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Biomedical subjects

G A Mackay

Publications and source records attributed to G A Mackay.

12 recordsLinked to original sources
PubMed2000

Domain one of the high affinity IgE receptor, FcepsilonRI, regulates binding to IgE through its interface with domain two.

The high affinity receptor for IgE, FcepsilonRI, binds IgE through the second Ig-like domain of the alpha subunit. The role of the first Ig-like domain is not well understood, but it is required for optimal binding of IgE to FcepsilonRI, either through a minor contact interaction or in a supporting structural capacity. The results reported here demonstrate that domain one of FcepsilonRI plays a major structural role supporting the presentation of the ligand-binding site, by interactions generated within the interdomain interface. Analysis of a series of chimeric receptors and point mutants indicated that specific residues within the A' strand of domain one are crucial to the maintenance of the interdomain interface, and IgE binding. Mutation of the Arg(15) and Phe(17) residues caused loss in ligand binding, and utilizing a homology model of FcepsilonRI-alpha based on the solved structure of FcgammaRIIa, it appears likely that this decrease is brought about by collapse of the interface and consequently the IgE-binding site. In addition discrepancies in results of previous studies using chimeric IgE receptors comprising FcepsilonRIalpha with either FcgammaRIIa or FcgammaRIIIA can be explained by the presence or absence of Arg(15) and its influence on the IgE-binding site. The data presented here suggest that the second domain of FcepsilonRI-alpha is the only domain involved in direct contact with the IgE ligand and that domain one has a structural function of great importance in maintaining the integrity of the interdomain interface and, through it, the ligand-binding site.

Amino Acid Sequence↗
PubMed2000

Pathogenic simian/human immunodeficiency virus SHIV(KU) inoculated into immunized macaques caused infection, but virus burdens progressively declined with time.

Using the simian immunodeficiency virus/human immunodeficiency virus (SHIV)-macaque model of AIDS, we had shown in a previous report that a live, nonpathogenic strain of SHIV, further attenuated by deletion of the vpu gene and inoculated orally into adult macaques, had effectively prevented AIDS following vaginal inoculation with pathogenic SHIV(KU). Examination of lymph nodes from the animals at 18 weeks postchallenge had shown that all six animals were persistently infected with challenge virus. We report here on a 2-year follow-up study on the nature of the persistent infections in these animals. DNA of the vaccine virus was present in the lymph nodes at all time points tested, as far as 135 weeks postchallenge. In contrast, the DNA of SHIV(KU) became undetectable in one animal by week 55 and in three others by week 63. These four macaques have remained negative for SHIV(KU) DNA as far as the last time point examined at week 135. Quantification of the total viral DNA concentration in lymph nodes during the observation period showed a steady decline. All animals developed neutralizing antibody and cytotoxic-T-lymphocyte responses to SHIV(KU) that persisted throughout the observation period. Vaccine-like viruses were isolated from two animals, and a SHIV(KU)-like virus was isolated from one of the two macaques that remained positive for SHIV(KU) DNA. There was no evidence of recombination between the vaccine and the challenge viruses. Thus, immunization with the live vaccine not only prevented disease but also contributed to the steady decline in the virus burdens in the animals.

AIDS Vaccines↗
PubMed1999

Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carcinoma.

Allergic reactions are mediated by IgE antibodies bound to high-affinity receptors on mast cells in peripheral tissues and are characterized by their immediacy and hypersensitivity. These properties could also be advantageous in immunotherapy against cancer growth in peripheral tissues. We have constructed chimeric IgE and IgG1 antibodies with murine V regions and human C regions corresponding to the MOv18 monoclonal antibody against the human ovarian tumor-associated antigen, folate binding protein. The antibodies exhibited the expected binding affinities for antigen and Fc receptors, and effector activities with human basophils and platelets in vitro. The protective activities of MOv18-IgE and MOv18-IgG1 were compared in a SCID mouse xenograft model of ovarian carcinoma. The beneficial effects of MOv18-IgE were greater and of longer duration than those of MOv18-IgG1. Our results suggest that the allergic reaction could be harnessed for the suppression of ovarian tumors.

Animals↗
PubMed1997

Participation of the N-terminal region of Cepsilon3 in the binding of human IgE to its high-affinity receptor FcepsilonRI.

The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcepsilonRI) expressed on mast cells and basophils is central to the development of an allergic reaction. Previous studies have implicated the third constant domain of IgE-Fc (Cepsilon3) as the site of the interaction with FcepsilonRI. We have prepared a series of site-directed mutants of human IgE-Fc, particularly focusing on the N-terminal "linker" region and AB loop of Cepsilon3. The kinetics of binding IgE and its Fc fragments to the immobilized receptor were determined by surface plasmon resonance (SPR), and two phases of binding were observed. We identified one mutation in the N-terminal linker region, R334S, that has a dramatic effect on binding. R334S lowers the affinity of IgE-Fc for FcepsilonRI by 120-fold, principally through an increase in the dissociation rate of the slower phase of the interaction. This mutation has a similar effect in Fcepsilon3-4, a truncated form of IgE-Fc which lacks the Cepsilon2 domain pair, and thus it does not exert its effect through altering the quaternary structure of IgE-Fc, firmly implicating Arg334 as a contact residue in the complex. However R334S has no effect on the binding of FcepsilonRII (CD23), the low-affinity receptor for IgE, demonstrating the structural integrity of the mutated IgE-Fc. Circular dichroism spectroscopy and thermal stability studies further indicate that the R334S mutation does not disorder or destabilize the structure of IgE-Fc or Fcepsilon3-4. These results demonstrate the importance of the N-terminal linker region of Cepsilon3 in the interaction of IgE with FcepsilonRI.

Animals↗
PubMed1997

Basis of the 1:1 stoichiometry of the high affinity receptor Fc epsilon RI-IgE complex.

A soluble fragment of the high-affinity IgE receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha) binds to the Fc fragment of IgE (IgE-Fc) as a 1:1 complex. IgE-Fc consists of a dimer of the C epsilon 2, C epsilon 3 and C epsilon 4 domains of the epsilon-heavy chain of IgE. This region of IgE has been modelled on the crystal structure of the Fc region of IgG1, which exhibits twofold rotational symmetry. This implies that IgE should be divalent with respect to its ligands. X-ray scattering studies reveal however that the twofold rotational symmetry of IgE-Fc is perturbed by a bend in the linker region between the C epsilon 2 and C epsilon 3 domains. The 1:1 stoichiometry could then arise from the conformational asymmetry or from steric occlusion of one of the sites by the overhanging C epsilon 2 domains. To test this hypothesis we have expressed a recombinant epsilon-chain fragment containing C epsilon 3 and C epsilon 4. This product, Fc epsilon 3-4, is secreted from cells as a disulphide linked dimer and binds with higher affinity than either IgE or IgE-Fc to cell surface Fc epsilon RI. Titration experiments, together with molecular mass measurements of the Fc epsilon 3-4/sFc epsilon RI alpha complex, reveal that Fc epsilon 3-4 binds only a single receptor molecule. This excludes the possibility that steric hindrance by C epsilon 2 accounts for the unexpected stoichiometry.

Binding Sites↗
PubMed1997

Functional analysis of histamine release from basophils and mast cells in subjects with the Ile-181-->Leu variant of Fc epsilon RI-beta.

1. Atopy is a genetically heterogeneous disorder, but there is now strong evidence that one important locus is on chromosome 11q13. Fc epsilon RI-beta at that location has been identified as a candidate gene and variants have been associated with atopy in population studies. 2. No information is available on the functional consequences of any of these variants, and defining this may prove difficult because of the complexity of the atopy phenotype and because Fc epsilon RI beta is expressed on a range of cells with different functions, including basophils, mast cells, eosinophils and antigen-presenting Langerhan's cells. 3. We have conducted a qualitative study of mast cell and basophil histamine release in nine atopic individuals with Ile-181-->Leu mutation of Fc epsilon RI beta, and ten unrelated similarly atopic individuals without Ile-181-->Leu mutation. There were non-significant trends for Ile-181-->Leu-positive atopic subjects to produce wheal responses at lower allergen challenge in skin prick tests, and to release more histamine from basophils following in vitro allergen challenge. 4. The data do not provide decisive evidence of functional differences between atopic subjects with Ile-181-->Leu and other atopic individuals; more discriminating functional experiments are required.

Adult↗
PubMed1996

Structure based design and characterization of peptides that inhibit IgE binding to its high-affinity receptor.

We have designed synthetic peptide inhibitors of the interaction between IgE and its high affinity receptor, Fc epsilon RI. The structure of the second domain of CD2 was used as a modelling template for the second alpha-chain domain of Fc epsilon RI, the C-C' loop of which has been implicated in the interaction with IgE. An L-amino acid peptide and a retro-enantiomeric D-amino acid peptide were designed to mimic the conformation of the C-C' region. Both peptides were cyclized by disulphide bond formation between terminal cysteine residues, and show mirror image symmetry by circular dichroism analysis. The C-C' peptide mimics act as competitive inhibitors of IgE binding. The cyclic L- and retro D-peptides exhibited KDs of approximately 3 microM and 11 microM, respectively, for IgE. Further, the peptides inhibit IgE-mediated mast cell degranulation, an in vitro model of an allergic response.

Amino Acid Sequence↗
PubMed1996

Extracellular guanosine 3',5'-cyclic monophosphate and disodium cromoglycate share a similar spectrum of activity in the inhibition of histamine release from isolated mast cells and basophils.

In comparison to adenosine 3',5'-cyclic monophosphate (cAMP), surprisingly little is known regarding the role of guanosine 3',5'-cyclic monophosphate (cGMP) in the functional modulation of mast cells and basophils. In the present study, the ability of cGMP, cAMP, and a range of related compounds, to inhibit immunologically induced histamine release from these cells was investigated and compared to the anti-asthmatic drug disodium cromoglycate (DSCG). Exogenously applied cGMP produced a potent inhibition of histamine release from rat peritoneal mast cells, but cAMP had a negligible effect. The attenuation noted with cGMP was markedly reduced if cells were pretreated with the nucleotide before stimulation. Similar results were obtained with DSCG. The inhibitory and tachyphylactic effects noted with cGMP were mimicked by direct derivatives of the compound but not by a range of other cyclic or guanosine nucleotides. The time courses of the induced tachyphylaxis seen with cGMP and DSCG were similar, and cross-tachyphylaxis between the two compounds was observed. In addition, both cGMP and DSCG showed a comparable spectrum of activity against mast cells isolated from the mouse, guinea pig, and human. The parallel effects of the two agents suggests that they may inhibit mediator release from mast cells through similar mechanisms.

Animals↗
PubMed1995

Stoichiometry and thermodynamics of the interaction between the Fc fragment of human IgG1 and its low-affinity receptor Fc gamma RIII.

IgG-Fc receptors, cell surface glycoproteins binding the Fc region of antibodies, play a crucial role in the immune system. To better understand the nature of the recognition process, we have examined the interaction between huIgG1-Fc and a soluble fragment of huFc gamma RIII (sCD16). Analytical ultracentrifugation experiments clearly demonstrate that IgG1-Fc and sCD16 interact weakly to form a 1:1 complex with an association constant of 1.7 x 10(5) M-1 in PBS at 22.0 degrees C. The thermodynamic parameters, obtained from the temperature dependence of the equilibrium binding constants, exhibit an enthalpy-entropy compensation with a favorable enthalpy at physiological temperatures. The value of -360 cal mol-1 K-1 for delta Cp zero possibly identifies the process as one in which local folding/rearrangement is coupled to complex formation. The 1:1 stoichiometry and thermodynamic parameters provide a basis for understanding the nature of the Fc gamma R-IgG interactions.

Amino Acid Sequence↗
PubMed1995

Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants.

We have constructed an expression vector that leads to secretion of the whole Fc of human immunoglobulin E (hIgE-Fc) from mammalian cells at levels up to 100 mg/l of culture. Two surface glycosylation sites at Asn265 and Asn371 have been changed to glutamine, to obtain a more homogeneous preparation of hIgE-Fc for structural studies. Comparison of wild-type and mutant products revealed that Asn371 is rarely glycosylated in Chinese hamster ovary cells. Both the double mutant and wild-type hIgE-Fc bind to the high-affinity IgE receptor, Fc epsilon RI, with about the same affinity as myeloma IgE (Ka in the range 10(10)-10(11) M-1), and were able to sensitize isolated human basophils for anti-IgE triggering of histamine release. However, only the double mutant hIgE-Fc approached the affinity of myeloma IgE for the low-affinity receptor, Fc epsilon RII (Ka = 7.3 x 10(7) M-1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (Ka = 4.1 x 10(6) M-1).

Amino Acid Sequence↗