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H G Boman

Publications and source records attributed to H G Boman.

At least 73 records · Page 4Linked to original sources
PubMed1985

On the primary structures of lysozyme, cecropins and attacins from Hyalophora cecropia.

Diapausing pupae of Cecropia respond to a bacterial infection by the selective synthesis of RNA and 15-20 hemolymph proteins. Of these we have purified lysozyme and two classes of antibacterial proteins called cecropins and attacins. The primary structure has been determined for the lysozyme, one attacin and five cecropins. We have also prepared a cDNA bank, isolated and sequenced clones corresponding to the lysozyme, the two main attacins and one cecropin. The results of these structural studies are briefly summarized. Finally we review the solid phase synthesis of cecropin A and B and 9 analogs of cecropin A.

Amino Acid Sequence↗
PubMed1985

Molecular cloning, cDNA sequencing, and chemical synthesis of cecropin B from Hyalophora cecropia.

Two cDNA clones containing coding information for cecropin B from the Cecropia moth (Hyalophora cecropia) were identified by means of a synthetic probe. Sequencing of the two inserts showed that cecropin B is processed from a 62-amino acid residue precursor molecule including a 26-residue leader peptide and a COOH-terminal glycine residue. The latter presumably donates the nitrogen of the amide group present on the COOH-terminal leucine residue of the mature cecropin B. The sequence deduced for the mature cecropin B differed in the COOH-terminal region from the tentative structure previously determined by carboxypeptidase digestion. To settle the discrepancy, cecropin B was synthesized according to the cDNA sequence with an amidated COOH-terminal leucine. Natural and synthetic cecropin B were found to be indistinguishable with respect to electrophoretic mobility and antibacterial activity against seven different bacteria. The COOH-terminal tetrapeptides were isolated from both natural and synthetic cecropin B and found to be indistinguishable. The correct sequence for cecropin B is (formula; see text).

Amino Acid Sequence↗
PubMed1984

Insect immunity. Isolation and sequence of two cDNA clones corresponding to acidic and basic attacins from Hyalophora cecropia.

The Cecropia moth has three known classes of antibacterial immune proteins, attacins, lysozyme and cecropins (earlier referred to as P5, P7 and P9, respectively). Six attacins with different isoelectric points have been purified. The N-terminal sequences for five of these forms imply that only two different genes exist. We have now isolated and sequenced two cDNA clones, one for the basic attacin and one for the acidic form. The two mature proteins show 76% homology at the nucleotide level, while the regions beyond the stop codons are 36% homologous. The differences in the content of aspartic acid accounts for the difference in net charge between the acidic and basic attacin. Further differences in charge can be obtained by post-translational removal of a lysine-containing tetrapeptide at the C-terminal end of the two proteins. Evidence for a prepro form of the basic attacin is presented.

Journal Article↗
PubMed1983

Insect immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia.

Six closely related antibacterial proteins, attacins A-F, were isolated from the hemolymph of immunized pupae of the Cecropia moth, Hyalophora cecropia. Chromatofocusing separated attacins A-F, with isoelectric points between 5.7 and 8.3. Immunological experiments show that the attacins constitute antibacterially active forms of the previously isolated inducible immune protein P5. Their mol. wts., 20-23 K, are similar to that of protein P5, but significantly lower than 28 K found for preP5 synthesized in vitro (see accompanying paper). The six attacins can be divided into two groups according to their amino acid composition and amino-terminal sequences, attacins A-D constitute a basic group and attacins E and F an acidic one. Within each group the forms are very similar. The attacins efficiently killed Escherichia coli and two other Gram-negative bacteria isolated from the gut of a silk worm but they did not act on other Gram-positive and Gram-negative bacteria tested. Only growing cells of E. coli were attacked; cells suspended in phosphate buffer were inert. Besides the cecropins and lysozyme, the attacins represent a third class of antibacterial proteins in the humoral immune system of H. cecropia.

Amino Acid Sequence↗
PubMed1983

Insect immunity. Isolation of cDNA clones corresponding to attacins and immune protein P4 from Hyalophora cecropia.

Diapausing pupae of the Cecropia moth (Hyalophora cecropia) respond to an injection of live bacteria by the selective synthesis of certain types of RNA and immune proteins (designated P1-P9). The in vitro translation products of RNA from both injured and infected pupae showed specific patterns with a defined number of extra bands. Some proteins characteristic of the normal RNA were reduced in the immune RNA translation products. Antibody reaction was used to show the selective synthesis of immune proteins P4 and P5 with mRNA from pupae subjected to injury or infection. The protein synthesized in vitro, which cross-reacted with P5 antibodies, is most likely a precursor of the attacins described in the preceding paper. A cDNA clone bank was prepared and two clones were isolated and shown to contain 750 bp corresponding to P4 and 250 bp of attacin information. These clones were used to estimate the sizes of the mRNAs by Northern blotting and to estimate, by RNA/DNA hybridization, the levels of P4 and P5 mRNA. In vivo incorporation of [35S]methionine into attacins and P4 during different conditions was compared with the levels of the corresponding mRNA.

Animals↗
PubMed1983

Solid-phase synthesis of cecropin A and related peptides.

Cecropin A, a 37-residue antibacterial peptide amide, was synthesized by the solid-phase method. It was shown to be homogeneous and totally indistinguishable from natural cecropin A by chemical and physical criteria, as well as by its antibacterial activity against several Gram-positive and Gram-negative organisms. The synthetic material was also used to establish unambiguously that the carboxyl-terminal blocking group of natural cecropin A is a primary amide as tentatively proposed earlier. The role of the amino terminus of cecropin A in antibacterial activity was investigated by the synthesis of two analogs.

Anti-Inflammatory Agents↗
PubMed1983

Escherichia coli mutants with an altered sensitivity to cecropin D.

Cecropins are a family of small, basic antibacterial polypeptides which can be isolated from pupae of immunized Lepidoptera. They are active against both gram-negative and gram-positive bacteria. We studied a mutant of Escherichia coli, strain SB1004, which is more sensitive to cecropin D than is the parental strain. The mutant was selected as resistant to a host range mutant of a Serratia marcescens phage. When the protein composition of the outer membrane was examined, strain SB1004 and some other phage-resistant mutants were found to be deficient in the OmpC protein. It was concluded that the OmpC protein is the receptor of the phage. Strain SB1004 was found to differ from other ompC mutants in being especially sensitive to hydrophobic antibiotics and to cecropin D. Furthermore, strain SB1004 has a tendency for spontaneous autolysis. A genetic analysis showed the mutations in strain SB1004 and a suppressor mutant to map in the ompC region. The activity of cecropin D against different strains of E. coli was specifically enhanced when divalent cations were absent. No such effect was found with cecropins A and B, which are less hydrophobic than the D form.

Animals↗
PubMed1982

Synthesis of the antibacterial peptide cecropin A (1-33).

Cecropin A(1-33) was synthesized by an improved stepwise solid-phase method. The synthesis was designed to give high coupling yields and minimal amounts of byproducts. All coupling steps were monitored for completion by a new ninhydrin procedure, and the fully protected peptide-resin was analyzed for deletion peptides by the solid-phase Edman preview technique. Both methods indicated that the average coupling yield was greater than 99.8%. The unpurified peptide mixture resulting from HF cleavage and extraction into 10% acetic acid was analyzed by reverse-phase high-pressure liquid chromatography, and 93% of the total product was shown to be the desired [Trp(For)2]cecropin A(1-33), indicating an average yield per synthetic cycle of 99.8%. Removal of the formyl group at pH 9, followed by ion-exchange chromatography, gave the purified product. Cecropin A(1-33) showed antibacterial activity against both Gram-positive and Gram-negative bacteria. Against Escherichia coli, the activity was only slightly lower than that of the natural 37-residue cecropin A when tested over a 100-fold concentration range; the minimum inhibitory concentration was approximately 1 microM. The formyl derivative was somewhat less effective in killing E. coli than the free 1-33 peptide. The antibacterial activity was discussed in terms of an amphipathic alpha-helix structure and the binding of the peptide to bacterial membranes.

Amino Acid Sequence↗
PubMed1982

Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae.

We have investigated low molecular weight antibacterial proteins from the Cecropia moth. Hyalophora cecropia. In addition to the previously described cecropins A and B, five new antibacterial proteins were discovered, the cecropins C, D, E and F, and the factor G. A scheme for the purification of these factors is presented. Cecropin D is a major cecropin, its amino acid sequence, WNPFKELEKVGQRVRDAVISAGPAVATVAQATALAK, shows homology to cecropin A and B. Like these cecropins, cecropin D has a block C-terminal. The previously tentative C-terminal sequence of cecropin A is also confirmed. It is concluded that the three major cecropins, A, B and D, are products of three different genes that are derived from a common ancestor. The cecropins C, E and F were present in very low amounts, and thus their primary structures could not be fully elucidated. Cecropin C has an amino acid sequence that up to residue 37 is identical to the sequence of A, though it lacks the C-terminal blocking group. It may be a precursor or degradation product of cecropin A. The minor cecropin E shows a similar relation to cecropin D. Cecropin F has a single amino acid replacement (17 Asp leads to Asn) compared to cecropin D, and is probably a product of an allele that is present at a low frequency in the population. The primary structure of the factor G could not be determined, however its amino acid composition is different from that of the cecropins. All the major cecropins were found to be efficient against several gram-positive and gram-negative bacterial strains. No significant difference was found between them in their activity against Escherichia coli, though against some less susceptible bacteria the most basic cecropins were more effective, the activity falling in the series B greater than A much greater than D.

Amino Acids↗
PubMed1982

Insect immunity: isolation and structure of cecropins B and D from pupae of the Chinese oak silk moth, Antheraea pernyi.

The immune system in the Chinese oak silk moth, Antheraea pernyi, has been compared with that of the Cecropia moth which has been characterized earlier. Antibacterial activity against Escherichia coli was induced in diapausing pupae by injection of viable E. coli or Enterobacter cloacae. The activity reached a maximum on day 7-8 after which the response gradually declined. The pupae produced a set of immune proteins with P4 and P5 as major labelled components similar to that earlier found in Cecropia. The major antibacterial factor in A. pernyi was cecropin D. A procedure is described for the isolation of cecropin B and D, which is in principle similar to the one used for the isolation of the corresponding cecropins from Cecropia pupae. Amino acid sequence analyses of the A. pernyi cecropins show the D form to contain 36 amino acid residues and that both cecropins have blocked C-termini. The general structure of cecropins having a charged N-terminal region (residues 1-21) followed by a long hydrophobic stretch (residues 22-32) is well conserved. Cecropin B and D from A. pernyi differ from the corresponding proteins in Cecropia by four and three conservative amino acid replacements, respectively. The homology between the cecropins from the two insects suggests that they orginate from a single ancestral gene. The antibacterial activity was tested against nine different bacterial species. Evolutionary aspects of the cecropins are discussed.

Amino Acids↗
PubMed1981

Sequence and specificity of two antibacterial proteins involved in insect immunity.

Immune responses have been described for many different insect species. However, it is generally acknowledged that immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropia moth, Hyalophora cecropia. The expression of this multicomponent system requires de novo synthesis of RNA and proteins and its broad antibacterial activity is due to at least three independent mechanisms, the most well known of which is the insect lysozyme. However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. WE recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacterial activity against Escherichia coli and several other Gram-negative bacteria. We believe that P9A and P9B plays an important part in the humoral immune responses described previously and that the P9 proteins represent a new class of antibacterial agents for which we propose the name cecropins. We describe here the primary structures of cecropins A and B. We also show that cecropin A is specific for bacteria in contrast to melittin, the main lytic component in bee venom which lyses both bacteria and eukaryotic cells.

Amino Acid Sequence↗
PubMed1980

Insect pathogenic properties of Serratia marcescens: phage-resistant mutants with a decreased resistance to Cecropia immunity and a decreased virulence to Drosophila.

A non-pigmented strain of Serratia marcescens (Db10) was isolated from moribund Drosophila flies. From this strain were isolated spontaneous mutants resistant to streptomycin (Db11) and nalidixic acid (Db12). Mutant Db11 was used for the isolation of two phages, phi J and phi K, which grew on Db10, Db11 and Db12, but not on three reference strains of S. marcescens. Mutant Db11 was demonstrated to fulfil koch's postulates. Strain Db10 and its antibiotic-resistant derivatives were lethal to Drosophila whether given in the food or by injection. Evidence for toxin(s) was found only in sterile supernatants from 7 d cultures. Such extracts contained proteolytic activity and inactivated the antibacterial activity in immune haemolymph from Cecropia. Phages phi J and phi K were used to isolate phage-resistant mutants of Db11. Three such mutants and their parental strain were investigated for their susceptibility to immune haemolymph from Cecropia. The parental strain was resistant to incubation with 90% haemolymph for 2 h at 37 degrees C; all phage-resistant mutants were susceptible to the immune haemolymph with "killing times" (i.e. the time required to kill 90% of the viable cells) ranging from 15 to 55 min. When the same strains were compared for their virulence to Drosophila, the phage-resistant mutants had significantly reduced virulence. It is concluded that resistance to insect immunity plays an important role in the overall pathogenicity of S. marcescens.

Animals↗
PubMed1980

Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia.

Three inducible bacteriolytic proteins, designated P7, P9A and P9B, from the hemolymph of immunized pupae of the giant silk moth Hyalophora cecropia have been purified using a two-step procedure with cation-exchange chromatography. Purified protein P7 has a molecular weight of 15000 and its amino acid composition shows a great similarity to that of the lysozyme from the wax moth Galleria mellonella. Moreover, heat stability, pH-rate profile and bacteriolytic specificity also indicate that protein P7 is a lysozyme. The other purified proteins, P9A and P9B, are highly potent against Escherichia coli and some other gram-negative bacteria. The amino acid compositions of proteins P9A and P9B are very similar, although the contents of glutamic acid and methionine were different. The molecular weights of these very basic proteins are around 7000. The P9 proteins are heat stable; their activities were retained after 30 min incubation at 100 degrees C. Both forms of protein P9 clearly differ from the lysozyme class of enzymes and they may represent a new type of bacteriolytic protein.

Amino Acids↗
PubMed1979

Virulence factors in Bacillus thuringiensis: purification and properties of a protein inhibitor of immunity in insects.

We have previously shown that Bacillus thuringiensis subsp. alesti, serotype 3, produces two extracellular inhibitors of the immune system of Saturniid pupae (designated inhibitors A and B; Edlund et al., 1976). Starting from the culture supernatant of a new mutant of B. thuringiensis with a decreased extracellular proteolytic activity, we have now purified immune inhibitor A(InA). The procedure described consists of three steps: ultrafiltration, precipitation with ammonium sulphate and chromatography on hydroxylapatite. Purified InA gave a single band on polyacrylamide gel electrophoresis using either a gel concentration of 7.5% (w/v) and reducing and denaturing conditions or a gradient gel and native conditions. In both cases the apparent molecular weight was 78 000. A certain amount of proteolytic activity was always co-purified with InA but the two activities could be dissociated by heat or EDTA treatment. Antiserum against purified InA gave only one sharp precipitation band on immunodiffusion against InA with or without EDTA. InA inhibited the in vitro killing of Escherichia coli by immune haemolymph but did not affect the killing of Bacillus subtilis. InA was toxic for Drosophila when injected into the abdomen of adult male flies.

Animals↗
PubMed1977

Insect immunity. III. Purification and partial characterization of immune protein P5 from hemolymph of Hyalophora cecropia pupae.

We previously showed that in pupae of Hyalophora cecropia, eight hemolymph proteins (P1 through P8) were selectively synthetized after immunization (Faye et al., Infect, Immun. 12:1426-1438, 1975). We also showed that a gross fractionation was obtained by a series of ammonium sulfate precipitations (designed A through D) and that protein P5 was enriched in fraction A. Starting from fraction A, we have now purified protein P5 by using dialysis, isoelectric focusing, and hydroxylapatite chromatography. The final product gave a single band in both gradient gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using the latter method, proteins P5 and P8 were found to be enriched in fraction A, but they were absent in fraction Ac prepared from nonimmunized pupae. Protein P5 was found to have a pI of 6.6 and a molecular weight of 24,000 in sodium dodecyl sulfate and 96,000 in tris (hydroxymethyl) aminomethane-borate, pH 9.0. These data suggest a structure for P5 composed of four subunits of equal size. Protein P5 stimulated the killing of Escherichia coli by hemolymph fractions B and D, but it had neither killing nor phenol oxidase activity of its own.

Animals↗
PubMed1976

Immunochemical studies on lipopolysaccharides from wild-type and mutants of Escherichia coli K-12.

Lipopolysaccharides from a number of mutants of Escherichia coli K-12 were investigated by means of chemical and serological methods. Inhibition of passive hemagglutination and inhibition of precipitation show that L-rhamnose is the immunodominant sugar in the lipopolysaccharide from wild-type E. coli K-12. The disaccharide rhamnosyl-KDO (where KDO is 3-deoxy-D-manno-octulosonic acid) was isolated and characterized after mild acid hydrolysis of the lipopolysaccharide. It is concluded that rhamnose is present in the innermost part of the core as a side-chain substituent on KDO. From crosses between an E. coli K-12 donor and E. coli O8, hybrids were obtained which contained either one or both of the donor rfa and rfb clusters. Serum absorption studies with lipopolysaccharides from these hybrids indicated that the histidine-linked rfb cluster is responsible for the presence of rhamnose in the K-12 core oligosaccharide. Using paper chromatography of 32P-labelled lipopolysaccharides we have found heterogeneous lipopolysaccharide in two strains as well as some differences between two wild-type strains. The latter difference is believed to be due to varying contents of KDO-linked ethanolamine phosphate. The overall results presented together with those described in the companion paper clearly show that the core oligosaccharide in E. coli K-12 has a structure different from the types previously described for other strains of E. coli (designed coli R1 to coli R4).

Alleles↗