Peggy L. Bunger

Sex Pheromone Receptor Specificity in the European Corn Borer Moth, Ostrinia nubilalis

Background The European corn borer (ECB), Ostrinia nubilalis (Hubner), exists as two separate sex pheromone races. ECB(Z) females produce a 97∶3 blend of Z11- and E11-tetradecenyl acetate whereas ECB(E) females produce an opposite 1∶99 ratio of the Z and E isomers. Males of each race respond specifically to their conspecific females blend. A closely related species, the Asian corn borer (ACB), O. furnacalis, uses a 3∶2 blend of Z12- and E12-tetradecenyl acetate, and is believed to have evolved from an ECB-like ancestor. To further knowledge of the molecular mechanisms of pheromone detection and its evolution among closely related species we identified and characterized sex pheromone receptors from ECB(Z). Methodology Homology-dependent (degenerate PCR primers designed to conserved amino acid motifs) and homology-independent (pyrophosphate sequencing of antennal cDNA) approaches were used to identify candidate sex pheromone transcripts. Expression in male and female antennae was assayed by quantitative real-time PCR. Two-electrode voltage clamp electrophysiology was used to functionally characterize candidate receptors expressed in Xenopus oocytes. Conclusion We characterized five sex pheromone receptors, OnOrs1 and 3–6. Their transcripts were 14–100 times more abundant in male compared to female antennae. OnOr6 was highly selective for Z11-tetradecenyl acetate (EC50 = 0.86±0.27 µM) and was at least three orders of magnitude less responsive to E11-tetradecenyl acetate. Surprisingly, OnOr1, 3 and 5 responded to all four pheromones tested (Z11- and E11-tetradecenyl acetate, and Z12- and E12-tetradecenyl acetate) and to Z9-tetradecenyl acetate, a behavioral antagonist. OnOr1 was selective for E12-tetradecenyl acetate based on an efficacy that was at least 5-fold greater compared to the other four components. This combination of specifically- and broadly-responsive pheromone receptors corresponds to published results of sensory neuron activity in vivo. Receptors broadly-responsive to a class of pheromone components may provide a mechanism for variation in the male moth response that enables population level shifts in pheromone blend use.

Single mutation to a sex pheromone receptor provides adaptive specificity between closely related moth species.

Sex pheromone communication, acting as a prezygotic barrier to mating, is believed to have contributed to the speciation of moths and butterflies in the order Lepidoptera. Five decades after the discovery of the first moth sex pheromone, little is known about the molecular mechanisms that underlie the evolution of pheromone communication between closely related species. Although Asian and European corn borers (ACB and ECB) can be interbred in the laboratory, they are behaviorally isolated from mating naturally by their responses to subtly different sex pheromone isomers, (E)-12- and (Z)-12-tetradecenyl acetate and (E)-11- and (Z)-11-tetradecenyl acetate (ACB: E12, Z12; ECB; E11, Z11). Male moth olfactory systems respond specifically to the pheromone blend produced by their conspecific females. In vitro, ECB(Z) odorant receptor 3 (OR3), a sex pheromone receptor expressed in male antennae, responds strongly to E11 but also generally to the Z11, E12, and Z12 pheromones. In contrast, we show that ACB OR3, a gene that has been subjected to positive selection (ω = 2.9), responds preferentially to the ACB E12 and Z12 pheromones. In Ostrinia species the amino acid residue corresponding to position 148 in transmembrane domain 3 of OR3 is alanine (A), except for ACB OR3 that has a threonine (T) in this position. Mutation of this residue from A to T alters the pheromone recognition pattern by selectively reducing the E11 response ∼14-fold. These results suggest that discrete mutations that narrow the specificity of more broadly responsive sex pheromone receptors may provide a mechanism that contributes to speciation.

Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences.

Neutrophils play an essential role in the cellular defense of the bovine mammary gland and compromised leukocyte function has been linked to the development of bovine mastitis. During mastitis, large numbers of leukocytes migrate into the mammary tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide aniongenerating complex known as the NADPH oxidase. The key membrane‐associated component of the NADPH oxidase is flavocytochrome b, which is a heterodimer of p22‐phox and gp91‐phox. Currently, only the human, porcine, murine, and rattus p22‐phox and the human, porcine, and murine gp91‐phox gene sequences are known. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and analyze expression of bovine flavocytochrome b. Using polymerase chain reaction cloning techniques and a bovine spleen cDNA library we have cloned both of the bovine flavocytochrome b subunits, p22‐phox and gp91‐phox. Comparison of the bovine sequences with those of other species also revealed important information regarding key structural features of gp91‐phox and p22‐phox, including location of putative glycosylation sites. This study greatly contributes to our understanding of the potential functional sites of the flavocytochrome b subunits as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases such as mastitis. J. Leukoc. Biol. 64: 114–123; 1998.

Platelet-activating factor induces a concentration-dependent spectrum of functional responses in bovine neutrophils.

We characterized the dose response of bovine neutrophils to platelet‐activating factor (PAF) with respect to the following functions: calcium flux and membrane potential changes, actin polymerization, degranulation, and the production and/or priming of the oxidative burst. PAF at very low concentrations (10 and 10 M) caused changes in intracellular calcium and membrane potential in bovine neutrophils, whereas moderate PAF concentrations (≥10–7 M) resulted in increased actin polymerization. Degranulation responses to PAF were more complex: low concentrations (10–9 M) caused secretory granule degranulation, moderate doses (≥ 10–7 M) caused specific granule degranulation, whereas azurophil degranulation only occurred at high (10–5 M) PAF concentrations. Treatment of bovine neutrophils with PAF at concentrations ≥10–7 M also caused up‐regulation of the adhesion molecules Mac‐1 and L‐selectin. PAF stimulation resulted in a very weak [compared to phorbol myristate acetate (PMA)] oxidative burst in bovine neutrophils, and only at high (10–6 M) concentrations. Unlike human neutrophils, bovine neutrophils were poorly primed by PAF treatment. Only high concentrations of PAF (10–5 M) caused an increased rate of PMA‐stimulated superoxide production, although lower doses of PAF did reduce the lag time preceding the PMA‐induced oxidative burst. The overall pattern that can be inferred is that lower concentrations of PAF promote neutrophil sensitivity and interaction by selective degranulation, up‐regulation of adhesion molecules, and increased actin polymerization. In contrast, higher PAF concentrations can promote, albeit weakly, more direct bactericidal responses, such as the release of reactive oxygen species and granule enzymes. The ability of PAF to modulate a graded response in bovine neutrophils would allow the cell to respond proportionally to the severity of a stimulus. J. Leukoc. Biol. 64: 817–827; 1998.

AP-1 is essential for p67phox promoter activity

The cytosolic NADPH oxidase cofactor p67phoxhas been shown to be one of the limiting factors in assembly andactivation of this multi‐protein enzyme complex and, therefore, must behighly regulated at the transcriptional level. In the present studies, we have further characterized the promoter for humanp67phox. Genomic sequence upstream of thetranslational start site (TLS; 2 kb) was cloned, and RACE was used toidentify and compare the transcriptional start site (TSS) in twomyeloid cell lines, HL‐60 and PLB‐985. Two major TSS were identifiedwithin the first intron for both cell lines, and one transcriptisolated from PLB‐985 cells started approximately 34 bp 5′ of exon 1and contained no intron 1 sequence. To identify regulatory regions ofthe promoter, a luciferase reporter was used to assay a series ofpromoter deletion constructs. The greatest transcriptional activity wasobserved for fragments containing at least 500 bp upstream of the TLS. Sequence analysis of the p67phox promoterrevealed consensus binding sites for previously described transcriptionfactors including AP‐1 and PU.1. Site‐directed mutagenesis of the AP‐1site demonstrated that this site was essential for basal transcription. EMSA, competition, and super‐shift assays showed that this site wasspecifically recognized by nuclear factors of the AP‐1 family. EMSAanalysis and promoter‐reporter assays with the PU.1 consensus sites atpositons ‐176, ‐283, and ‐328 demonstrate that PU.1 binds the site atposition ‐283 with high affinity. Mutagenesis of any one of the PU.1sites reduced the basal transcriptional activity by approximately 50%,demonstrating that, although none of these sites is singularlyresponsible for the basal transcriptional activity, all three sitesplay some role in the transcriptional activity of thep67phox promoter. In support of thisconclusion, mutagenesis of all three sites completely abrogatedtranscriptional activity.

Cloning and sequencing of rabbit leukocyte NADPH oxidase genes reveals a unique p67phox homolog

The NADPH oxidase plays an important role in immune and nonimmune cell functions. Because rabbits represent an established model for studying a number of important disease processes that involve NADPH oxidase activity, we carried out studies to clone and sequence all five rabbit leukocyte NADPH oxidase genes. Comparison of the rabbit sequences with those of other species showed that, with the exception of p67phox, the rabbit phox proteins were highly conserved. In contrast, rabbit p67phox had a very divergent C‐terminus and was 17 amino acids longer than any other known p67phox homolog. This was surprising, given the high degree of conservation among all of the phox proteins sequenced previously. To evaluate the functional consequences of this difference, wild‐type rabbit p67phox and a mutated rabbit p67phox missing the C‐terminal 17 amino acids were expressed and analyzed in a cell‐free assay. Our results show that the full‐length and truncated rabbit p67phox proteins were able to support oxidase activity, although the truncated form reproducibly supported a higher level of activity than full‐length p67phox. These studies contribute to our understanding of the nature of the leukocyte NADPH oxidase in different species and will be valuable in future research using the rabbit model.

Cloning and expression of bovine p47-phox and p67-phox: comparison with the human and murine homologs.

Neutrophils play an essential role in bovine cellular host defense, and compromised leukocyte function has been linked to the development of respiratory and mucosal infections. During the host defense process, neutrophils migrate into infected tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion‐generating complex known as the NADPH oxidase. Two of the essential cytosolic components of the NADPH oxidase are p47‐phox and p67‐phox. Currently, only the human and murine homologs of these proteins have been sequenced. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and express bovine p47‐phox and p67‐phox. Using polymerase chain reaction (PCR) cloning techniques and a bovine bone marrow cDNA library, we have cloned both of these bovine NADPH oxidase cytosolic components. Comparison of the bovine sequences with those of the human and murine homologs showed that they were highly conserved, but also revealed important information regarding key structural features of p47‐phox and p67‐phox, including location of putative phosphorylation sites. Functional expression of bovine p47‐phox and p67‐phox showed that these proteins could substitute for the human proteins in reconstituting NADPH oxidase activity in a cell‐free assay system, again demonstrating the high degree of conservation between human and bovine homologs. This study greatly contributes to our understanding of the potential structural/functional regions of p47‐phox and p67‐phox as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases. J. Leukoc. Biol. 67: 63–72; 2000.

Molecular analysis of the bovine anaphylatoxin C5a receptor

Recruitment of phagocytes to inflammatory sites involves the coordinated action of several chemoattractants, including the anaphylatoxin C5a. While the C5a receptor (C5aR) has been well characterized in humans and rodents, little is known about the bovine C5aR. Here, we report cloning of bovine C5R1, the gene encoding bovine C5aR. We also analyzed genomic sequence upstream of the C5R1 translation start site. Although the bovine C5aR amino acid sequence was well conserved among species, significant differences in conserved features were found, including major differences in the N terminus, intracellular loop 3, and transmembrane domain VII. Analysis of C5aR expression by flow cytometry and confocal microscopy demonstrated high levels of C5aR on all bovine neutrophils and a subset of bovine monocytes. C5aR was not expressed on resting or activated bovine lymphocytes, although C5aR message was present in these cells. C5aR was also expressed on a small subset of bovine mammary epithelial cells. Pharmacological analysis of bovine C5aR‐mediated responses showed that bovine C5a and C5adesArg both induced dose‐dependent calcium fluxes and chemotaxis in bovine neutrophils, with similar efficacy for both agonists. Treatment of bovine neutrophils with C5a or C5adesArg resulted in homologous desensitization of bovine C5aR and cross‐desensitization to interleukin 8 (IL‐8) and platelet‐activating factor (PAF); whereas, treatment with IL‐8 or PAF did not cross‐desensitize the cells to C5a or C5adesArg. Overall, these studies provide important information regarding distinct structural and functional features that may contribute to the unique pharmacological properties of bovine C5aR.

Identification of a novel tumor necrosis factor α-responsive region in the NCF2 promoter

The phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is a multiprotein enzyme that catalyzes the production of microbicidal oxidants. Although oxidase assembly involves association of several membrane and cytosolic oxidase proteins, one of the cytosolic cofactors, p67phox, appears to play a more prominent role in final activation of the enzyme complex. Based on the importance of p67phox, we investigated transcriptional regulation of the p67phox gene [neutrophil cytosolic factor 2 (NCF2)] and demonstrated previously that activator protein‐1 (AP‐1) was essential for basal transcriptional activity. As p67phox can be up‐regulated by tumor necrosis factor α (TNF‐α), which activates AP‐1, we hypothesized that TNF‐α might regulate NCF2transcription via AP‐1. In support of this hypothesis, we show here that NCF2 promoter‐reporter constructs are up‐regulated by TNF‐α but only when AP‐1 factors were coexpressed. Consistent with this observation, we also demonstrate that NCF2 mRNA and p67phox protein are up‐regulated by TNF‐α in various myeloid cell lines as well as in human monocytes. It was surprising that mutagenesis of the AP‐1 site in NCF2 promoter constructs did not eliminate TNF‐α induction, suggesting additional elements were involved in this response and that AP‐1 might play a more indirect role. Indeed, we used NCF2 promoter‐deletion constructs to map a novel TNF‐α‐responsive region (TRR) located between −56 and −16 bp upstream of the translational start site and demonstrated its importance in vivo using transcription factor decoy analysis. Furthermore, DNase footprinting verified specific binding of factor(s) to the TRR with AP‐1 binding indirectly to this region. Thus, we have identified a novel NCF2 promoter/enhancer domain, which is essential for TNF‐α‐induced up‐regulation of p67phox.

Molecular analysis of the bison phagocyte NADPH oxidase: cloning and sequencing of five NADPH oxidase cDNAs

During the host defense process, neutrophils migrate into infected tissues where they become activated, resulting in the assembly of a superoxide anion-generating complex known as the NADPH oxidase. Despite the importance of this system in animal host defense, almost nothing is known about the NADPH oxidase in neutrophils from wild ruminant species. In the present studies, we provide a molecular analysis of the bison leukocyte NADPH oxidase. Using reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends, we cloned and sequenced the full-length cDNAs for five bison NADPH oxidase components: p22(phox), p40(phox), p47(phox) and p67(phox), and gp91(phox). When compared to other species, the deduced amino acid sequences of the bison homologs were most similar to those of bovine. Interestingly, a bison p40(phox) alternative splice product was isolated, which was similar to that observed for human p40(phox) in that the cDNAs contained sequence from intron 8. Consistent with the high degree of similarity between bison and bovine amino acid sequences, immunoblot analysis showed that the bison homologs migrated similarly to their bovine counterparts. Overall, these studies show that the bison and bovine NADPH oxidase genes are highly conserved between these two species, despite their divergence from a common ancestor over 1 million years ago.