Biomedical subjects

Kenneth H Roux

Publications and source records attributed to Kenneth H Roux.

At least 19 recordsLinked to original sources

PubMed2006

Cloning and characterization of profilin (Pru du 4), a cross-reactive almond (Prunus dulcis) allergen.

BACKGROUND: The identity of allergenic almond proteins is incomplete. OBJECTIVE: Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen. METHODS: An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin. RESULTS: The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins. CONCLUSION: Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. CLINICAL IMPLICATIONS: Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.

Amino Acid Sequence↗

PubMed2006

Distribution and three-dimensional structure of AIDS virus envelope spikes.

Envelope glycoprotein (Env) spikes on AIDS retroviruses initiate infection of host cells and are therefore targets for vaccine development. Though crystal structures for partial Env subunits are known, the structure and distribution of native Env spikes on virions is obscure. We applied cryoelectron microscopy tomography to define ultrastructural details of spikes. Virions of wild-type human immunodeficiency virus 1 (HIV-1) and a mutant simian immunodeficiency virus (SIV) had approximately 14 and approximately 73 spikes per particle, respectively, with some clustering of HIV-1 spikes. Three-dimensional averaging showed that the surface glycoprotein (gp120) 'head' of each subunit of the trimeric SIV spike contains a primary mass, with two secondary lobes. The transmembrane glycoprotein 'stalk' of each trimer is composed of three independent legs that project obliquely from the trimer head, tripod-like. Reconciling available atomic structures with the three-dimensional whole spike density map yields insights into the orientation of Env spike structural elements and possible structural bases of their functions.

Acquired Immunodeficiency Syndrome↗

PubMed2006

Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch] proteins: effects of thermal treatments and in vitro digestion.

Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods.

Antigens↗

PubMed2006

Nature of nonfunctional envelope proteins on the surface of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies are thought be distinguished from nonneutralizing antibodies by their ability to recognize functional gp120/gp41 envelope glycoprotein (Env) trimers. The antibody responses induced by natural HIV-1 infection or by vaccine candidates tested to date consist largely of nonneutralizing antibodies. One might have expected a more vigorous neutralizing response, particularly against virus particles that bear functional trimers. The recent surprising observation that nonneutralizing antibodies can specifically capture HIV-1 may provide a clue relating to this paradox. Specifically, it was suggested that forms of Env, to which nonneutralizing antibodies can bind, exist on virus surfaces. Here, we present evidence that HIV-1 particles bear nonfunctional gp120/gp41 monomers and gp120-depleted gp41 stumps. Using a native electrophoresis band shift assay, we show that antibody-trimer binding predicts neutralization and that the nonfunctional forms of Env may account for virus capture by nonneutralizing antibodies. We hypothesize that these nonfunctional forms of Env on particle surfaces serve to divert the antibody response, helping the virus to evade neutralization.

Animals↗

PubMed2005

A 50 kDa maize gamma-zein has marked cross-reactivity with the almond major protein.

Cross-reactivity of antibodies against almond major protein (AMP, a legumin), the major almond allergen, with cereal proteins may cause problems in detecting almond contaminants in cereal products when antibody-based assays are used. Rabbit polyclonal IgG antiserum produced against AMP was used to test cross-reactivity with protein extracts from maize, a cereal commonly found in breakfast and snack foods. Gradient SDS-PAGE followed by Western blotting was performed, and two cross-reactive proteins were detected by chemiluminescence. A fraction of maize proteins purified by elution from an IgG anti-AMP affinity column followed by electrophoreseis and immunoblotting showed a high degree of cross-reactivity with a minor 50 kDa protein of maize, as well as low cross-reactivity with the 27 kDa gamma-zein. The 50 kDa cross-reactive protein was identified as the 50 kDa gamma-zein by immunoreaction with anti-50 kDa gamma-zein antiserum. Notably, the 50 kDa maize gamma-zein also reacted with IgE from pooled human sera from patients with self-reported severe almond allergies. The high immunoreactivity of the 50 kDa gamma-zein should be considered in maize quality improvement programs, and such notable cross-reactivity is of relevance in the design of antibody-based assays for almond allergen detection.

Allergens↗

PubMed2005

Effects of food processing on the stability of food allergens.

The ubiquitous presence of allergens in the human food supply coupled with increased awareness of food allergies warrants undertaking appropriate preventive measures to protect sensitive consumers from unwanted exposure to offending food allergens. Attempts to reduce or eliminate food allergenicity through food processing have met with mixed results. The rationale for using food processing to reduce/eliminate allergenicity and limitations to using this approach are discussed.

Allergens↗

PubMed2005

Ana o 3, an important cashew nut (Anacardium occidentale L.) allergen of the 2S albumin family.

BACKGROUND: Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. We have previously cloned and characterized major cashew allergens belonging to the vicilin and legumin families of seed storage proteins. OBJECTIVE: Here we set out to describe a third major cashew allergen, a 2S albumin. METHODS: The recombinant cashew 2S albumin was amplified from a cDNA library by means of PCR, sequenced, and expressed in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition immunoblotting was used to identify the corresponding native cashew nut proteins. The mass of affinity-purified native allergen was determined by means of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectroscopy. Patients' sera were used to probe solid-phase 2S albumin peptides to identify linear epitopes. RESULTS: The cloned allergen, designated Ana o 3, was identified as 2S albumin. MALDI-TOF mass spectroscopy of native Ana o 3 yielded a molecular mass of 12,598 d. Immunoblot analysis showed 21 (81%) of 26 sera from patients with cashew allergy were reactive. Three native Ana o 3 large-subunit isoforms with molecular weights ranging from approximately 6 to 10 kd were identified. Probing of overlapping synthetic Ana o 3 peptides with patients' sera identified 16 reactive peptides, 4 of which gave strong signals and one of which positionally overlaps linear epitopes in mustard and walnut allergenic 2S albumins. The overlapping cashew and walnut epitopes also share considerable homology. CONCLUSIONS: We conclude that this 2S albumin protein is a major allergen in cashew nut and demonstrates a possible basis for cross-reactivity with walnut 2S albumin.

Adolescent↗

PubMed2005

Soluble mimetics of human immunodeficiency virus type 1 viral spikes produced by replacement of the native trimerization domain with a heterologous trimerization motif: characterization and ligand binding analysis.

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, mediates binding to the viral receptors and, along with the transmembrane glycoprotein gp41, is a major target for neutralizing antibodies. We asked whether replacing the gp41 fusion/trimerization domain with a stable trimerization motif might lead to a more stable gp120 trimer that would be amenable to structural and immunologic analysis. To obtain stable gp120 trimers, a heterologous trimerization motif, GCN4, was appended to the C terminus of YU2gp120. Biochemical analysis indicated that the gp120-GCN4 trimers were superior to gp140 molecules in their initial homogeneity, and trilobed structures were observable by electron microscopy. Biophysical analysis of gp120-GCN4 trimers by isothermal titration calorimetry (ITC) and ultracentrifugation analyses indicated that most likely two molecules of soluble CD4 could bind to one gp120-GCN4 trimer. To further examine restricted CD4 stoichiometric binding to the gp120-GCN4 trimers, we generated a low-affinity CD4 binding trimer by introducing a D457V change in the CD4 binding site of each gp120 monomeric subunit. The mutant trimers could definitively bind only one soluble CD4 molecule, as determined by ITC and sedimentation equilibrium centrifugation. These data indicate that there are weak interactions between the gp120 monomeric subunits of the GCN4-stabilized trimers that can be detected by low-affinity ligand sensing. By similar analysis, we also determined that removal of the variable loops V1, V2, and V3 in the context of the gp120-GCN4 proteins allowed the binding of three CD4 molecules per trimer. Interestingly, both the gp120-GCN4 variants displayed a restricted stoichiometry for the CD4-induced antibody 17b of one antibody molecule binding per trimer. This restriction was not evident upon removal of the variable loops V1 and V2 loops, consistent with conformational constraints in the wild-type gp120 trimers and similar to those inherent in the functional Env spike. Thus, the gp120-GCN4 trimers demonstrate several properties that are consistent with some of those anticipated for gp120 in the context of the viral spike.

Amino Acid Motifs↗

PubMed2004

Differential segmental flexibility and reach dictate the antigen binding mode of chimeric IgD and IgM: implications for the function of the B cell receptor.

Mature, naive B cells coexpress IgD and IgM with identical binding sites. In this study, the binding properties of such IgM and IgD are compared to determine how size and shape may influence their ability to bind Ag and thus function as receptors. To dissect their intrinsic binding properties, recombinant IgM and IgD were produced in soluble form as monomers of the basic H(2)L(2) Ab architecture, each with two Ag binding sites. Since these sites are connected with a hinge region in IgD and structural Ig domains in IgM, the two molecules differ significantly in this region. The results show that IgD exhibited the larger angle and longer distance between its binding sites, as well as having the greater flexibility. Relative functional affinity was assessed on two antigenic surfaces with high or low epitope density, respectively. At high epitope density, IgM had a higher functional affinity for the Ag compared with IgD. The order was reversed at low epitope density due to a decrease in the functional affinity of IgM. Studies of binding kinetics showed similar association rates for both molecules. The dissociation rate, however, was slower for IgM at high epitope density and for IgD at low epitope density. Taken together, the results show that IgM and IgD with identical Ag binding regions have different Ag binding properties.

Amino Acid Sequence↗

PubMed2003

Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions.

We used electron tomography to directly visualize trilobed presumptive envelope (env) glycoprotein structures on the surface of negatively stained HIV type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions. Wild-type HIV-1 and SIV virions had an average of 8-10 trimers per virion, consistent with predictions based on biochemical evidence. Mutant SIVs, biochemically demonstrated to contain high levels of the viral env proteins, averaged 70-79 trimers per virion in tomograms. These correlations strongly indicate that the visualized trimers represent env spikes. The env trimers were without obvious geometric distribution pattern or preferred rotational orientation. Combined with biochemical analysis of gag/env ratios in virions, these trimer counts allow calculation of the number of gag molecules per virion, yielding an average value of approximately 1400. Virion and env dimensions were also determined. Image-averaging analysis of SIV env trimers revealed a distinct chirality and strong concordance with recent molecular models. The results directly demonstrate the presence of env trimers on the surface of AIDS virus virions, albeit at numbers much lower than generally appreciated, and have important implications for understanding virion formation, virus interactions with host cells, and virus neutralization.

AIDS Vaccines↗

PubMed2003

Antibody domain exchange is an immunological solution to carbohydrate cluster recognition.

Human antibody 2G12 neutralizes a broad range of human immunodeficiency virus type 1 (HIV-1) isolates by binding an unusually dense cluster of carbohydrate moieties on the "silent" face of the gp120 envelope glycoprotein. Crystal structures of Fab 2G12 and its complexes with the disaccharide Manalpha1-2Man and with the oligosaccharide Man9GlcNAc2 revealed that two Fabs assemble into an interlocked VH domain-swapped dimer. Further biochemical, biophysical, and mutagenesis data strongly support a Fab-dimerized antibody as the prevalent form that recognizes gp120. The extraordinary configuration of this antibody provides an extended surface, with newly described binding sites, for multivalent interaction with a conserved cluster of oligomannose type sugars on the surface of gp120. The unique interdigitation of Fab domains within an antibody uncovers a previously unappreciated mechanism for high-affinity recognition of carbohydrate or other repeating epitopes on cell or microbial surfaces.

Amino Acid Sequence↗

PubMed2003

A sensitive sandwich ELISA for the detection of trace amounts of cashew (Anacardium occidentale L.) nut in foods.

Trace amounts of cashew nut protein can provoke severe allergic reactions in sensitive patients. Consequently, commercial food processors and regulatory agencies must be vigilant to prevent cashew nut cross-contamination among foods and ensure proper labeling. Toward this end, we have developed a sandwich enzyme-linked immunosorbent (ELISA) to detect the predominant cashew protein fraction (anacardein or cashew major protein, CMP) that can be extracted in aqueous buffer from food matrixes. Protein G-purified goat antiwhole cashew extract IgG and rabbit anti-CMP IgG were used as capture and secondary antibodies, respectively. Immunoadsorption against several nut and seed proteins significantly minimized the inherent cross-reactivity of these reagents. Food samples spiked with cashew flour and CMP were extracted and tested in a sandwich ELISA where standard curves were based on reactivity with CMP. The assay was optimized to detect as little as 20 ng/mL (0.02 ppm) of CMP and was successfully used to quantify CMP, and thus cashew, in various food matrixes.

Allergens↗

PubMed2003

Tree nut allergy.

Tree nuts are clinically associated with severe immunoglobulin E-mediated systemic allergic reactions independent of pollen allergy and with reactions that are usually confined to the oral mucosa in patients with immunoglobulin E directed toward cross-reacting pollen allergens. The latter reactions can progress to severe and life-threatening episodes in some patients. Many patients with severe tree nut allergy are co-sensitized to peanut. Clinical studies on cross-reactivity between the tree nuts are few in number, but based on reports to date, avoidance of the other tree nuts once sensitivity is diagnosed appears prudent unless specific challenges are performed to ensure clinical tolerance. Even then, great care must be taken to avoid cross-contamination. As with other severe food allergies, a recurrent problem in clinical management is the failure of physicians to prescribe self-injectable epinephrine to patients who are at risk of anaphylaxis.

Clinical Trials as Topic↗

PubMed2003

Purification, characterization, and immunogenicity of a soluble trimeric envelope protein containing a partial deletion of the V2 loop derived from SF162, an R5-tropic human immunodeficiency virus type 1 isolate.

The envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the major target of neutralizing antibody responses and is likely to be a critical component of an effective vaccine against AIDS. Although monomeric HIV envelope subunit vaccines (gp120) have induced high-titer antibody responses and neutralizing antibodies against laboratory-adapted HIV-1 strains, they have failed to induce neutralizing antibodies against diverse heterologous primary HIV isolates. Most probably, the reason for this failure is that the antigenic structure(s) of these previously used immunogens does not mimic that of the functional HIV envelope, which is a trimer, and thus these immunogens do not elicit high titers of relevant functional antibodies. We recently reported that an Env glycoprotein immunogen (o-gp140SF162DeltaV2) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162, when used in a DNA priming-protein boosting vaccine regimen in rhesus macaques, induced neutralizing antibodies against heterologous subtype B primary isolates as well as protection to the vaccinated animals upon challenge with pathogenic SHIV(SF162P4) virus. Here we describe the purification of this protein to homogeneity, its characterization as trimer, and its ability to induce primary isolate-neutralizing responses in rhesus macaques. Optimal mutations in the primary and secondary protease cleavage sites of the env gene were identified that resulted in the stable secretion of a trimeric Env glycoprotein in mammalian cell cultures. We determined the molecular mass and hydrodynamic radius (R(h)) using a triple detector analysis (TDA) system. The molecular mass of the oligomer was found to be 324 kDa, close to the expected M(w) of a HIV envelope trimer protein (330 kDa), and the hydrodynamic radius was 7.27 nm. Negative staining electron microscopy of o-gp140SF162DeltaV2 showed that it is a trimer with considerable structural flexibility and supported the data obtained by TDA. The structural integrity of the purified trimeric protein was also confirmed by determinations of its ability to bind the HIV receptor, CD4, and its ability to bind a panel of well-characterized neutralizing monoclonal antibodies. No deleterious effect of V2 loop deletion was observed on the structure and conformation of the protein, and several critical neutralization epitopes were preserved and well exposed on the purified o-gp140SF162DeltaV2 protein. In an intranasal priming and intramuscular boosting regimen, this protein induced high titers of functional antibodies, which neutralized the vaccine strain, i.e., SF162. These results highlight a potential role for the trimeric o-gp140SF162DeltaV2 Env immunogen in a successful HIV vaccine.

Amino Acid Sequence↗

PubMed2003

Tree nut allergens.

Allergic reactions to tree nuts can be serious and life threatening. Considerable research has been conducted in recent years in an attempt to characterize those allergens that are most responsible for allergy sensitization and triggering. Both native and recombinant nut allergens have been identified and characterized and, for some, the IgE-reactive epitopes described. Some allergens, such as lipid transfer proteins, profilins, and members of the Bet v 1-related family, represent minor constituents in tree nuts. These allergens are frequently cross-reactive with other food and pollen homologues, and are considered panallergens. Others, such as legumins, vicilins, and 2S albumins, represent major seed storage protein constituents of the nuts. The allergenic tree nuts discussed in this review include those most commonly responsible for allergic reactions such as hazelnut, walnut, cashew, and almond as well as those less frequently associated with allergies including pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, coconut, Nangai nut, and acorn.

Allergens↗

PubMed2003

Ana o 2, a major cashew (Anacardium occidentale L.) nut allergen of the legumin family.

BACKGROUND: We recently cloned and described a vicilin and showed it to be a major cashew allergen. Additional IgE-reactive cashew peptides of the legumin group and 2S albumin families have also been reported. Here, we attempt to clone, express and characterize a second major cashew allergen. METHODS: A cashew cDNA library was screened with human IgE and rabbit IgG anti-cashew extract antisera, and a reactive nonvicilin clone was sequenced and expressed as a fusion protein in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition of immunoblotting was used to identify the corresponding native peptides in cashew nut extract. The identified allergen was subjected to linear epitope mapping using SPOTs solid-phase synthetic peptide technology. RESULTS: Sequence analysis showed the selected clone, designated Ana o 2, to encode for a member of the legumin family (an 11S globulin) of seed storage proteins. By IgE immunoblotting, 13 of 21 sera (62%) from cashew-allergic patients were reactive. Immunoblot inhibition data showed that the native Ana o 2 constitutes a major band at approximately 33 kD and a minor band at approximately 53 kD. Probing of overlapping synthetic peptides with pooled human cashew-allergic sera identified 22 reactive peptides, 7 of which gave strong signals. Several Ana o 2 epitopes were shown to overlap those of the peanut legumin group allergen, Ara h 3, in position but with little sequence similarity. Greater positional overlap and identity was observed between Ana o 2 and soybean glycinin epitopes. CONCLUSIONS: We conclude that this legumin-like protein is a major allergen in cashew nut.

Adult↗

PubMed2002

Characterization of the soluble allergenic proteins of cashew nut (Anacardium occidentale L.).

The allergens associated with cashew food allergy have not been well-characterized. We sought to identify the major allergens in cashew nut by performing IgE immunoblots to dissociated and reduced or nonreduced cashew protein extracts, followed by sequencing of the peptides of interest. Sera from 15 subjects with life-threatening reactions to cashews and 8 subjects who tolerate cashews but have life-threatening reactions to other tree nuts were compared. An aqueous cashew protein extract containing albumin/globulin was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to IgE immunoblotting using patient sera. Selected IgE reactive bands were subjected to N-terminal amino acid sequencing. Each of the 15 sera from cashew-allergic subjects showed IgE binding to the cashew protein extract. The dominant IgE-binding antigens in the reduced preparations included peptides in the 31-35 kD range, consistent with the large subunits of the major storage 13S globulin (legumin-like protein). Low-molecular-weight polypeptides of the 2S albumin family, with similarity to the major walnut allergen Jug r 1, also bound IgE. The sera from eight patients who tolerate cashew but displayed allergies to other tree nuts showed only minimal or no IgE binding to cashew. Cashew food allergy is associated with the presence of IgE directed against the major seed storage proteins in cashew, including the 13S globulin (legumin group) and 2S albumins, both of which represent major allergen classes in several plant seeds. Thus, the legumin-group proteins and 2S albumins are again identified as major food allergens, which will help further research into seed protein allergenicity.

Adult↗

PubMed2002

Biochemical characterization of amandin, the major storage protein in almond (Prunus dulcis L.).

The almond major storage protein, amandin, was prepared by column chromatography (amandin-1), cryoprecipitation (amandin-2), and isoelectric precipitation (amandin-3) methods. Amandin is a legumin type protein characterized by a sedimentation value of 14S. Amandin is composed of two major types of polypeptides with estimated molecular weights of 42-46 and 20-22 kDa linked via disulfide bonds. Several additional minor polypeptides were also present in amandin. Amandin is a storage protein with an estimated molecular weight of 427,300 +/- 47,600 Da (n = 7) and a Stokes radius of 65.88 +/- 3.21 A (n = 7). Amandin is not a glycoprotein. Amandin-1, amandin-2, and amandin-3 are antigenically related and have similar biochemical properties. Amandin-3 is more negatively charged than either amandin-1 or amandin-2. Methionine is the first essential limiting amino acid in amandin followed by lysine and threonine.

Amino Acid Sequence↗