Biomedical subjects

S W Ludmerer

Publications and source records attributed to S W Ludmerer.

15 recordsLinked to original sources

PubMed2003

RdlDv, a novel GABA-gated chloride channel gene from the American dog tick Dermacentor variabilis.

The American dog tick Dermacentor variabilis is a major transmitter of bacterial and viral pathogens in human and animal populations, and compounds active against this species would benefit both human and animal health. Invertebrate GABA-gated chloride channels are validated targets of commonly used insecticides and acaricides. We cloned a novel member of the invertebrate GABA-gated chloride channel gene family from Dermacentor variabilis, RdlDv. The closest homologue of the predicted gene product of RdlDv is the RDL protein encoded by the GABA-gated chloride channel gene Drosophila Rdl (Resistance to Dieldrin), with which it shares 64% amino acid identity. When expressed in Xenopus oocytes, RdlDv produces GABA-activated currents blocked by the known insecticides and RDL antagonists fipronil and picrotoxinin. These results suggest that RdlDv encodes a GABA-gated chloride channel subunit, making it a potential target for compounds active against the tick D. variabilis.

Amino Acid Sequence↗

PubMed2001

Hybrid papillomavirus L1 molecules assemble into virus-like particles that reconstitute conformational epitopes and induce neutralizing antibodies to distinct HPV types.

Human papillomavirus (HPV) hybrid virus-like particles (VLPs) were prepared using complementary regions of the major capsid L1 proteins of HPV-11 and -16. These hybrid L1 proteins were tested for assembly into VLPs, for presentation and mapping of conformational neutralizing epitopes, and as immunogens in rabbits and mice. Two small noncontiguous hypervariable regions of HPV-16 L1, when replaced into the HPV-11 L1 backbone, produced an assembly-positive hybrid L1 which was recognized by the type-specific, conformationally dependent HPV-16 neutralizing monoclonal antibody (N-MAb) H16.V5. Several new N-MAbs that were generated following immunization of mice with wild-type HPV-16 L1 VLPs also recognized this reconstructed VLP, demonstrating that these two hypervariable regions collectively constituted an immunodominant epitope. When a set of hybrid VLPs was tested as immunogens in rabbits, antibodies to both HPV-11 and -16 wild-type L1 VLPs were obtained. One of the hybrid VLPs containing hypervariable FG and HI loops of HPV-16 L1 replaced into an HPV-11 L1 background provoked neutralizing activity against both HPV-11 and HPV-16. In addition, conformationally dependent and type-specific MAbs to both HPV-11 and HPV-16 L1 VLP were obtained from mice immunized with hybrid L1 VLPs. These data indicated that hybrid L1 proteins can be constructed that retain VLP-assembly properties, retain type-specific conformational neutralizing epitopes, can map noncontiguous regions of L1 which constitute type-specific conformational neutralizing epitopes recognized by N-MAbs, and trigger polyclonal antibodies which can neutralize antigenically unrelated HPV types.

Animals↗

PubMed2001

A novel human papillomavirus type 6 neutralizing domain comprising two discrete regions of the major capsid protein L1.

We have mapped the binding sites on human papillomavirus (HPV) type 6 for three HPV 6-specific neutralizing monoclonal antibodies (mAbs). The critical binding residues were first identified by making HPV 11-like amino acid substitutions in the HPV 6 major capsid protein L1 and assaying the resulting virus-like particles (VLPs) for reactivity with the mAbs. To confirm the relevance of these residues for mAb binding, we demonstrated that HPV 6 type-specificity could be transferred to HPV 11 VLPs by making the appropriate HPV 6-like amino acid substitutions in the HPV 11 L1. Two binding regions were found. For one mAb, all critical residues are centered at residue 53, while for the other two mAbs, type-specific binding also requires a second site located more than 100 residues distal to the first. Both binding sites coincide with regions of L1 where the sequences of the closely related HPV 6 and 11 diverge. These regions are where the L1 sequences are the least well conserved among all HPV types and they have been implicated in type-specific binding for other HPV types. This suggests that clusters of diverged residues, surrounded by conserved L1 sequences, are presented on the surface of assembled particles and are responsible for eliciting critical humoral immune responses to the virus.

Amino Acid Sequence↗

PubMed2000

Drug-resistant Drosophila indicate glutamate-gated chloride channels are targets for the antiparasitics nodulisporic acid and ivermectin.

The fruit fly Drosophila melanogaster was used to examine the mode of action of the novel insecticide and acaricide nodulisporic acid. Flies resistant to nodulisporic acid were selected by stepwise increasing the dose of drug in the culture media. The resistant strain, glc(1), is at least 20-fold resistant to nodulisporic acid and 3-fold cross-resistant to the parasiticide ivermectin, and exhibited decreased brood size, decreased locomotion, and bang sensitivity. Binding assays using glc(1) head membranes showed a marked decrease in the affinity for nodulisporic acid and ivermectin. A combination of genetics and sequencing identified a proline to serine mutation (P299S) in the gene coding for the glutamate-gated chloride channel subunit DmGluClalpha. To examine the effect of this mutation on the biophysical properties of DmGluClalpha channels, it was introduced into a recombinant DmGluClalpha, and RNA encoding wild-type and mutant subunits was injected into Xenopus oocytes. Nodulisporic acid directly activated wild-type and mutant DmGluClalpha channels. However, mutant channels were approximately 10-fold less sensitive to activation by nodulisporic acid, as well as ivermectin and the endogenous ligand glutamate, providing direct evidence that nodulisporic acid and ivermectin act on DmGluClalpha channels.

Animals↗

PubMed2000

HPV11 mutant virus-like particles elicit immune responses that neutralize virus and delineate a novel neutralizing domain.

Characterization of the regions of human papillomaviruses (HPVs) that elicit neutralizing immune responses supports studies on viral infectivity and provides insight for the development and evaluation of prophylactic vaccines. HPV11 is a major etiologic agent of genital warts and a likely vaccine candidate. A conformationally dependent epitope for the binding of three neutralizing monoclonal antibodies (mAbs) has been mapped to residues G(131)T(132) of the L1 major capsid protein. The mAbs bind L1 only when it is assembled into virions or into virus-like particles (VLPs) that mimic the capsid structure. We were interested in identifying other domains of L1 that elicit neutralizing responses. To this end, we have generated a panel of mAbs against VLPs derived from HPV11 L1 harboring a G131S substitution. The new mAbs are unlike the neutralizing mAbs previously mapped to residues G(131)T(132) in that they bind both prototype and HPV11:G131S mutant VLPs. Some of the new mAbs neutralized virus in vitro. We have mapped epitopes for three of these new mAbs, as well as a neutralizing mAb generated against HPV11 virions, by measuring binding to HPV6 VLPs substituted with HPV11-like amino acids. Two regions are critical: one defined by HPV11 L1 residues 263-290 and the other by residues 346-349. mAbs H11.H3 and H11.G131S.G3 bind HPV6 VLPs with substitutions derived from the 346-349 region; in addition, H11.G131S.G3 binds HPV6 VLPs with substitutions derived only from the 263-290 region. Although H11.H3 does not bind HPV6 VLPs with substitutions derived from the 263-290 region, binding to HPV6 VLPs is enhanced when both sets of substitutions are present. mAbs H11.G131S.I1 and H11.G131S.K5 bind HPV6 VLPs with the 263-290 substitutions, but show little binding to HPV6 VLPs with the 346-349 substitutions. However, binding to HPV6 VLPs is enhanced when substitutions at both regions are present. The 346-349 region has not previously been described as eliciting a neutralizing response for any HPV type. In addition, the work demonstrates a complex binding site contributed by two distinct regions of L1.

Animals↗

PubMed1997

A neutralizing epitope of human papillomavirus type 11 is principally described by a continuous set of residues which overlap a distinct linear, surface-exposed epitope.

A panel of monoclonal antibodies (MAbs) which neutralize human papillomavirus type 11 (HPV11) in the athymic mouse xenograph neutralization assay and bind HPV11 virus-like particles (VLPs) has been described. We recently presented evidence that the Gly131-Tyr132 residues of the major capsid protein L1 confer type 11-specific binding. However, residues distally located on the primary L1 sequence also were shown to affect binding. This poses the question whether the epitope is principally centered in the region of Gly131-Tyr132 or, alternatively, is comprised of diversely located residues which come into proximity only upon proper assembly. We analyzed the result of numerous substitutions located between Tyr123 and Val142 of the HPV11 L1 sequence. We show that substitutions at five positions result in loss of binding for one or more of these MAbs by an enzyme-linked immunosorbent assay which measures antibody binding to VLPs. We demonstrate that binding of these MAbs is redirected to HPV16 VLPs which harbor eight type 11-like substitutions within the homologous region. Three of these substitutions did not affect binding when individually substituted in HPV11 but yet were still required to transfer binding to substituted HPV16 VLPs. The results demonstrate that the epitope for this class of neutralizing MAbs, although conformational and requiring VLP assembly for presentation, principally lies along a 20-residue stretch of the L1 major capsid protein. This targets the region for evaluation of the possibility of receptor binding and suggests possibilities for the design of peptide inhibitors of virus infectivity.

Amino Acid Sequence↗

PubMed1996

Two amino acid residues confer type specificity to a neutralizing, conformationally dependent epitope on human papillomavirus type 11.

Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism.

Amino Acid Sequence↗

PubMed1996

Rapid, high-level transient expression of papillomavirus-like particles in insect cells.

Empirical scanning of natural or engineered peptide sequences for functional residues is inherently dependent upon efficient expression of large numbers of individual sequence variants to assay their relative functional potency. The insect baculovirus system has been widely used for expression of viral coat proteins, but it generally requires prior isolation and expansion of a plaque-purified recombinant viral stock to generate useful quantities of self-assembled virus-like particles. In search of a more rapid means of expression of analytical levels of the L1 coat protein of cottontail rabbit and human type 11 papilloma-viruses, we found that even brief transient cotransfection of insect cells with baculovirus plasmid transfer vectors and viral DNA yielded assembled particles that were immunologically indistinguishable from particles obtained with plaque-purified viral stocks. Within six days of plasmid/viral DNA cotransfection of Sf9 cells, at least 1-2 micrograms of assembled L1 particles/100-mm plate could be demonstrated, which proved more than sufficient to assay functionality. Transient cotransfection of insect cells should provide general utility for rapid high-level expression of sets of protein sequence variants, as well as other sequence-scanning applications such as sequence optimization in protein engineering.

Animals↗

PubMed1995

Use of a novel mutagenesis strategy, optimized residue substitution, to decrease the off-rate of an anti-gp120 antibody.

We have developed a novel strategy to decrease the antibody:antigen off-rate which we call optimized residue substitution. This strategy employs alanine substitution to first identify residues non-optimal for binding, as evidenced by a decrease in off-rate upon alanine replacement. These positions are then individually randomized to all amino acids, and the best replacement for each position determined. Finally, a construct which combines all optimized substitutions is generated and evaluated. We applied this strategy to the heavy chain CDR3 of P5Q, a scFv antibody which recognizes an epitope on the V3 loop of HIV gp120. We identified two amino acid substitutions that together decrease the off-rate by nearly ten-fold. The contributions by the two substitutions were near additive, indicative of independent affects on binding. We suggest that this strategy can be generalized to strengthen protein:ligand and protein:protein interactions in other systems.

Alanine↗

PubMed1994

The C-terminus of the B cell activator Oct-2 functions as an activation domain in yeast.

Oct-1 and Oct-2 are human transcriptional activators that bind to the same DNA element but activate distinct sets of genes. We expressed these factors in S. cerevisiae and observed greater than 5-fold stimulation of a lacZ reporter gene only with Oct-2. Transfer of the Oct-2 C-terminal domain onto either Oct-1 (Oct1.2) or a nonactivating DNA-binding domain from GAL4 created activators capable of greater than 15 and 10-fold stimulation of activity, respectively. Thus, the C-terminus of Oct-2 is sufficient to confer activation potential to nonactive DNA-binding fragments in yeast.

B-Lymphocytes↗

PubMed1993

Purification of glutamine tRNA synthetase from Saccharomyces cerevisiae. A monomeric aminoacyl-tRNA synthetase with a large and dispensable NH2-terminal domain.

Glutamine tRNA synthetase from Saccharomyces cerevisiae has been purified to homogeneity and shown to be a monomer of 91 kDa. The size of the polypeptide agrees with that predicted from the previously reported translated DNA sequence. Mild tryptic digestion removes an amino-terminal domain and releases a fragment of 65 kDa which begins at Ser205. This tryptic fragment is similar in size and sequence to Escherichia coli glutamine tRNA synthetase and shows a modest increase from the full-length yeast enzyme in the Km values for glutamine and ATP and no difference in the kcat for aminoacylation or the Km for tRNA. Thus, the removal of the NH2-terminal domain appears to indirectly affect the ATP- and glutamine-binding sites in the nucleotide-binding fold domain to which the NH2-terminal domain is fused. A monoclonal antibody directed against the NH2-terminal domain of the full-length enzyme has little effect upon the aminoacylation activity. Therefore, over 200 amino acids of the NH2 terminus of the full-length enzyme form a domain that operationally has only a modest influence on the catalytic core of the protein. These studies reinforce the concept that eukaryotic synthetases have quasi-independent domains not found in their prokaryotic counterparts which may confer a function distinct from aminoacylation.

Amino Acid Sequence↗

PubMed1987

Gene for yeast glutamine tRNA synthetase encodes a large amino-terminal extension and provides a strong confirmation of the signature sequence for a group of the aminoacyl-tRNA synthetases.

The gene for the yeast Saccharomyces cerevisiae glutamine tRNA synthetase is shown here to encode a protein of 809 amino acids. This contrasts with the 551 amino acids of the Escherichia coli glutamine tRNA synthetase. The yeast GLN4 transcripts have 5' termini that start approximately 25 nucleotides in front of the long open reading frame. Much of the extra size of the yeast enzyme is due to a large amino-terminal extension. At codon 225, the yeast enzyme aligns with the amino terminus of the E. coli protein. From this point on, the two sequences have an average of 40% identity, with a few small gaps for alignment, until their respective carboxyl termini. At codon 254 of the yeast and codon 30 of the E. coli enzyme, however, there starts an exact 15-amino acid match between the two proteins. This match encompasses and is partially the same as a short sequence which is a signature sequence for the amino acid group of the bacterial aminoacyl-tRNA synthetases which are specific for different amino acids. This is the strongest sequence match found between any yeast cytoplasmic or mitochondrial aminoacyl-tRNA synthetase with its bacterial homologue. This region of the structure is associated with a nucleotide fold. The result provides strong validation of the signature sequence, especially for sequences where the homology relationships are less dramatic than in this example. Because the 224-amino acid extension of the yeast enzyme does not align with any part of the E. coli enzyme, we propose that it is not associated directly with the catalytic function of the enzyme. Its possible function is investigated in the accompanying paper.

Amino Acid Sequence↗

PubMed1987

Construction and analysis of deletions in the amino-terminal extension of glutamine tRNA synthetase of Saccharomyces cerevisiae.

GLN4 of Saccharomyces cerevisiae encodes an amino-terminal extension of 224 amino acids. This is connected to a polypeptide which is colinear with and 40% identical to Escherichia coli glutamine tRNA synthetase. We examined the potential significance of the amino-terminal extension. Two single base and five multiple base frame shift deletions were constructed in this segment. Each of these mutations is associated with a lethal phenotype. This suggests that the coding sequence for the amino-terminal extension is translated. It also implies that there are no translation restarts downstream of the coding region for the amino-terminal extension which produce active enzyme. Three internal deletions of various sizes, and which preserve the correct reading frame, were constructed in the coding region of the amino-terminal extension. Cells which harbor such in-frame deletions on a multi copy plasmid are viable, even when a deletion construct is the only source of GLN4-encoded activity. Extracts of cells which have one of these deletions have reduced, but measurable, glutamine tRNA synthetase activity. We conclude that the catalytic activity resides with the segment which is homologous to the E. coli enzyme and that the amino-terminal extension itself is dispensable for aminoacylation activity. Each of the internal in-frame deletion constructions is respiration-proficient. The amino-terminal extension, therefore, is not used for an essential mitochondrial function of the GLN4 gene product. Within the accuracy of the measurements, activities of four other aminoacyl-tRNA synthetases are not affected by the presence of a GLN4 internal deletion allele as the only source of GLN4-encoded activity. This suggests that the amino-terminal extension does not stabilize a complex which includes one or more of these four enzymes and whose activity depends on proper assembly of the complex.

Alleles↗

PubMed1985

Cloning of GLN4: an essential gene that encodes glutaminyl-tRNA synthetase in Saccharomyces cerevisiae.

The structural gene for glutaminyl-tRNA synthetase has been isolated from a gene bank of Saccharomyces cerevisiae chromosomal DNA. Cloning was achieved by complementation of a recently described yeast strain that is auxotrophic for glutamine. A multicopy recombinant plasmid with a 5-kilobase-pair genomic insert conferred sixfold elevation in glutaminyl-tRNA synthetase activity and restored a Gln+ phenotype to strains that were Gln- by virtue of a mutant gln4 allele. Subfragments of the 5-kilobase insert directed integration of URA3 to GLN4. Further experiments established that GLN4 is an essential gene that is located on chromosome XV. RNA blots with a GLN4-specific probe detected a single transcript of approximately 2,900 nucleotides.

Amino Acyl-tRNA Synthetases↗

PubMed1984

Identification of a glutaminyl-tRNA synthetase mutation Saccharomyces cerevisiae.

Saccharomyces cerevisiae glutaminyl-tRNA synthetase mutants were isolated through systematic screening of tight Gln- derivatives of a leaky glutamine auxotroph. These mutations define a single nuclear gene, GLN4. The gln4-1 mutation is specific for Gln-tRNA synthetase and shows a dosage effect in heterozygous diploids. The wild-type Gln-tRNA synthetase exhibits a Km for glutamine of 25 microM; the gln4-1 mutation increases this value 20-fold. These observations strongly suggest that GLN4 encodes the Gln-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗