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L Berthiaume

Publications and source records attributed to L Berthiaume.

At least 19 recordsLinked to original sources
PubMed1995

Strain variability and localization of important epitopes on the major structural protein (VP2) of infectious pancreatic necrosis virus.

Infectious pancreatic necrosis virus (IPNV), a birnavirus, is an important pathogen in fish farms. Analyses of viral proteins showed that VP2 is the major structural and immunogenic polypeptide of the virus. All neutralizing monoclonal antibodies (mAbs) against IPNV are specific to VP2 and bind to continuous or discontinuous epitopes. In order to determine which parts of the protein are involved in antigenic variations, five IPNV strains were sequenced over the VP2 coding region. Comparison of the sequences obtained with three previously published strains revealed a central variable domain (positions 183 to 335) which encompasses two hydrophilic hypervariable segments. Viral mutants which escaped neutralization were then selected with anti-VP2 mAbs directed against discontinuous epitopes. Sequencing of three mutants revealed a single amino acid mismatch in each of them. All of these substitutions occurred in the hypervariable segments, suggesting that these regions are involved in the formation of a discontinuous epitope. Finally, expression of different truncated VP2s in Escherichia coli allowed localization of the binding site for neutralizing mAbs which recognize continuous epitopes. One of these mAbs bound to the region adjacent to the C-terminus of the variable domain of VP2, while two others reacted with the central and C-terminal parts of the variable domain. No antibody reacted with the N-terminus of VP2. These results suggest that the variable domain of VP2 and the 20 adjacent amino acids of the conserved C-terminal part are the most important in inducing an immune response for the protection of animals.

Amino Acid Sequence↗
PubMed1995

Biochemical characterization of a palmitoyl acyltransferase activity that palmitoylates myristoylated proteins.

Dynamic regulation of signal transduction by reversible palmitoylation-depalmitoylation cycles has been recently described. However, further understanding of fatty acylation reactions has been hampered by our lack of knowledge about the specific transferases and thioesterases involved. Here, we describe an assay for the palmitoyl acyltransferase (PAT) that palmitoylates "myrGlyCys" containing members of the Src family of protein tyrosine kinases (PTKs). Since N-myristoylation of Fyn PTK, a member of the Src family, has been shown to be a prerequisite for palmitolylation, a new single plasmid vector that allows overexpression of myristoylated Fyn substrate in Escherichia coli was developed. Purified myristoylated protein substrates were incubated with [125I]iodopalmitoyl CoA, a palmitoyl CoA analog, in the presence of bovine brain lysates. Transfer of radiolabel to the Fyn substrate was detected by SDS-polyacrylamide gel electrophoresis and autoradiography. This assay was used to partially purify and characterize PAT activity from bovine brain. Here, we demonstrate that PAT is a membrane-bound enzyme, which palmitoylates myristoylated Fyn substrates containing a cysteine residue in position three. The PAT activity attached palmitate to Fyn proteins via a thioester linkage and exhibited a fatty acyl CoA preference for long chain fatty acids. It is likely that palmitoylation of Fyn and other Src family members by PAT regulates PTK localization and signaling functions.

Acyl Coenzyme A↗
PubMed1995

Evidence of a major neutralizable conformational epitope region on VP2 of infectious pancreatic necrosis virus.

A collection of neutralizing monoclonal antibodies (MAbs) produced against the LWVRT 60.1, Jasper and N1 strains of infectious pancreatic necrosis virus (IPNV) were selected for the analysis of VP2 epitopes. Previous characterization of the LW and JA MAbs allowed the identification of continuous and discontinuous epitopes but the topological localization of these sites remained obscure. The ability of these MAbs to differentiate individual epitopes was evaluated by additivity and competition assays using antigen-coated plates and by surface plasmon resonance (SPR), an automated biosensor system that is able to retain the conformation integrity of proteins. IPNV-neutralizing MAbs defined a major, conformational-dependent and immunodominant area where continuous epitopes represent portions of a larger discontinuous epitope. Moreover, weakly neutralizing MAbs could interact with internal sites, located within the foldings of the polypeptide chain. Anti-VP2 MAbs prepared against the European serotype N1 recognized and competed for epitopes present on the North American strain LWVRT 60.1 and Jasper. Attempts to establish the proximity of VP2 and VP3 epitopes have been made by SPR. Results indicate that these major structural proteins do not overlap in the virion.

Antibodies, Monoclonal↗
PubMed1995

Characterization of the small open reading frame on genome segment A of infectious pancreatic necrosis virus.

The genome of infectious pancreatic necrosis virus (IPNV) is composed of two segments of dsRNA. The larger segment contains a small ORF partly overlapping the 5' end of the polyprotein reading frame. Yet very little is known about this possible new gene, which presumably codes for a 17 kDa polypeptide (VP5). The region of the viral genome which encompasses the small ORF was reverse-transcribed and amplified by PCR before cloning and sequencing. Analysis of the sequences obtained from five different virus strains revealed that the small ORF is not found on one of them, and that it is truncated on two others. Moreover, the deduced amino acid sequences did not appear to be well conserved. Despite the large variations between IPNV strains at the genomic level, all predicted VP5 are arginine-rich basic polypeptides. To verify whether the small ORF is translated into protein in fish cells, the 17 kDa polypeptide of the VR-299 strain was expressed as fusion protein in a prokaryotic expression vector and used to produce a specific antiserum. This antiserum reacted with concentrated virus in an immunodot assay indicating that VP5 is synthesized in infected cells, but probably only in small quantities. When tested with 12 other IPNV strains, results were less conclusive than those obtained with strain VR-299. Nevertheless, three of the 12 viruses gave a clearly negative signal in the immunodot assay, suggesting that possibly more than one viral strain lacks the small ORF.

Amino Acid Sequence↗
PubMed1994

Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization.

The Src family consists of nine related tyrosine protein kinases with a common domain structure, including a myristylated N-terminal glycine residue. In this report, we identify cysteine residues within the N-terminal region of the Src family member Fyn which serve as sites for palmitylation. To facilitate detection of protein fatty acylation, p59fyn was overexpressed in COS cells and incubated with radioiodinated fatty acid analogs of myristate (IC13) or palmitate (IC16). Incorporation of both fatty acids into p59fyn was readily observed. Acylation with the palmitate analog was prevented when Gly-2 was mutated to alanine, implying that N-myristylation is required for palmitylation, and when either Cys-3 or Cys-6 was mutated to serine. Palmitylation was shown to alter the distribution of p59fyn between membrane-bound and soluble fractions. In contrast, no incorporation of the palmitate analog into pp60v-src, which lacks N-terminal cysteine residues, was observed. Mutation of Ser-3 of Src to cysteine, but not Ser-6, resulted in incorporation of the palmitate analog. These results serve to delineate sequence elements important for dual acylation of proteins, and further illustrate the utility of radioiodinated fatty acid analogs for studies of protein fatty acid acylation.

Amino Acid Sequence↗
PubMed1994

Regulation of enzymatic activity by active site fatty acylation. A new role for long chain fatty acid acylation of proteins.

Methylmalonate semialdehyde dehydrogenase (MMSDH) is a mitochondrial enzyme which can be acylated by myristoyl-CoA analogs (Deichaite, I., Berthiaume, L., Peseckis, S. M., Patton, W. F., and Resh, M. D. (1993) J. Biol. Chem. 268, 13788-13747). Here we describe the mechanisms which mediate regulation of the enzymatic activity of bovine MMSDH by long chain fatty acylation. The substrate specificity of the acylation reaction was measured in vitro using purified MMSDH and the coenzyme A derivative of an 125I-labeled long chain fatty acid (13-iodotridecanoate), an analog of myristoyl-CoA. Long chain fatty acyl CoAs (> 8 carbons) were able to inhibit radiolabeling of MMSDH. In order to study the physiological role of the acylation process in vivo, a system using highly purified mitochondria from COS-1 cells overexpressing MMSDH was exploited. MMSDH was shown to be processed properly, targeted to the mitochondrial fraction, and enzymatically active. The extent of fatty acylation of MMSDH as well as of other mitochondrial proteins was correlated with the mitochondrial energy level. Biochemical evidence as well as site-specific mutagenesis of cysteine 319 revealed that this highly conserved active site cysteine of MMSDH was the target of the fatty acylation. Another member of the aldehyde dehydrogenase family, yeast aldehyde dehydrogenase was also covalently modified by [125I]13-iodotridecanoyl-CoA and thereby inactivated. Furthermore, we demonstrate that glutamate dehydrogenase, an enzyme that has been previously shown to be strongly inhibited by palmitoyl-CoA, is fatty acylated by the 125I-labeled myristoyl-CoA analog. Our data suggest that attachment of long chain fatty acids to proteins is a new and potentially widespread type of enzyme regulation mechanism that we denote active site fatty acylation.

Acyl Coenzyme A↗
PubMed1993

Novel use of an iodo-myristyl-CoA analog identifies a semialdehyde dehydrogenase in bovine liver.

We describe here the identification, purification, and characterization of a semialdehyde dehydrogenase with a novel fatty acid binding function. The coenzyme A derivative of an 125I-labeled long chain saturated fatty acid (13-iodo-tridecanoate) was used to tag proteins which bind myristoyl-CoA. A prominent 57 kDa band was identified, which was isolated from bovine liver by a high salt extraction followed by ammonium sulfate precipitation. Sequential chromatographic separation using phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, Mono Q, and Fast Flow S resins resulted in a purified protein that migrated as a single band of 57 kDa on denaturing gels. Sephacryl-200 gel filtration provided a native molecular mass estimation of 118 kDa suggesting that this protein exists as a dimer. Two-dimensional gel analysis resolved three isoform variants with pI values of 7.4, 7.7, and 7.9, respectively, and established that the pI = 7.9 form has the highest propensity for fatty acid binding. We proceeded to generate tryptic peptides from the purified protein and subjected several peptides to microchemical sequencing. Degenerate oligonucleotide probes were designed and polymerase chain reaction was used to generate a unique nucleotide sequence. Subsequent screening of a bovine liver cDNA library yielded a 1.7-kilobase clone which encodes a protein of 537 amino acids (58 kDa) with 95% identity to mammalian methylmalonate semialdehyde dehydrogenase (MMSDH). In vitro assays confirmed that the purified 57-kDa protein exhibited MMSDH activity, and that preincubation of the enzyme with fatty acyl-CoA inhibited its dehydrogenase activity. The myristyl-CoA analog therefore serves as an affinity label for MMSDH. We propose that fatty acyl CoAs may have the potential to function as enzyme regulators in vivo.

Acyl Coenzyme A↗
PubMed1993

Differential usage of the carboxyl-terminal region among aldolase isozymes.

Sequence homology among nonconserved residues 357-362 of the COOH-terminal region in fructose-1,6-bisphosphate aldolases correlates with isozyme classification of aldolases. Recombinant chimers of human liver and maize aldolases were constructed by exchanging residues 357-362 with those from muscle, maize, and liver isozyme and by insertion in the maize sequence at position 349 rabbit muscle and liver residues 346-349. Activity variation among the chimers relative to native controls ranged from less than 10% to greater than 300% of Vm. Exchange of residues 357-362 significantly affected both Vm and Km without modifying catalytic efficiency kcat/Km, whereas insertion of residues 346-349 modified Vm and Km and increased catalytic efficiency. Steady state carbanion oxidation rates varied inversely with activity and were differentially affected with respect to equilibrium oxidation rates. Sequence exchange of residues 357-362 appears to modulate carbanion proton exchange, whereas sequence insertion of residues 346-349 modifies substrate and aldehyde interaction with C6 phosphate binding locus. Low intrinsic susceptibility to carboxypeptidase A degradation of the COOH terminus in liver aldolase is consistent with tight association of this COOH terminus in a conformation unfavorable for promoting high catalytic activity. Efficient carbanion protonation promoted by specific sequences 357-362 represents a mechanistic feature which distinguishes catalytically active maize and muscle isozymes from less active liver isozyme. Conservation of active site residues among aldolases suggests that isozyme diversity among aldolases arose from divergent evolution of the COOH-terminal sequence.

Amino Acid Sequence↗
PubMed1993

Comparison of amino acid sequences deduced from a cDNA fragment obtained from infectious pancreatic necrosis virus (IPNV) strains of different serotypes.

Infectious pancreatic necrosis is an important viral disease of salmonid fish reared in hatchery. Its etiological agent, IPNV, showed a high degree of antigenic heterogeneity. Up to 10 serotypes and 2 serogroups were proposed. Yet, very little is known about genomic variations among viruses of different origin. In order to investigate these variations, a 310-bp cDNA fragment was prepared from 17 IPNV strains by reverse transcription of the viral genome and amplification by the polymerase chain reaction. These fragments were then cloned and sequenced. Comparison of the 17 sequences obtained with 3 previously published ones, at the amino acid level, showed that serologically related viruses are highly homologous (over 96% homology) but some strains which were reported to belong to different serotypes also appeared closely related. Thus, only three major groups, clearly distinct from each other, could be formed. Apart from this, a search for the exact cleavage site of the unprocessed polyprotein of IPNV was done since the amplified fragment used for sequencing was localized at the junction between two polypeptides of the virus, pVP2 and NS. No obvious sequence or dipeptide appeared conserved in all birnaviruses.

Amino Acid Sequence↗
PubMed1993

Antigenic characterization of serogroup 'A' of infectious pancreatic necrosis virus with three panels of monoclonal antibodies.

Monoclonal antibodies (MAbs) were produced against three serotypes of infectious pancreatic necrosis virus (IPNV): A1 (LWVRT 60-1, U.S.A.), A2 (d'Honnincthun, France) and A9 (Jasper, Canada). Each panel of MAbs (identified as LW, HF and JA) was analysed by ELISA with the 10 proposed serotypes of IPNV and their specificity defined by immunoprecipitation and Western immunoblotting analysis. A first group of MAbs, directed against the outer capsid protein VP2, reacted with linear or conformational epitopes. A second group of MAbs, directed against the internal protein VP3, reacted with linear epitopes. There was no relationship between the neutralizing property of anti-VP2 MAb and the configuration of the epitope that it recognized. The MAbs were used for antigenic characterization of serogroup A. Each panel of MAbs showed a characteristic pattern of reactivity. The European HF series was predominantly cross-reactive and detected conserved epitopes among the 10 serotypes for both VP2 and VP3. The North American LW and JA series identified a group of conserved epitopes on VP3 and new specific epitopes on VP2 and VP3. The higher variability observed for VP2 in comparison with VP3 is one example of how external pressures may promote natural selection of those epitopes required for virus survival. Our results are consistent with an ancestral relationship of the European to the North American strains, the latter having developed new antigenic determinants upon evolution in their new geographical location.

Animals↗
PubMed1992

Direct expression of mature bovine adrenodoxin in Escherichia coli.

Site-directed mutagenesis was utilized to enable direct expression of the mature form of bovine adrenodoxin cDNA using the pKK223-3 expression vector in Escherichia coli. Expression was under control of the "tac" promoter and resulted in a direct expression of soluble mature bovine adrenodoxin (greater than 15 mg per liter). Chromatographic behavior of recombinant adrenodoxin did not differ from that reported for mature native adrenodoxin. The purified recombinant protein was identical to native mitochondrial adrenodoxin on the basis of molecular weight, NH2 terminal sequencing and immunoreactivity. E. coli lysates were brown in color, and the purified protein possessed a visible absorbance spectra identical to native bovine adrenodoxin consistent with incorporation of a [2Fe-2S] cluster in vivo. Recombinant bovine adrenodoxin was active in cholesterol side-chain cleavage when reconstituted with adrenodoxin reductase and cytochrome P450scc and exhibited kinetics reported for native bovine adrenodoxin. The presence of the adrenodoxin amino terminal presequence does not appear to be essential for correct folding of mature recombinant adrenodoxin in E. coli. This expression system should prove useful for overexpression of adrenodoxin mutants in future structure/function studies. The approach described herein can potentially be used to directly express the mature form of any protein in bacteria.

Adrenal Glands↗
PubMed1992

Ultrastructure of lymphocystis disease virus (LDV) as compared to frog virus 3 (FV3) and chilo iridescent virus (CIV): effects of enzymatic digestions and detergent degradations.

Ultrastructure of fish lymphocystis disease virus (LDV), the largest of all known icosahedral viruses, has been studied under electron microscopy using enzymatic digestions and detergent degradations. LDV structure appeared roughly the same as those of frog virus 3 (FV3) and chilo iridescent virus (CIV), two other well known viruses of the family Iridoviridae, although the great flexibility of its capsid as observed on negatively stained and shadow cast particles, and its three electron dense layers visualized in ultrathin sections, differed from observations made with the two other viruses. Specific degradation of the virions with enzymes or detergents revealed that the composition of the three iridoviruses was very much alike. In fact, their capsid was composed of two layers as observed in negative staining: an external one, which was removed following digestion with proteinase K, and an internal one which could be digested with phospholipase A2. Thus, the outermost layer is probably made of surface protein units, more or less tightly bound to each other, while the internal one would be a lipoprotein membrane. Consequently, these three iridoviruses appeared structurally related.

Animals↗
PubMed1992

Evidence of genomic variations between infectious pancreatic necrosis virus strains determined by restriction fragment profiles.

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of an important disease in hatchery-reared salmonid fish in North America, Europe and Japan. It belongs to the family Birnaviridae and shows a high degree of antigenic heterogeneity. However, genomic variations between the 10 identified serotypes have not yet been studied. In order to correlate genomic heterogeneity with the different serotypes, oligonucleotides were synthesized according to the published sequence of the Jasper strain (serotype A9). They were used as primers for the amplification of a 359 bp cDNA fragment of the viral genome using the polymerase chain reaction. Fragments amplified from 37 strains were digested with five different restriction enzymes. Restriction fragment profiles obtained an agarose gels showed heterogeneity not only between strains of different serotypes, but also among those belonging to serotype A1. A cluster analysis of the restriction patterns showed that IPNV strains can be divided into three major groups, corresponding approximately to serotypes A1, A2 and A3, and 10 subgroups which do not correlate with the serotyping of the strains.

Animals↗
PubMed1992

Antigenic and genomic differences of two Jasper strains of infectious pancreatic necrosis virus.

Strains of infectious pancreatic necrosis virus (IPNV-Jasper) obtained from two different laboratories were compared serologically with polyclonal and monoclonal antibodies. Nucleotide sequence and restriction endonuclease patterns of 359-bp fragment of genome segment A cDNA were also compared. Substantial differences were found in both analyses that will support the fact that the two Jasper strains are not identical.

Animals↗
PubMed1991

Carboxyl terminus region modulates catalytic activity of recombinant maize aldolase.

Site-directed mutagenesis was utilized to study the functional role of the COOH-terminal region in recombinant maize aldolase. A single mutation was created in each of the last nine amino acids of the COOH terminus and characterized kinetically. Point mutations in the COOH-terminal region were found to influence both the rate of fructose 1,6-bisphosphate and fructose 1-phosphate cleavage. Catalytic efficiency, kcat/Km, was not affected by the mutations within experimental error consistent with this region of the COOH terminus modulating product release. Concentrations of the carbanion-enamine enzyme intermediate complex produced upon substrate cleavage increased with the severity of the point mutation. A condensation assay was developed to directly measure fructose 1,6-bisphosphate synthesized by aldolases in the presence of high triose phosphate concentrations. The maximal rate of aldol condensation of triose phosphates, D-glyceralehyde-3-P and dihydroxyacetone-P, was affected by the point mutations to the same extent as the maximal rate of substrate cleavage. Interpretation of the data is consistent with point mutations in the COOH terminus predominantly affecting the proton exchange with the dihydroxyacetone-P enzymatic complex at the carbanion-enamine step and that this step is probably rate-limiting in the catalytic mechanism of recombinant maize aldolase. The role of the COOH-terminal region in aldolases is thus consistent with a sequence dependent modulation of catalytic activity.

Amino Acid Sequence↗
PubMed1989

Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications.

Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle. Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture. The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively. Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence. Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases. Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases. Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme. Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology. The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.

Amino Acid Sequence↗
PubMed1989

A new bacteriophage of Corynebacterium glutamicum isolated from swine waste.

A bacteriophage for Corynebacterium glutamicum strain LP-6 was isolated from swine waste. It belongs to the Siphoviridae family or Bradley morphologic group B, has a narrow host range, and is sensitive to chloroform and resistant to carbon tetrachloride. The phage is unstable (96% inactivation) in swine waste stored for 4 months at 22 C. The DNA has a molecular weight of approximately 20 Md, cohesive ends, and numerous restriction endonuclease sites. The phage differs from other known C glutamicum phages.

Animals↗