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Bacillus anthracis is surrounded by an anti-phagocytic capsule that is entirely composed of γ-linked D-glutamic acid (γDPGA). γDPGA is required for virulence and is produced in large quantities following spore germination. We have previously described the isolation of several γDPGA-reactive mAbs. The reagents are effective in both immunoprotection and diagnostic applications. The current work was done to further investigate the specificity of γDPGA-reactive mAbs. The specificity of each mAb was characterized using surface plasmon resonance. Our results indicate that each mAb is stereoselective for binding to D-glutamic acid oligomers, but to varying degrees. In particular, mAb F26G3 is highly selective for γDPGA; alterations in stereochemistry disrupted recognition. These differences in mAb reactivity suggest that binding of γDPGA by mAb F26G3 is more specific than non-directional ionic interactions between a negatively charged antigen and a positively charged antibody., (Published by Elsevier Ltd.)

Bacterial capsules are common targets for antibody-mediated immunity. The capsule of Bacillus anthracis is unusual among capsules because it is composed of a polymer of poly-γ-d-glutamic acid (γdPGA). We previously generated murine IgG3 monoclonal antibodies (mAbs) to γdPGA that were protective in a murine model of pulmonary anthrax. IgG3 antibodies are characteristic of the murine response to polysaccharide antigens. The goal of the present study was to produce subclass switch variants of the γdPGA mAbs (IgG3 → IgG1 → IgG2b → IgG2a) and assess the contribution of subclass to antibody affinity and protection. Subclass switch antibodies had identical variable regions but differed in their heavy chains. The results showed that a switch from the protective IgG3 to IgG1, IgG2b or IgG2a was accompanied by i) a loss of protective activity ii) a change in mAb binding to the capsular matrix, and iii) a loss of affinity. These results identify a role for the heavy chain constant region in mAb binding. Hybrid mAbs were constructed in which the CH1, CH2 or CH3 heavy chain constant domains from a non-protective, low binding IgG2b mAb were swapped into the protective IgG3 mAb. The IgG3 mAb that contained the CH1 domain from IgG2b showed no loss of affinity or protection. In contrast, swapping the CH2 or CH3 domains from IgG2b into IgG3 produced a reduction in affinity and a loss of protection. These studies identify a role for the constant region of IgG heavy chains in affinity and protection against an encapsulated bacterial pathogen.

Ipsdienone (2-methyl-6-methylene-2,7-octadien-4-one) is an important intermediate in the biosynthesis of pheromonal ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol) in male pine engraver beetles, Ips pini (Say). A novel ipsdienol dehydrogenase (IDOLDH) with a pheromone-biosynthetic gene expression pattern was cloned, expressed, functionally characterized, and its cellular localization analyzed. The cDNA has a 762nt ORF encoding a 253 amino acid predicted translation product of 28kDa and pI 5.8. The protein has conserved motifs of the Cp2 subfamily of "classical" short-chain dehydrogenases. Transcript levels were highest in pheromone producing tissue: the anterior midgut of fed males. The protein was detected only in male midguts and localized in the cytosolic fraction of midgut cells. Recombinant IDOLDH was produced in Sf9 cells using a baculovirus expression system. Enzyme assays of protein preparations showed IDOLDH used both NAD⁺ and NADP⁺ as coenzymes with specific activities in the nanomole range. Enzyme assays and GC/MS analysis showed that IDOLDH catalyzed the oxidation of racemic ipsdienol and (4R)-(-)-ipsdienol to form ipsdienone, while (4S)-(+)-ipsdienol was not a substrate. These data strongly implicate IDOLDH as an enzyme involved in terminal pheromone-biosynthetic steps, likely functioning to "tune" ipsdienol enantiomeric ratios., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Eclosion hormone (EH) is an integral component in the cascade regulating the behaviors culminating in emergence of an insect from its old exoskeleton. Little is known regarding the EH solution structure; consequently, we utilized a computational approach to generate a hypothetical structure for Manduca sexta EH. The de novo algorithm exploited the restricted conformational space of disulfide bonds (Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49) and predicted secondary structure elements to generate a thermodynamically stable structure characterized by 55% helical content, an unstructured N-terminus, a helical C-terminus, and a solvent-exposed loop containing Trp28 and Phe29. Both the strain and pseudo energies of the predicted peptide compare favorably with those of known structures. The 62-amino acid peptide was synthesized, folded, assayed for activity, and structurally characterized to confirm the validity of the model. The helical content is supported by circular dichroism and hydrogen-deuterium exchange mass spectrometry. Fluorescence emission spectra and acrylamide quenching are consistent with the solvent exposure predicted for Trp28, which is shielded by Phe29. Furthermore, thermodynamically stable conformations that deviated only slightly from the predicted Manduca EH structure were generated in silico for the Bombyx mori and Drosophila melanogaster EHs, indicating that the conformation is not species-dependent. In addition, the biological activities of known mutants and deletion peptides were rationalized with the predicted Manduca EH structure, and we found that, on the basis of sequence conservation, functionally important residues map to two conserved hydrophobic clusters incorporating the C-terminus and the first loop.

Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full-length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three-dimensional structure of coleopteran FPPS was determined and compared to the X-ray crystal structure of avian FPPS. The alpha-helical fold is conserved in AgFPPS and the size of the active site cavity is consistent with the enzyme being a FPPS., ((c) 2009 Wiley Periodicals, Inc.)

Topologically, platelet factor-4 kinocidins consist of distinct N-terminal extended, C-terminal helical, and interposing gamma-core structural domains. The C-terminal alpha-helices autonomously confer direct microbicidal activity, and the synthetic antimicrobial peptide RP-1 is modeled upon these domains. In this study, the structure of RP-1 was assessed using several complementary techniques. The high-resolution structure of RP-1 was determined by NMR in anionic sodium dodecyl sulfate (SDS) and zwitterionic dodecylphosphocholine (DPC) micelles, which approximate prokaryotic and eukaryotic membranes, respectively. NMR data indicate the peptide assumes an amphipathic alpha-helical backbone conformation in both micelle environments. However, small differences were observed in the side-chain orientations of lysine, tyrosine, and phenylalanine residues in SDS versus DPC environments. NMR experiments with a paramagnetic probe indicated differences in positioning of the peptide within the two micelle types. Molecular dynamics (MD) simulations of the peptide in both micelle types were also performed to add insight into the peptide/micelle interactions and to assess the validity of this technique to predict the structure of peptides in complex with micelles. MD independently predicted RP-1 to interact only peripherally with the DPC micelle, leaving its spherical shape intact. In contrast, RP-1 entered deeply into and significantly distorted the SDS micelle. Overall, the experimental and MD results support a preferential specificity of RP-1 for anionic membranes over zwitterionic membranes. This specificity likely derives from differences in RP-1 interaction with distinct lipid systems, including subtle differences in side chain orientations, rather than gross changes in RP-1 structure in the two lipid environments.

Myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation is likely one of the final reactions involved in the production of the Ips spp. (Coleoptera: Scolytidae) aggregation pheromone components, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol). To gain insight into the evolution of pheromone production, we isolated a full-length cDNA from the pinyon ips, Ips confusus (LeConte), that encodes a pheromone-biosynthetic cytochrome P450, I. confusus CYP9T1 (IcCYP9T1). The recovered cDNA is 1.70 kb, and the open reading frame encodes a 532 amino acid protein. IcCYP9T1 is 94% identical to the pine engraver, Ips pini (Say), CYP9T2 ortholog that hydroxylates myrcene. Quantitative real-time PCR experiments showed that IcCYP9T1, as does CYP9T2, has an expression pattern similar to other pheromone-biosynthetic genes in I. pini. Basal expression levels were higher in males than females, and expression was significantly induced in male, but not in female, anterior midguts by feeding on host phloem. Microsomes, prepared from Sf9 cells co-expressing baculoviral-mediated recombinant IcCYP9T1 and house fly (Musca domestica) NADPH-cytochrome P450 reductase, converted myrcene to ~85%-(R)-(-)-ipsdienol. These results are consistent with IcCYP9T1 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway. Since the I. confusus pheromone blend contains >90%-(S)-(+)-ipsdienol, these results confirm further that Ips spp. myrcene hydroxylases do not control the final ipsdienol enantiomeric blend. Other enzymes are required following myrcene hydroxylation to achieve the critical quantity and enantiomeric composition of pheromonal ipsenol and ipsdienol used by different Ips spp.

Candida albicans is the most common cause of hematogenously disseminated and oropharyngeal candidiasis. Both of these diseases are characterized by fungal invasion of host cells. Previously, we have found that C. albicans hyphae invade endothelial cells and oral epithelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the fungal surface protein and host cell receptors that mediate this process. We found that the C. albicans Als3 is required for the organism to be endocytosed by human umbilical vein endothelial cells and two different human oral epithelial lines. Affinity purification experiments with wild-type and an als3delta/als3delta mutant strain of C. albicans demonstrated that Als3 was required for C. albicans to bind to multiple host cell surface proteins, including N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. Furthermore, latex beads coated with the recombinant N-terminal portion of Als3 were endocytosed by Chinese hamster ovary cells expressing human N-cadherin or E-cadherin, whereas control beads coated with bovine serum albumin were not. Molecular modeling of the interactions of the N-terminal region of Als3 with the ectodomains of N-cadherin and E-cadherin indicated that the binding parameters of Als3 to either cadherin are similar to those of cadherin-cadherin binding. Therefore, Als3 is a fungal invasin that mimics host cell cadherins and induces endocytosis by binding to N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. These results uncover the first known fungal invasin and provide evidence that C. albicans Als3 is a molecular mimic of human cadherins.

Mammalian platelets contain an array of antimicrobial peptides, termed platelet microbicidal proteins (PMPs). Human and rabbit PMPs include known chemokines, such as platelet factor-4 (hPF-4); PMP-1 is the rabbit orthologue of hPF-4. Chemokines that also exert direct antimicrobial activity have been termed kinocidins. A consensus peptide domain library representing mammalian PF-4 family members was analyzed to define structural domains contributing to antimicrobial activity against a panel of human pathogens. Secondary conformations were assessed by circular dichroism spectrometry, and molecular modeling was employed to investigate structural correlates of antimicrobial efficacy. Antimicrobial activity against isogenic peptide-susceptible or -resistant Staphylococcus aureus, Salmonella typhimurium, and Candida albicans strain pairs mapped to the C-terminal hemimer (38-74) and modular domains thereof (49-63 and 60-74). Increasing electrostatic charge and steric bulk were general correlates of efficacy. Structural data corroborated spatial distribution of charge, steric bulk and putative secondary structure with organism-specific efficacy. Microbicidal efficacies of the cPMP antimicrobial hemimer and C-terminal peptide (60-74) were retained in a complex human-blood biomatrix assay. Collectively, these results suggest that modular determinants arising from structural components acting independently and cooperatively govern the antimicrobial functions of PF-4 family kinocidins against specific target pathogens.

Chemokines are small (8-12 kDa) effector proteins that potentiate leukocyte chemonavigation. Beyond this role, certain chemokines have direct antimicrobial activity against human pathogenic organisms; such molecules are termed kinocidins. The current investigation was designed to explore the structure-activity basis for direct microbicidal activity of kinocidins. Amino acid sequence and 3-dimensional analyses demonstrated these molecules to contain iterations of the conserved gamma-core motif found in broad classes of classical antimicrobial peptides. Representative CXC, CC and C cysteine-motif-group kinocidins were tested for antimicrobial activity versus human pathogenic bacteria and fungi. Results demonstrate that these molecules exert direct antimicrobial activity in vitro, including antibacterial activity of native IL-8 and MCP-1, and microbicidal activity of native IL-8. To define molecular determinants governing its antimicrobial activities, the IL-8 gamma-core (IL-8gamma) and alpha-helical (IL-8alpha) motifs were compared to native IL-8 for antimicrobial efficacy in vitro. Microbicidal activity recapitulating that of native IL-8 localized to the autonomous IL-8alpha motif in vitro, and demonstrated durable microbicidal activity in human blood and blood matrices ex vivo. These results offer new insights into the modular architecture, context-related deployment and function, and evolution of host defense molecules containing gamma-core motifs and microbicidal helices associated with antimicrobial activity.

Bacillus anthracis is surrounded by a polypeptide capsule composed of poly-gamma-d-glutamic acid (gammaDPGA). In a previous study, we reported that a monoclonal antibody (MAb F26G3) reactive with the capsular polypeptide is protective in a murine model of pulmonary anthrax. The present study examined a library of six MAbs generated from mice immunized with gammaDPGA. Evaluation of MAb binding to the capsule by a capsular "quellung" type reaction showed a striking diversity in capsular effects. Most MAbs produced a rim type reaction that was characterized by a sharp increase followed directly by a decrease in refractive index at the capsular edge. Some MAbs produced a second capsular reaction well beneath the capsular edge, suggesting complexity in capsular architecture. Binding of MAbs to soluble gammaDPGA was assessed by a fluorescence perturbation assay in which a change in the MAb intrinsic fluorescence produced by ligand binding was used as a reporter for antigen-antibody interaction. The MAbs differed considerably in the complexity of the binding curves. MAbs producing rim type capsule reactions typically produced the more complex binding isotherms. Finally, the protective activity of the MAbs was compared in a murine model of pulmonary anthrax. One MAb was markedly less protective than the remaining five MAbs. Characteristics of the more protective MAbs included a relatively high affinity, an immunoglobulin G3 isotype, and a complex binding isotherm in the fluorescence perturbation assay. Given the relatively monotonous structure of gammaDPGA, the results demonstrate a striking diversity in the antigen binding behavior of gammaDPGA antibodies.

The final steps in the pheromone-biosynthetic pathway of the pine engraver beetle, Ips pini (Say) (Coleoptera: Scolytidae) are unknown, but likely involve myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation to produce the aggregation pheromone component, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol). We have isolated a full-length I. pini cDNA encoding a cytochrome P450, CYP9T2. The recovered cDNA is 1.83kb and the open reading frame encodes a 532 amino acid protein. CYP9T2 is regulated by the same physiological factors that induce pheromone production. Quantitative real-time PCR experiments showed that feeding on host phloem induced CYP9T2 expression in males, but not females, and that basal expression levels are highest in male midguts, similar to other I. pini pheromone-biosynthetic genes. Microsomes prepared from Sf9 cells co-expressing baculoviral-mediated recombinant CYP9T2 and housefly (Musca domestica) NADPH-cytochrome P450 reductase converted myrcene to ipsdienol. The product identified by coupled GC-MS was mostly (4R)-(-)-ipsdienol, an important aggregation pheromone component for western North American I. pini. These results are consistent with CYP9T2 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway.

HIV attachment via the CD4 receptor is an important target for developing novel approaches to HIV chemotherapy. Cyclotriazadisulfonamide (CADA) inhibits HIV at submicromolar levels by specifically down-modulating cell-surface and intracellular CD4. An effective five-step synthesis of CADA in 30% overall yield is reported. This synthesis has also been modified to produce more than 50 analogues. Many tail-group analogues have been made by removing the benzyl tail of CADA and replacing it with various alkyl, acyl, alkoxycarbonyl and aminocarbonyl substituents. A series of sidearm analogues, including two unsymmetrical compounds, have also been prepared by modifying the CADA synthesis, replacing the toluenesulfonyl sidearms with other sulfonyl groups. Testing 30 of these compounds in MT-4 cells shows a wide range of CD4 down-modulation potency, which correlates with ability to inhibit HIV-1. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) approaches. The X-ray crystal structures of four compounds, including CADA, show the same major conformation of the central 12-membered ring. The solid-state structure of CADA was energy minimized and used to generate the remaining 29 structures, which were similarly minimized and aligned to produce the 3D-QSAR models. Both models indicate that steric bulk of the tail group, and, to a lesser extent, the sidearms mainly determine CD4 down-modulation potency in this series of compounds.

Geranyl diphosphate synthase (GPPS) catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to form geranyl diphosphate. Geranyl diphosphate is the precursor of monoterpenes, a large family of natural occurring C(10) compounds predominantly found in plants. Similar to plants but unique to animals, some bark beetle genera (Coleoptera: Scolytidae) produce monoterpenes that function in intraspecific chemical communication as aggregation and dispersion pheromones. The release of monoterpene aggregation pheromone mediates host colonization and mating. It has been debated whether these monoterpene pheromone components are derived de novo through the mevalonate pathway or result from simple modifications of dietary precursors. The data reported here provide conclusive evidence for de novo biosynthesis of monoterpene pheromone components from bark beetles. We describe GPPS in the midgut tissue of pheromone-producing male Ips pini. GPPS expression levels are regulated by juvenile hormone III, similar to other mevalonate pathway genes involved in pheromone biosynthesis. In addition, GPPS transcript is almost exclusively expressed in the anterior midgut of male I. pini, the site of aggregation pheromone biosynthesis. The recombinant enzyme was functionally expressed and produced geranyl diphosphate as its major product. The three-dimensional model structure of GPPS shows that the insect enzyme has the sequence structural motifs common to E-isoprenyl diphosphate synthases.

A water-soluble conjugate (1) with intact carboxyl groups was prepared by addition of poly(ethylene glycol) thiol (MPEG-SH) regiospecifically to the exo vinyl group of bilirubin. (1)H and (13)C NMR and absorbance spectroscopy in CDCl(3) and DMSO-d(6) confirmed the assigned structure and showed that pegylation did not disrupt the hydrogen-bonded ridge-tile conformation of the pigment moiety. Aqueous solutions of 1 were optically clear, but NMR signals were seen only from the MPEG portion and none from the tetrapyrrole, consistent with dissolved assemblies containing aggregated bilirubin cores within mobile polyether chains. On alkalinization (pH >12), signals from the pigment moiety reappeared. Titrimetric measurements on 1 in water showed the pK(a)'s of the two carboxyl groups to be similar (average 6.42). Control studies with pegylated half-esters of succinic, suberic, brassylic, thapsic, and 1,20-eicosanedioic acid showed that pegylation per se has little, if any, effect on carboxyl ionization. However, aggregation increases the apparent pK(a) by approximately 1-2 units. The molecularity of bilirubin in solution was further characterized by ultracentrifugation. Over the pH range 8.5-10 in buffer, bilirubin formed multimers with aggregation numbers ranging from approximately 2-7. Bilirubin is monomeric in DMSO or CHCl(3) at approximately 2 x 10(-)(5) M, but aggregation occurred when the CHCl(3) was contaminated with trace adventitious (perhaps lipoidal) impurities. These observations show that aggregation increases the pK(a)'s of aliphatic carboxylic acids relative to their monomer values in water. They are consistent with earlier (13)C NMR-based estimates of approximately 4.2 and approximately 4.9 for the aqueous pK(a)'s of bilirubin and similar studies of bilirubin in micellar bile-salt solutions. Together with earlier work, they confirm that the pK(a)'s of bilirubin are about normal for aliphatic carboxyls and suggest that the high (>7.5) values occasionally reported, including those based on CHCl(3) partitioning, are artifacts of aggregation or technique.

Mammalian platelets release platelet microbicidal proteins (PMPs) as components of their antimicrobial armamentarium. The present studies defined the structure of PMP-1 and examined its structure-activity relationships. Amino acid sequencing and mass spectroscopy demonstrated that distinct N-terminal polymorphism variants of PMP-1 isolated from nonstimulated or thrombin-stimulated platelets arise from a single PMP-1 propeptide. Sequence data (NH(2)-[S]D(1)DPKE(5)SEGDL(10)HCVCV(15)KTTSL(20) . . .) enabled cloning of PMP-1 from bone marrow and characterization of its full-length cDNA. PMP-1 is translated as a 106-amino-acid precursor and is processed to yield 73-residue (8,053 Da) and 72-residue (7,951-Da) variants. Searches with the BLAST program and sequence alignments demonstrated the homology of PMP-1 to members of the mammalian platelet factor 4 (PF-4) family of proteins. On the basis of phylogenetic relatedness, congruent sequence motifs, and predicted three-dimensional structures, PMP-1 shares the greatest homology with human PF-4 (hPF-4). By integration of its structural and antimicrobial properties, these results establish the identity of PMP-1 as a novel rabbit analogue of the microbicidal chemokine (kinocidin) hPF-4. These findings advance the hypothesis that stimuli in the setting of infection prompt platelets to release PF-4-class or related kinocidins, which have structures consistent with their likely multiple roles that bridge molecular and cellular mechanisms of antimicrobial host defense.

Bacterial indole-3-acetyl-l-aspartic acid (IAA-Asp) hydrolase has shown very high substrate specificity compared with similar IAA-amino acid hydrolase enzymes found in Arabidopsis thaliana. The IAA-Asp hydrolase also exhibits, relative to the Arabidopsis thaliana-derived enzymes, a very high Vmax (fast reaction rate) and a higher Km (lower substrate affinity). These two characteristics indicate that there are fundamental differences in the catalytic activity between this bacterial enzyme and the Arabidopsis enzymes. By employing a computer simulation approach, a catalytic residue, His-385, from a non-sequence-related zinc-dependent exopeptidase of Pseudomonas was found to structurally match His-405 of IAA-Asp hydrolase. The His-405 residue is conserved in all related sequences of bacteria and Arabidopsis. Point mutation experiments of this His-405 to seven different amino acids resulted in complete elimination of enzyme activity. However, point mutation on the neighboring His-404 to eight other residues resulted in reduction, to various degrees, of enzyme activity. Amino acid substitutions for His-404 also showed that this residue influenced the minor activity of the IAA-Asp hydrolase for the substrates IAA-Gly, IAA-Ala, IAA-Ser, IAA-Glu and IAA-Asn. These results show the value and potential of structural modeling for predicting target residues for further study and for directing bioengineering of enzyme structure and function.

The human fungal pathogen Candida albicans colonizes and invades a wide range of host tissues. Adherence to host constituents plays an important role in this process. Two members of the C. albicans Als protein family (Als1p and Als5p) have been found to mediate adherence; however, the functions of other members of this family are unknown. In this study, members of the ALS gene family were cloned and expressed in Saccharomyces cerevisiae to characterize their individual functions. Distinct Als proteins conferred distinct adherence profiles to diverse host substrates. Using chimeric Als5p-Als6p constructs, the regions mediating substrate-specific adherence were localized to the N-terminal domains in Als proteins. Interestingly, a subset of Als proteins also mediated endothelial cell invasion, a previously unknown function of this family. Consistent with these results, homology modeling revealed that Als members contain anti-parallel beta-sheet motifs interposed by extended regions, homologous to adhesins or invasins of the immunoglobulin superfamily. This finding was confirmed using circular dichroism and Fourier transform infrared spectrometric analysis of the N-terminal domain of Als1p. Specific regions of amino acid hypervariability were found among the N-terminal domains of Als proteins, and energy-based models predicted similarities and differences in the N-terminal domains that probably govern the diverse function of Als family members. Collectively, these results indicate that the structural and functional diversity within the Als family provides C. albicans with an array of cell wall proteins capable of recognizing and interacting with a wide range of host constituents during infection.

Manduca sexta juvenile hormone diol kinase (JHDK) catalyzes the conversion of juvenile hormone (JH) diol to JH diol phosphate. JHDK may be the first example of a phosphotransferase directly involved in the catabolism and inactivation of a lipid-soluble hormone. JHDK is an enzyme crucial for secondary metabolism of JH and possesses high specificity and catalytic efficiency for JH diol. In this study, the purification and characterization of native JHDK are described; its enzymatic properties are examined; and its role in cellular JH metabolism is explored. Using a variety of potential substrates, we show that JHDK has a preference for ATP, but will catalyze the formation of JH diol phosphate with GTP as the phosphate donor. JHDK has a nanomolar K(m) for JH I diol and a low micromolar value for MgATP. JH II and III diols also serve as phosphate acceptors with low micromolar K(m), whereas other diol derivatives of terpenoid esters structurally similar to JH metabolites are not phosphorylated. The reaction proceeds via a sequential Bi Bi mechanism. JHDK is active as a homodimer with a subunit molecular mass of 20 kDa. JHDK binds 5'-p-fluorosulfonylbenzoyladenosine and is inhibited by micromolar levels of Ca2+.

The gene sequence of Manduca sexta juvenile hormone diol kinase (JHDK) codes for an enzyme that has 59% sequence identity to Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2). JHDK and dSCP2 are similar to G-proteins with three conserved sequence elements involved in purine nucleotide binding. Both proteins contain two pairs of EF-hand motifs. Characterization and partial purification of the D. melanogaster homolog of M. sexta JHDK from adult D. melanogaster gave material with JHDK activity. This activity has an experimental pI and molecular mass that are nearly identical to those of dSCP2. Moreover, D. melanogaster phosphotransferase activity has very similar chromatographic retention in three systems compared with M. sexta JHDK. Substrate docking to three-dimensional models of JHDK has shown that the three conserved nucleotide-binding elements surround the putative substrate-binding site and align with conserved sequence elements of p21(Ras) and adenylate kinase. D. melanogaster dSCP2 is a homolog of M. sexta JHDK, and these proteins constitute a novel kinase family that binds nucleotides using the scaffold of an SCP (Protein Data Bank code ).

Cytotoxic lymphocytes kill virally infected cells with specialized cytotoxic granules containing perforin, a protein that forms toxic pores in the target cell membrane. These specialized cytotoxic granules also contain calreticulin, an endoplasmic reticulum chaperone protein. The calcium-independent association of perforin and calreticulin prompted our evaluation of calreticulin's potential to function as a regulatory molecule that protects cytotoxic lymphocytes from their own perforin. We report here that 10(-7) M calreticulin blocked perforin-mediated lysis in the hemolytic model system using erythrocytes as targets. Previously, we found that millimolar levels of calcium in the hemolytic assays dissociate high-affinity perforin-calreticulin complexes, which makes it unlikely that perforin associates with calreticulin in solution when hemolysis is blocked. Calreticulin may affect perforin at the erythrocyte membrane. We observed calcium-dependent binding of calreticulin to erythrocyte membranes with a Kd of 2.7 x 10(-7) M and a saturation average of 10(5) molecules calreticulin per erythrocyte. At concentrations that blocked hemolysis, calreticulin occupied many of the calreticulin membrane-binding sites and was in molar excess of perforin. These observations open the possibilities that membrane-bound calreticulin prevents hydrophobic entry of perforin into membranes and (or) prevents perforin from assembling into polyperforin pores.

A novel membrane-bound fatty acid synthetase (FAS) associated with the microsomal fraction from the housefly, Musca domestica, was solubilized and purified to homogeneity. The microsomal FAS was solubilized by 0.75 M KCl in phosphate buffer and was purified to homogeneity by the sequential use of ammonium sulfate precipitation followed by Sepharose CL-6B, DEAE Sephacel and Red Agarose (dye ligand affinity) chromatography. The specific activity of the microsomal FAS was increased 1,440-fold to 6,522 U/mg during purification. The cytosolic FAS from the housefly was also purified by similar methods and the specific activity increased 183-fold to 7,533 U/mg. The relative molecular mass of the microsomal and cytosolic FAS are 419 +/- 22 kDa and 405 +/- 18 kDa, respectively, for the dimers as determined by gel permeation chromatography. The microsomal and the cytosolic FAS yield different tryptic digestion maps and have slightly different amino acid compositions, which demonstrate structural differences between the two FASs. In addition, there are differences between the two FASs in their kinetic characteristics and their ability to incorporate methylmalonylCoA into the growing fatty acyl chain.

The activity of the 1-acyl-sn-glycero-3-phosphocholine acyltransferase enzyme (E.C. 2.3.1.??) was measured with three radically different acceptor substrates: 1-palmitoyl-sn-glycero-3-phosphocholine (P-sn-G3PC), 1-palmitoyl-sn-glycero-2-phosphocholine (P-sn-G2PC), and 1-hexadecyl-sn-glycero-3-phosphocholine (He-sn-G3PC). It was found that the enzyme had similar activity with P-sn-G3PC, the natural acceptor substrate, and with P-sn-G2PC. The enzyme showed no detectable activity toward He-sn-G3PC. These results are much different than would be expected from simple examination of the structures. Computer-assisted molecular modeling was done to study the geometrical configurations and to focus upon the similarities and differences of the three substrate acceptor molecules. Three bond distances were selected as important for enzyme recognition: the distance between the oxygen of the acceptor hydroxyl group and 1) the phosphorus; 2) the nitrogen; and 3) the oxygen bridge to the hydrocarbon chain. There were striking similarities for the bond distances of two of the three acceptor substrates, P-sn-G3PC and P-sn-G2PC. These were the two molecules that were shown to have activity with the enzyme. The bond distances found for the enzymically inactive acceptor substrate, He-sn-G3PC, differed significantly from P-sn-G3PC and P-sn-G2PC. Therefore, this latter molecule probably does not fit into the active site of the enzyme. The modeling data are also consistent with the experimental observation that He-sn-G3PC is not an inhibitor.

Monoclonal antibodies that react with the capsular polysaccharide, termed glucuronoxylomannan (GXM), of Cryptococcus neoformans have potential roles in the diagnosis, monitoring of disease progress, and immunotherapy of cryptococcal GXM of all four serotypes. A molecular model of the Fab fragment of monoclonal antibody 439 was constructed from the amino acid sequence and a template antibody molecule, Fab 4-4-20. A tryptophan is present on the surface between light chain CDR3 and heavy chain CDR3 in the putative binding site. This tryptophan residue proved to be a reporter group, and a fluorescence study of Fab 439 was performed to analyze the interaction between cryptococcal GXM and Fab 439. Binding of the polysaccharide enhanced the intrinsic fluorescence and caused a blue shift in the emission maximum, indicating that the environment of a tryptophan changes from a polar to less polar environment. This is consistent with the loss of water from the binding site caused by the binding of antigen. This interpretation was confirmed by acrylamide quenching, which showed that 1 less tryptophan was exposed to solvent in the Fab-GXM complex than in free Fab. Fluorescence titration was used to determine binding and dissociation constants (KD). The apparent KD values for serotypes A-C were approximately the same; the KD for serotype D GXM was 5-11-fold greater. De-O-acetylation of serotype A GXM produced a 31-fold increase in the KD, indicating that the O-acetyl groups are important, but not essential, for binding. Carboxyl groups appear to be essential for strong binding because the KD for carboxyl-reduced GXM was so large that it could not be determined.

The methylation reactions which convert phosphatidylethanolamine (PE) to phosphatidylcholine (PC) have been studied kinetically using exogenously added intermediates and crude membrane preparations from brain. The addition of exogenous PE resulted in no change in the methylation rates compared to that of endogenous PE. The addition of the two intermediates, monomethylphosphatidylethanolamine (PMME) and dimethylphosphatidylethanolamine (PDME), resulted in significantly increased rates of methylation and allowed the kinetic analysis of these latter two methylation reactions. The mechanism for this enzyme appears to be similar to human RBC (Reitz et al. (1989) J. Biol. Chem. 264, 8097-8106) which was a rapid-equilibrium random Bi-Bi sequential mechanism. There were some slight differences between the brain enzyme and that from the RBC, but there is little reason to suggest a fundamentally different mechanism. It is more likely that the differences may relate to an additional dead-end complex for the enzyme from brain such that saturation with AdoMet cannot eliminate AdoHcy inhibition. The KM values for the two phospholipid substrates were 41-44 microM and 39 microM for the methylation of PMME and PDME, respectively. The KM for S-adenosylmethionine (AdoMet) was 7-9 microM with PMME and 4 microM with PDME as the other substrates. The Ki(lipid) varied from 54 microM with PMME to 225 microM with PDME, and the Ki(AdoMet) was 11 microM with PMME and 21 microM with PDME. The product from the use of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a noncompetitive inhibitor of both lipid substrates as well as AdoMet. The methylation of PMME was somewhat higher in cerebellum and brain stem compared to cortex and striatum, but the methylation of PDME was similar in cerebellum, brain stem and cortex.

Encapsulated and nonencapsulated cryptococci exhibit quantitative and qualitative differences in their activation of the complement system. We examined the kinetics for the rapid amplification phase in which C3 was activated and bound to encapsulated cryptococci, nonencapsulated cryptococci, and zymosan particles. Yeast cells were incubated in normal human serum containing 125I-labeled C3, and bound C3 fragments were measured after 1 to 64 min of incubation. A kinetic analysis showed that the apparent first-order rate constant (k') for binding of C3 to nonencapsulated cryptococci did not differ significantly from k' for binding of C3 to zymosan particles (P greater than 0.05). However, the rate constant for binding of C3 to encapsulated cryptococci was significantly (P less than 0.001) greater than k' for binding of C3 to nonencapsulated cryptococci and zymosan particles. A plot of C3 molecules bound to encapsulated cryptococci versus time cubed was nearly linear, suggesting that accumulation of C3 in the cryptococcal capsule follows the kinetics predicted by an expanding sphere. In contrast, the plot of C3 molecules bound to nonencapsulated cryptococci or zymosan particles against time was nearly linear, but those plots against time squared or time cubed were not. This result indicates that the rate-limiting step for the addition of C3 fragments to these latter yeast cells follows the kinetics of neither the perimeter of an expanding circle nor the surface of an expanding sphere. Taken together, the results indicate that the high rate of accumulation of C3 in the cryptococcal capsule is consistent with the expected geometry of an expanding sphere of bound C3 within the three-dimensional matrix of the capsule.

The successive methylations of phosphatidylethanolamine to form phosphatidylcholine were measured using exogenously added intermediates and membrane preparations from human red blood cells. The addition of phosphatidylethanolamine resulted in no increase in methylation rate over that with endogenous substrate; however, the addition of monomethylphosphatidylethanolamine (PME) and dimethylphosphatidylethanolamine (PDE) markedly increased the reaction rate and allowed studies into the kinetic mechanism for the second and third methylation reactions. The data are consistent with catalysis of the last two methylations being by a single enzyme with a random Bi-Bi sequential mechanism. Analysis of PDE:phosphatidylcholine product ratios indicates that the enzyme can conduct multiple methylations of enzyme-bound phospholipid. The nature of the acyl chain (16:0 versus 18:1) of the phospholipid had only a small effect on the value of the kinetic constants. The maximal velocities obtained with the 18:1 substrate were less than 5% lower than those obtained with the 16:0 substrate. The Km values for the two phospholipids were 20-45 and 10-14 microM for the methylation of PME and PDE, respectively. The Km for S-adenosylmethionine (AdoMet) was 5-9 microM with PME and 4 microM with PDE as substrates. Depending on the acyl chain and the phospholipid, the Ki(AdoMet) varied from 8 to 19 microM, the Ki(PME) from 41 to 82 microM, and the Ki(PDE) from 35 to 61 microM. The Ki for S-adenosylhomocysteine (AdoHcy) was between 1.0 and 1.4 microM depending upon the variable substrate. The endogenous concentrations of PME and PDE in red blood cell membranes were estimated to be 0.49 and 0.24 mumol/liter packed cells, respectively. The product from the utilization of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a competitive inhibitor of its precursor, AdoMet, and a noncompetitive inhibitor of the two phospholipid substrates.

Herpesviruses have been isolated from white-tailed, white-bearded and blue wildebeest, as well as from Jimela topi and Cape hartebeest. These animals are members of the sub-family Alcelaphinae of the family Bovidae. Viruses isolated from wildebeest cause malignant catarrhal fever (MCF) in susceptible ruminant species. Alcelaphine herpesviruses (AHV) isolated from wildebeest replicate in both fetal aoudad sheep kidney (FAK) cells and bovine embryonic lung (BEL) cells. However, virus isolates from topi and hartebeest, which have not been linked to clinical MCF, replicate only in FAK cells. Buoyant density analysis by analytical ultracentrifugation, restriction endonuclease analysis and blot hybridization of virus genomic DNA from both alcelaphine herpesviruses as well as from bovine herpesviruses 1, 2, and 4 demonstrate that there are two types of alcelaphine herpesviruses, each distinct and different from the other bovine herpesviruses. Genomic size of both alcelaphine herpesviruses, estimated from DNA restriction fragments, is approximately 110 kilobase pairs. Alcelaphine herpesvirus DNA resembles Herpesvirus saimiri DNA during equilibrium sedimentation in that the majority of the DNA bands as a light (L) fraction with a minor heavy (H) component. Polyacrylamide gel analysis of virion proteins indicates that both viruses have distinct patterns, each consisting of 36 polypeptides ranging in molecular weight from 12,000 to 275,000. Virus isolates from wildebeest have been designated AHV-1, while viruses isolated from topi and hartebeest have been designated AHV-2.