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B A Helm

Publications and source records attributed to B A Helm.

At least 19 recordsLinked to original sources
PubMed1998

Amino acid residues that influence Fc epsilon RI-mediated effector functions of human immunoglobulin E.

Immunoglobulin E (IgE) mediates its effector functions via the Fc region of the molecule. IgE binding to and subsequent aggregation of the high-affinity receptor (Fc epsilon RI) by allergen plays a pivotal role in type I hypersensitivity responses. Earlier studies implicated the C epsilon 2 and 3 interface and the A-B loop in C epsilon 3 in the IgE-Fc epsilon RI interaction. These regions and glycosylation sites in C epsilon 3 were now targeted by site-specific mutagenesis. IgE binding to Fc epsilon RI was compared with surface plasmon resonance (SPR) measurements, which assessed the binding of the soluble extracellular domain of Fc epsilon RI to IgE. Kinetic analysis based on a pseudo-first-order model agrees with previous determinations. A more refined SPR-based kinetic analysis suggests a biphasic interaction. A model-free empirical analysis, comparing the binding strength and kinetics of native and mutant forms of IgE, identified changes in the kinetics of IgE-Fc epsilon RI interaction. Conservative substitutions introduced into the A-B loop have a small effect on binding, suggesting that the overall conformation of the loop is important for the complementary interaction, but multiple sites across the C epsilon 3 domain may influence IgE-Fc epsilon RI interactions. Asn394 is essential for the generation of a functional IgE molecule in mammalian cells. A role of Pro333 in the maintenance of a constrained conformation at the interface between C epsilon 2-3 emerged by studying the functional consequences of replacing this residue by Ala and Gly. These substitutions cause a dramatic decrease in the ability of the ligand to mediate stimulus secretion coupling, although only small changes in the association and dissociation rates are observed. Understanding the molecular basis of this phenomenon may provide important information for the design of inhibitors of mast cell degranulation.

Amino Acids↗
PubMed1998

Protein and cell engineering of components of the human immunoglobulin E receptor/effector system: applications for therapy and diagnosis.

Adaptive immune responses characterised by the synthesis of antibodies of the immunoglobulin E (IgE) isotype play an important role in type I hypersensitivity disorders and parasitic infestations, diseases which have an significant socioeconomic impact world-wide. This paper considers potential applications of recent advances in our understanding of the origin of isotype specific immune responses which emerged as a result of cell and protein engineering studies on components of the human IgE/receptor/effector system. Furthermore, the identification of the receptor binding regions in IgE as a result of the development of a stable assay system has important applications for the design of rational therapeutic interventions in allergy and asthma, the treatment of mast cell tumours, and the establishment of procedures for the selective isolation of cells expressing the high-affinity receptor for IgE for functional studies.

Animals↗
PubMed1996

Identification of the high affinity receptor binding region in human immunoglobulin E.

We have investigated the capacity of N- and C-terminally truncated and chimeric human (h) IgE-derived peptides to inhibit the binding of 125I-labeled hIgE, and to engage cell lines expressing high and low affinity receptors (Fc-epsilon-RI/II). The peptide sequence Pro343-Ser353 of the hC-epsilon-3 domain is common to all h-epsilon-chain peptides that recognize hFc-epsilon-RI. This region in IgE is homologous to the A loop in C-gamma-2 that engages the rat neonatal IgG receptor. Optimum Fc-epsilon-RI occupancy by hIgE occurs at pH 6.4, with a second peak at 7.4. N- or C-terminal truncation has little effect on the association rate of the ligands with this receptor. Dissociation markedly increases following C-terminal deletion, and hFc-epsilon-RI occupancy at pH 6.4 is diminished. His residue(s) in the C-terminal region of the epsilon-chain may thus contribute to the high affinity of interaction. Grafting the homologus rat epsilon-chain sequence into hIgE maintains hFc-epsilon-RI interaction without conferring binding to rat Fc-epsilon-RI. hFc-epsilon-RII interaction is lost, suggesting that these residues also contribute to hFc-epsilon RII binding. h-epsilon-chain peptides comprising only this sequence do not block hIgE/hFc-epsilon-RI interaction or engage the receptor. Therefore, sequences N- or C-terminal to this core peptide provide structures necessary for receptor recognition.

Animals↗
PubMed1996

Potential allergens stimulate the release of mediators of the allergic response from cells of mast cell lineage in the absence of sensitization with antigen-specific IgE.

A number of structurally diverse antigens preferentially stimulate the synthesis of IgE antibodies, but no unifying principle has been proposed that explains the nature of isotype selection. In the present study, we show that common allergens present in bee venom, house dust mite emanations and parasite proteins induce mast cell and basophil degranulation and stimulate interleukin-4 synthesis, and secretion in the absence of antigen-specific IgE. These data point to a linkage between the initial activation of cells of the innate immune system and subsequent adaptive immune responses. They suggest that IgE-independent mast cell and basophil degranulation is predictive of potential allergenicity and can be evaluated by means of a cellular assay. Our study indicates that non-immunological degranulation by prototypic allergens, such as bee venom phospholipase A2 or proteases associated with house dust mite emanations, is critically dependent on enzymatic activity. These findings have potentially important implications for vaccine design in allergic and parasitic disease.

Allergens↗
PubMed1995

A link between catalytic activity, IgE-independent mast cell activation, and allergenicity of bee venom phospholipase A2.

The molecular and cellular mechanisms controlling Ab isotype selection following encounter of a given Ag are still unclear, although the regulatory role of cytokines is established. In the present study we explored the possibility that the nonimmunologic interaction of an allergen with cells of the innate immune system might result in a release of mediators that promote IgE isotype selection in adaptive responses. Using the bee venom allergen phospholipase A2 (PLA2) and a mutant variant lacking enzymatic function, we show that PLA2, but not its catalytically inactive variant, is able to induce IgE-independent mediator release, including IL-4, from rodent mast cells. Assessing the in vivo relevance of these observations, we find that repeated injections of low doses of active enzyme into mice induce the synthesis of high levels of PLA2-specific IgE, while immunization with the inactive form yields no detectable IgE response. Both Ags were similarly immunogenic when high doses of Ag were used for immunization. These findings suggest that mast cells might be a source of IL-4 at the onset of specific immunity against sources of allergens such as bee venom that contain PLA2 and support the concept that the biologic action of an Ag on cells of the innate immune system can play a role in determining adaptive immune responses.

Allergens↗
PubMed1994

Defective protein phosphorylation and Ca2+ mobilization in a low secreting variant of the rat basophilic leukemia cell line.

High and low secreting variants of the rat basophilic leukemia cell line represent powerful tools to study the molecular basis of stimulus/secretion coupling via the high affinity receptor (Fc epsilon R1) complex for immunoglobulin E since an identification of the differences between these subclones may produce important information concerning the signaling pathways involved. A comparison between a variant supporting high mediator secretion (> 50%) and one with a 10-fold reduced response to antigen shows that the latter is associated with a defect in threonine and tyrosine phosphorylation of the subunits of the Fc epsilon R1 complex. The delayed onset and reduced mediator release in the low secretor facilitated a slow motion study of the early events following receptor activation. It showed that tyrosine phosphorylation of a 72-kDa protein is an early event preceding threonine and subsequent tyrosine phosphorylation of the gamma-chain. This points to the activation of both protein-tyrosine kinases and protein kinase(s) C as early events in signal transduction. The retarded onset and low intensity of phosphorylation in the low secreting variant is associated with reduced levels of inositol phosphate production, and this and the lack of the Ca2+ mobilization from intracellular stores indicate a defect upstream of teh activation of phospholipase C.

Animals↗
PubMed1993

Human IgE mediates stimulus secretion coupling in rat basophilic leukemia cells transfected with the alpha chain of the human high-affinity receptor.

Cell surface expression of the alpha chain of the human (h) Fc epsilon receptor type 1 (Fc epsilon R1) can be observed following stable transfection of RBL-2H3 cells with the h alpha chain of the Fc epsilon R1 complex. Association and dissociation constants for hIgE are similar to those previously reported for the ligand and its receptor. The demonstration of mediator release following the cross-linking of hFc epsilon R1 alpha by hIgE and antigen or anti-hFc epsilon R1 alpha antibody suggests that this system will assist the identification of structural motifs and specific amino acids that are involved in the generation of the signal(s) that will initiate and/or propagate the secretion of mediators from mast cells and basophils.

Adenosine↗
PubMed1993

Modeling of the lectin-homology domains of the human and murine low-affinity Fc epsilon receptor (Fc epsilon RII/CD23).

Models of the lectin-homology domains of the human and murine low-affinity receptors for IgE (Fc epsilon RII/CD23) were built on the basis of sequence similarity with rat mannose-binding protein, the structure of which is known. The sites on Fc epsilon RII/CD23 that are possibly involved in the interaction with IgE and with another ligand, CD21/CR2, are proposed. The models may assist the design of protein engineering experiments for the study of the reactivity of these molecules.

Amino Acid Sequence↗
PubMed1993

Characterization of defective phorbol ester responses in a low secreting rat basophilic leukemia (RBL-2H3) cell variant.

The role of protein kinase C (PKC) in the signaling mechanism that stimulates the release of mediators from rat mast cells, for which the RBL-2H3 cell line is a model, is at present unresolved. Current evidence suggests that PKC activation alone is an insufficient stimulus, although it can modulate mast cell exocytosis induced by other agents. In this article we characterize a variant of the RBL-2H3 cell line that has a reduced capacity for mediator secretion in response to an IgE-mediated antigen-induced stimulation. The outcome of our study suggests that at least two PKC isotypes are active in RBL-2H3 cells, and affect the positive and negative modulation of the secretory response.

Actins↗
PubMed1992

A modeling study of the alpha-subunit of human high-affinity receptor for immunoglobulin-E.

The extracellular portion of the alpha-subunit of human high-affinity receptor for immunoglobulin-E (IgE), which contains two immunoglobulin (Ig) domains, was modeled on the basis of sequence similarity with antibody domains of known three-dimensional structure. Each receptor domain contains 86 amino acid residues, and both domains were modeled as bilayer structures. In both domains, one layer is made up of three anti-parallel beta-strands and the other of four strands, with the two layers linked by a disulfide bridge. The two domains show significant sequence similarity with each other (22 identities) and with the homologous domains of the murine and rat high-affinity receptors for IgE and the Fc gamma receptors from various species. Two plausible modes of association of the domains were considered: In the first, the two domains were positioned end-to-end, with essentially only longitudinal interactions between them; in the second, the molecule is more bent, with more lateral interactions between the two domains. The models will be useful in the design of protein engineering studies of this and homologous receptors to delineate the site of interaction with ligand. Furthermore, they may lend themselves as possible probes in crystallographic analyses by molecular replacement techniques.

Amino Acid Sequence↗
PubMed1991

The nature and importance of the inter-epsilon chain disulfide bonds in human IgE.

IgE antibodies are best known for their pathological role in allergy. The class-specific effector sites are located in the epsilon chains; these form covalent dimers via two cystine residues (Cys241 and Cys328) linking opposite C epsilon 2 domains. The nature and biological significance of the inter-epsilon chain disulfide-bond arrangement is unresolved. For structural and functional analysis site-specific mutations were introduced into the C epsilon 2 domain of recombinant human IgE. The introduction of an additional cyanogen bromide cleavage site (His246----Met) facilitated the identification of parallel disulfide bond pairing. This linkage was also confirmed for myeloma IgE PS by sequence determination of disulfide-linked C epsilon 2 dimers. Substitution of Cys241 and Cys328 by Ser does not destroy receptor binding, but reductive alkylation, or the replacement of Cys328 by Met, leads to loss of activity. This shows that covalent dimerization is not essential for IgE/receptor interaction and points to the importance of the structural integrity of the site surrounding Cys328, visualized in a new model of human Fc epsilon.

Amino Acid Sequence↗