with SN15. However, the substitution of Asp residues in place of phosphoserines (SNA15), restores the binding affinity and crystal growth inhibition properties, suggesting that the negative charge density at the N-terminal rather than any specific interaction of the phosphate group is important for HAP surface interactions. The C-terminal SC15 and SC25 fragments elicit a much higher affinity for HAP surface than that of the middle sequence (SM15), indicating that hydrogen bonding potential of the C-terminal sequence also contributes to the interaction of statherin with HAP. CD studies provide evidence that the N-terminal SN15 fragment has a strong tendency to adopt an ordered helical conformation, whereas the shorter N-terminal sequence, middle, and C-terminal fragments are structurally flexible and prefer to adopt scattered turn structures or unordered random conformations in organic and aqueous solutions. Collectively, the data indicate that the negative charge density, sequence (1-15), and helical conformation at the N-terminal region of statherin are important for its surface interaction with HAP.     

Binding of myotoxin a to sarcoplasmic reticulum Ca(2+)-ATPase: a structural study.

The interaction of myotoxin alpha with intact sarcoplasmic reticulum (SR) components was investigated, and two SR proteins were identified that associated with myotoxin a. One of the proteins has an apparent molecular weight similar to the Ca(2+)-ATPase, the major SR protein responsible for calcium loading. Ca(2+)-ATPase was purified, and its interaction with myotoxin a was studied. Evidence for specific binding of myotoxin a to Ca(2+)-ATPase was established by isolating chemically cross-linked myotoxin a-Ca(2+)-ATPase complexes and further proving their association with anti-myotoxin a antibodies. The binding region of myotoxin a was further delineated by cleaving the protein with cyanogen bromide (CNBr) into two fragments, a larger N-terminal fragment of 28 residues and a smaller C-terminal fragment of 14 residues. Competition experiments with 125I-myotoxin a showed that the C-terminal fragment competed better against 125I-myotoxin a than the N-terminal fragment for SR protein binding. Two overlapping peptides covering the sequence of the N-terminal fragment were synthesized to clarify the interaction of the N-terminal fragment of myotoxin a with SR proteins. A 16-residue peptide corresponding to residues 1-16 competed strongly with 125I-myotoxin a, while a second peptide (residues 13-28) did not.     

Amino acid sequence of the diazooxonorleucine binding site of Acinetobacter and Pseudomonas 7A glutaminase--asparaginase enzymes

Acinetobactor glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine, reduced with sodium borohydride, and cleaved with cyanogen bromide. Radioactivity was present only in a 96-residue-N-terminal peptide which eluted as the second peptide peak on Sephadex G-50. Radioactivity was released with the threonine in position 12 during automatic sequencing of this peptide. The amino acid sequence of a 60-residue tn-terminal segment and a 16-residue C-terminal segment of this peptide was determined. Pseudomonas 7 A glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine and reduced with sodium borohydride. Radioactivity was released with the threonine in residue 20 during automatic sequencing of the whole enzyme. Analysis of 26 N-terminal residues showed that an 8-residue segment containing the radioactive threonine was identical with that in Acinetobacter glutaminase-asparaginase and in Escherichia coli asparaginase. Additional identical residues were noted in the N-terminal regions of these enzymes.     

Characteristics of protein splicing in trans mediated by a semisynthetic split intein

Protein splicing in trans results in the ligation of two protein or peptide segments linked to appropriate intein fragments. We have characterized the trans-splicing reaction mediated by a naturally expressed, approximately 100-residue N-terminal fragment of the Mycobacterium tuberculosis intein and a synthetic peptide containing the 38 C-terminal intein residues, and found that the splicing reaction was very versatile and robust. The efficiency of splicing was nearly independent of temperature between 4 and 37 degrees C and pH between 6.0 and 7.5, with only a slight decline at pH values as high as 8.5. In addition, there was considerable flexibility in the choice of the C-terminal intein fragment, no significant difference in protein ligation efficiency being observed between reactions utilizing the N-terminal fragment and either the naturally expressed 107-residue C-terminal portion of the intein, much smaller synthetic peptides, or the 107-residue C-terminal intein fragment modified by fusion of a maltose binding protein domain to its N-terminus. The ability to use different types of the C-terminal intein fragments and a broad range of reaction conditions make protein splicing in trans a versatile tool for protein ligation.     

Primary and tertiary structures of the first domain of Panulirus interruptus hemocyanin and comparison of arthropod hemocyanins

The amino acid sequence of the first domain (positions 1-175) of Panulirus interruptus hemocyanin subunit a has been determined. The sequence of residues 1-158 (18-kDa fragment obtained by limited proteolysis) was derived from peptides obtained by digestion of this fragment with CNBr and trypsin and by subdigestion of these peptides with other enzymes. The peptides were sequences automatically or manually. The amino acid sequence has been fitted into the electron-density map at 0.32-nm resolution. The residues of domain 1 are folded into a large, mainly helical, globular part, containing one disulfide bridge, and a smaller part near the molecular twofold axis. The latter part consists of an alpha helix and a beta strand which contains a covalently attached carbohydrate moiety. The sites susceptible to limited proteolytic cleavage of the subunit are discussed. Comparison of the N-terminal sequence with those of other arthropod hemocyanins revealed, besides an N-terminal extension of five residues, the presence of a 21-residue loop (positions 22-42) in the crustacean sequences. This loop contains helix 1.2, a less defined region in the electron-density map. It is absent in chelicerate sequences. Strong evidence is presented that: (a) the structure of the first 21 residues (including helix 1.1) is the same in all arthropod hemocyanins with known amino acid sequence; (b) a stretch containing about 15 residues (including part of helix 1.3) following the 21-residue loop has a different structure in crustaceans and chelicerates; (c) the rest of domain 1 has the same structure again. It is shown that all conserved residues are in the contact region with the other two domains.     

Purification of twelve cyanogen bromide fragments from bovine plasma fibronectin and the amino acid sequence of eight of them. Overlap evidence aligning two plasmic fragments, internal homology in gelatin-binding region and phosphorylation site near C terminus

Twelve cyanogen bromide fragments (CB1-12) from bovine plasma fibronectin have been isolated and eight of these completely sequenced. Altogether they account for 502 of the total expected 1880 residues in each of the two chains of fibronectin. Four of these fragments (CB1-4) constitute residues 1-289 in fibronectin with CB4 overlapping the N-terminal 29-kDa plasmic fragment to the second plasmic fragment, of 170-kDa in fibronectin. Fragments CB 5-9 are all contained within a 45-kDa gelatin-binding region, which is N-terminal in the 170-kDa fragment. The sequence of two of these five fragments in the 45-kDa fragment (CB7-8) contains two mutually homologous stretches with 57% sequence identity. Another two fragments (CB10-11) are derived from the heparin-binding region of the 170-kDa fragment. CB12 constitutes the C-terminal 13-residue stretch in fibronectin and contains a partly phosphorylated serine residue in the C-terminal sequence: -Arg-Glu-Asp-Ser(P)-Arg-Glu.     

Specific recognition of human pi glutathione transferase by an antipeptide antibody

Antibodies were raised in a rabbit by using a 12-residue synthetic peptide, corresponding to fragment 2-13 of rat placental glutathione S-transferase, as the immunogen. The antiserum appeared to react with the fragment as well as with the corresponding human enzyme (GST-pi), which shares with the rat transferase a 92% sequence homology at the N terminus. In addition, the binding of the antibody to the protein was completely inhibited by small amounts of peptide. The enzymatic activity of glutathione transferase was not affected by the antibody. This might indicate that the N-terminal fragment is not involved in the catalytic activity of the enzyme. This antibody of predetermined specificity might thus find a useful application for the detection and approximate quantitation of this marker in human preneoplastic lesions.     

Genotyping the bovine kappa-casein locus using polymerase chain reaction]

Polymerase chain reaction (PCR) was used for detecting kappa-casein (kappa-casein) genotype in the synthetic breed (Jersey x Black and White x Holstein-Friesian) dairy cattle. The amplified 228 bp fragment includes a region, where relevant mutations lead to both the appearance of different kappa-casein alleles associated with amino acid substitutions and the appearance of new TaqI and HindIII restriction sites in ae-casein B gene. The specificity of the kappa-casein gene fragment amplification was supported by restriction analysis and Southern blot hybridization. Digestion of amplified fragment with endonucleases PstI, HindIII and/or TaqI allows detection of AA-, AB- and BB genotypes of kappa-casein. A total of 32 animals with known (18 samples) and unknown (14 samples) kappa-casein phenotypes were tested using PCR and blot hybridization. In all known cases the detected genotype confirmed the phenotype. Frequencies of the B allele and of the AB genotype in the breeding population are rather high (53.1 +/- 8.8 and 43.7%, respectively). The possibility of effective use of the PCR analysis for genotyping kappa-casein locus in bulls and their offspring has been shown. The advantages of the PCR method in large breeding programs and linkage analysis have been discussed.     

Primary structure of the casein macropeptide of kappa casein of buffalo]

The complete amino acid sequence of Italian water buffalo (Bubalus arnee) caseinomacropeptide, the C-terminal fragment released from kappa-casein by chymosin, has been determined. It contains 64 amino acid residues including one phosphoserine and differs from its bovine (Bos taurus) B counterpart by 10 amino acid substitutions. The sequence of the last 11 amino acid residues of para-kappa-casein is also reported. In relation to the Ala148/Asp substitution which is responsible for the different electrophoretic behaviour of bovine kappa-caseins B and A, water buffalo kappa-casein is homologous to the bovine variant B. It is suggested that a variant Thr136-Ala148 might be the wild type of the Bos genus.     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

Expression of kappa-casein in normal and neoplastic rat mammary gland is under the control of prolactin

An 820-nucleotide-long cDNA clone for the kappa-casein (the casein micelle-stabilizing protein) from rat mammary gland was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence from the nucleotide sequence revealed a signal peptide, 21 amino acids long, and a mature protein of 157 amino acids. The signal peptide of rat kappa-casein was highly homologous to that of the precursor to ovine kappa-casein. However, little homology was apparent when the mature kappa-casein protein sequences from ovine or bovine sources were compared with rat kappa-casein. The kappa-casein mRNA content of the mammary tissue was found to increase during its functional differentiation. Prolactin appears to modulate the production of kappa-casein mRNA. Mammary glands of virgin females had no detectable kappa-casein mRNA; however, a marked induction of kappa-casein mRNA was obtained by intravenous infusion of prolactin. Mammary carcinomas did not follow the same pattern. 7,12-Dimethylbenz[a]anthracene-induced mammary carcinomas had normally low levels of kappa-casein mRNA, but intravenous prolactin infusion increased the levels by 2-fold. The MTW9 mammary carcinoma that grows only in the presence of high levels of mammotropic hormones had kappa-casein mRNA content equivalent to that in 10-day lactating rat mammary gland. Continuous venous infusion of prolactin to MTW9 mammary carcinoma did not modify the kappa-casein mRNA levels. Nitrosomethylurea-induced mammary carcinomas had no detectable kappa-casein mRNA, and intravenous prolactin infusion was unable to induce it.     

Limited proteolysis and ''in vitro'' mutagenesis of bovine brain inositol monophosphatase identifies an N-terminal region important for activity.

Bovine brain inositol monophosphatase is rapidly cleaved by endoprotease lys-C at a single site in the absence of SDS. Further sites are revealed only after prolonged incubation with high concentrations of protease. The initial cleavage occurs near one end of the enzyme, generating an N-terminally-derived 36-residue peptide, which is blocked, and a large 28 kDa fragment bearing a free N-terminus. The start sequence of this fragment was found to be Xaa-Ser-Pro-Ala-Asp-Leu-Val, consistent with the cDNA sequence, and Lys-36-Ser-37 was identified as the cleavage site. The activity of the cleaved enzyme was markedly decreased to 3% of that of the native enzyme, although its dimeric structure was preserved. The 36-residue peptide was not covalently associated with the large fragment after proteolytic cleavage, although the possibility of non-covalent association could not be excluded. Finally, the epitope for the inhibitory monoclonal antibody G-2A4 [Gee, Howell, Ryan & Ragan (1989) Biochem J. 264. 793-798] was found to lie proximal to the endoprotease lys-C cleavage site. In vitro mutagenesis further mapped the epitope for monoclonal antibody G-2A4 to residues around Cys-8 of the enzyme. These results suggest that the N-terminal region of the enzyme is important for activity.     

Nucleotide sequence of the gene encoding the Streptomyces albus G beta-lactamase precursor

A 1400-base DNA fragment, which contains the gene encoding the extracellular active-site serine beta-lactamase of Streptomyces albus G previously cloned into Streptomyces lividans [Dehottay et al. (1986) Gene 42, 31-36], was sequenced. The gene codes for a 314-amino-acid precursor, the N-terminal region of which has the characteristics of a signal peptide. The beta-lactamase as excreted by the host strain S. lividans PD6 has a ragged N-terminus, indicating either the presence of a leader peptidase of poor specificity or the action of an aminopeptidase. The primary structure (as deduced from the nucleotide sequence) was confirmed by amino acid sequencing of a 16-residue stretch at the amino terminus of the protein, a 12-residue stretch containing the active-site serine [De Meester et al. (1987) Biochem. J. 244, 427-432] and a 23-residue stretch obtained by trypsin digestion of the protein. The beta-lactamase belongs to class A, has three half-cystine residues (one of which occurs on the amino side of the active-site serine) and is inactivated by thiol reagents. Putative ribosome binding site and terminator region were identified.     

Purification, identification, and cDNA cloning of Jun a 2, the second major allergen of mountain cedar pollen

The second major allergen of Juniperus ashei (mountain cedar) pollen, Jun a 2, has been purified and its cDNA cloned. The purified protein has a molecular mass of 43 kDa and its N-terminal 9-residue amino acid sequence is highly homologous to those of Cry j 2 and Cha o 2, the second major allergen of Cryptomeria japonica and Chamaecyparis obtusa pollen, respectively. cDNA clones encoding Jun a 2 were isolated after PCR based amplification, and their nucleotide sequences were determined. The cDNA contains an open reading frame of 507 amino acid residues, and encodes a putative 54-residue signal sequence and a 453-residue intermediate, which releases a C-terminal fragment upon maturation. Three possible N-linked glycosylation sites and 20 cystein-residues are found in the deduced amino acid sequence. The amino acid sequence of Jun a 2 shows 70.7 and 82.0% identity with those of Cry j 2 and Cha o 2, respectively. Immunological observations that IgE antibodies in sera of Japanese pollinosis patients bind not only to Cry j 2 and Cha o 2 but also to Jun a 2 strongly suggest that Jun a 2 is an allergen of mountain cedar pollen, and that allergenic epitopes of these three allergens are similar.     

Prepeptide sequence of cinnamycin (Ro 09-0198): the first structural gene of a duramycin-type lantibiotic

The tetracyclic polypeptide antibiotic cinnamycin (Ro 90-0198) belongs to the duramycin-type lantibiotics and contains the unusual amino acids threo-3-methyl-lanthionine, meso-lanthionine, lysinoalanine and 3-hydroxyaspartic acid. Its structural gene, referred to as cinA, has been identified on isolated chromosomal DNA of the Ro 09-0198-producing strain Streptoverticillium griseoverticillatum via a 39-residue oligonucleotide probe derived from fragment 7-19 of the hypothetical prolantibiotic sequence CRQSCSFGPFTFVCDGNTK. This propeptide part was then found within an open reading frame of 77 amino acids. In contrast to the nisin-type prelantibiotics, this first duramycin-type prelantibiotic has an unusually long leader sequence of 58 amino acids. it also differs in the processing site and the direction of the formation of the threo-3-methyl-lanthionine bridges is from N-terminal cysteine to C-terminal dehydrated threonine residues, whereas the meso-lanthionine and lysinoalanine bridges are formed by addition reactions from C-terminal cysteine or lysine to N-terminal dehyrated serine residues.     

Nucleotide sequence and characterization of the gene for secreted alkaline phosphatase from Lysobacter enzymogenes.

Lysobacter enzymogenes produces an alkaline phosphatase which is secreted into the medium. The gene for the enzyme (phoA) was isolated from a recombinant lambda library. It was identified within a 4.4-kb EcoRI-BamH1 fragment, and its sequence was determined by the chain termination method. The structural gene consists of an open reading frame which encodes a 539-amino-acid protein with a 29-residue signal sequence, followed by a 119-residue propeptide, the 281-residue mature phosphatase, and a 110-residue carboxy-terminal domain. The roles of the propeptide and the carboxy-terminal peptide remain to be determined. A molecular weight of 30,000 was determined for the mature enzyme from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence was compared with sequences available in the current protein data base, and a region of the sequence was found to show considerable homology with sequences in mammalian type 5 iron-containing purple acid phosphatases.     

Proteasomes begin ornithine decarboxylase digestion at the C terminus

Proteasomes denature folded protein substrates and thread them through a narrow pore that leads to the sequestered sites of proteolysis. Whether a protein substrate initiates insertion from its N or C terminus or in a random orientation has not been determined for any natural substrate. We used the labile enzyme ornithine decarboxylase (ODC), which is recognized by the proteasome via a 37-residue C-terminal tag, to answer this question. Three independent approaches were used to assess orientation as follows. 1) The 461-residue ODC protein chain was interrupted at position 305. The C-terminal fragment was degraded by purified proteasomes, but because processivity requires continuity of the polypeptide chain, the N-terminal fragment was spared. 2) A proteasome-inhibitory viral sequence prevented degradation when introduced near the C terminus but not when inserted elsewhere in ODC. 3) A bulky tightly folded protein obstructed in vivo degradation most effectively when positioned near the C terminus. These data demonstrate that the proteasome initiates degradation of this native substrate at the C terminus. The co-localization of entry site and degradation tag to the ODC C terminus suggests that recognition tags determine the site for initiating entry. Flexibility of a polypeptide terminus may promote the initiation of degradation.     

Avian thymic hormone (ATH) is a parvalbumin

Amino acid sequence analysis of a protein from chicken thymus tissue which promotes immunological maturity in chicken bone marrow cells in culture has established sequences of a 45-residue fragment, a 24-residue fragment and a 9-residue and an 8-residue peptide. Independent comparison of the 45- and 24-residue fragments with known amino acid sequences by computer search has unequivocally identified avian thymic hormone as a parvalbumin. This is the first demonstration that a protein previously identified by a biological function is a parvalbumin.