Search PubMed⌕ Search

Biomedical subjects

J M Hexham

Publications and source records attributed to J M Hexham.

13 recordsLinked to original sources
PubMed1999

A human immunoglobulin (Ig)A calpha3 domain motif directs polymeric Ig receptor-mediated secretion.

Polymeric immunoglobulins provide immunological protection at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR). Using a panel of human IgA1/IgG1 constant region "domain swap" mutants, the binding site for the pIgR on dimeric IgA (dIgA) was localized to the Calpha3 domain. Selection of random peptides for pIgR binding and comparison with the IgA sequence suggested amino acids 402-410 (QEPSQGTTT), in a predicted exposed loop of the Calpha3 domain, as a potential binding site. Alanine substitution of two groups of amino acids in this area abrogated the binding of dIgA to pIgR, whereas adjacent substitutions in a beta-strand immediately NH2-terminal to this loop had no effect. All pIgR binding IgA sequences contain a conserved three amino acid insertion, not present in IgG, at this position. These data localize the pIgR binding site on dimeric human IgA to this loop structure in the Calpha3 domain, which directs mucosal secretion of polymeric antibodies. We propose that it may be possible to use a pIgR binding motif to deliver antigen-specific dIgA and small-molecule drugs to mucosal epithelia for therapy.

Amino Acid Sequence↗
PubMed1996

Localization of the binding site for the monocyte immunoglobulin (Ig) A-Fc receptor (CD89) to the domain boundary between Calpha2 and Calpha3 in human IgA1.

Immunoglobulin (Ig) A serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities of blood. In playing its role in humoral immunity, IgA interacts with a variety of effector molecules present both in serum and on the surfaces of immune and inflammatory cells. To study these interactions, we previously established expression of human IgA1 in insect cells using recombinant baculoviruses and showed that the expressed antibody is a structurally and functionally intact polypeptide useful for examining the molecular properties of IgA. Indeed, since the C alpha 2 N-linked glycosylation site lies near the Fab-distal pole of C alpha 2, the inability of a mutant IgA1 lacking C alpha 2 N-glycosylation to bind its cognate receptor suggested that the monocyte Fc alpha receptor (mFcalphaR) recognizes IgA at a hinge-distal site encompassing the boundary between the C alpha 2 and C alpha 3 domains. In this report, we utilize both domain-swapped IgA/IgG and point-mutated IgA chimeras to verify the above hypothesis. Using an antigen-specific rosetting assay and a mFc alpha R-expressing cell line, we show that (a) C alpha 2 and C alpha 3 together are necessary and sufficient for binding; (b) neither the IgA hinge nor the tailpiece is necessary for binding; (c) mutations away from the interdomain boundary do not affect binding; and (d) mutations located near the three-dimensional boundary between C alpha 2 and C alpha 3 completely disrupt binding. Taken together, these results localize the mFc alpha R recognition site on IgA to the boundary region between the second and third constant domains--a site analogous to that recognized by Staphylococcus aureus protein A on IgG. The use of this hinge-distal site is, to date, unique among Fc receptors of the Ig superfamily.

Amino Acid Sequence↗
PubMed1994

Cloning and characterisation of TPO autoantibodies using combinatorial phage display libraries.

Thyroid lymphocyte RNA from a Hashimoto patient with high serum levels of autoantibodies to thyroid peroxidase (TPO) was used to construct a phage display antibody library in the phagemid vector pComb3. The library (100,000cfu) encoded IgG1 heavy chains together with kappa light chains. Selection of the phages displaying TPO antibody on TPO-coated ELISA plates yielded a phage population enriched for surface expression of TPO antibody Fabs. 3 different Fabs specific for TPO were subsequently isolated with affinities in the region of 10(9) molar-1. 2 of the Fabs recognised the same, or closely related, epitopes on TPO whereas the third Fab recognised a different epitope. These 2 epitopes were recognised by TPO autoantibodies in the serum of the lymphocyte donor and a series of 10 patient sera. Available sequence data showed that several non-self antibodies and non-thyroid autoantibodies use the same V kappa and VH germline genes as TPO autoantibodies. There appeared to be no clear relationship between gene sequence or gene family usage by TPO autoantibodies of the same or similar epitope specificity.

Amino Acid Sequence↗
PubMed1992

Cloning of a human autoimmune response: preparation and sequencing of a human anti-thyroglobulin autoantibody using a combinatorial approach.

Thyroid lymphocyte RNA from a Hashimoto patient exhibiting high titre serum IgG autoantibodies against thyroglobulin (Tg) has been used to construct a Fab library in phage lambda. Screening of this library with radioiodinated Tg has permitted the cloning of an anti-Tg antibody (MH52) with an affinity of 4.5 x 10(9) molar-1 as determined by inhibition ELISA. Sequence analysis showed MH52 to be an authentic antibody of the IgG1/K isotype with variable region genes from the VHI and VKIII families in combination with the JH3, DK4 and JK2 gene segments. The MH52 light chain gene showed high sequence homology (93%) with the germline gene used by several rheumatoid factors and some DNA autoantibodies. Greater divergence from the germline was observed in the case of the MH52 heavy chain gene which showed 86% homology with a germline heavy chain gene isolated from human liver. Overall the similarity between the genes coding for MH52 and the genes coding for some other autoantibodies of non-related specificity might suggest that similar regulatory processes control the formation of these different autoantibodies.

Amino Acid Sequence↗
PubMed1989

A protein kinase C pseudosubstrate peptide inhibits phosphorylation of the CD3 antigen in streptolysin-O-permeabilized human T lymphocytes.

Activation of human T lymphocytes leads to the phosphorylation of the CD3-antigen gamma polypeptide. We have investigated a possible role for protein kinase C (PKC) in mediating this phosphorylation event by using T cells permeabilized with streptolysin-O in the presence of 120 mM-K+ buffers containing Ca2+-EGTA. The gamma-chain was phosphorylated by [gamma-32P]ATP in permeabilized T lymphoblasts in the presence of phorbol 12,13-dibutyrate (Pdbu) or phytohaemagglutinin (PHA). Ca2+ alone in the range 0.5-1.0 microM also induced gamma-chain phosphorylation in some T-lymphoblast preparations; that in Jurkat-6 cells occurred at lower concentrations (50-500 nM). Two experimental approaches were used to investigate the possible involvement of PKC. Firstly, when permeabilization was carried out in buffer lacking free Ca2+, PKC was lost from the cells, and gamma-chain phosphorylation could then no longer be induced on subsequent addition of Pdbu or PHA in 400 nM-Ca2+, or 800 nM-Ca2+ alone, to permeabilized cells. However, when permeabilization was carried out in the presence of these three agents, PKC was translocated to intracellular membranes, and subsequent addition of [gamma-32P]ATP to these cells then resulted in gamma-chain phosphorylation. In the second approach, induction of gamma-chain phosphorylation by Pdbu, 1-oleoyl-2-acetylglycerol, 1,2-diolein, PHA or Ca2+ alone was effectively blocked by permeabilizing T cells in the presence of a PKC pseudosubstrate peptide (50 microM). Pseudosubstrate concentrations in the range 7-20 microM inhibited gamma-chain phosphorylation by 50%. In contrast, addition of four other 'irrelevant' basic peptides (50 microM) did not result in detectable inhibition, and 50 microM-pseudosubstrate did not inhibit the phosphorylation of 17 other polypeptides isolated from permeabilized T cells. These data suggest that Pdbu-, 1,2-diacylglycerol-, PHA- and Ca2+-induced phosphorylation of the CD3-antigen gamma chain in permeabilized T cells is mediated by PKC.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

The association of type 1, type 2A and type 2B phosphatases with the human T lymphocyte plasma membrane.

Several putative plasma-membrane-associated components of the T-lymphocyte signal-transduction pathway are phosphorylated during the initial events of cellular activation. Little is known about the control of dephosphorylation of these components. We have shown by immunoblotting that the type 1 phosphatase, the type 2A phosphatase and type 2B phosphatase (calcineurin) are associated with the plasma membrane of normal human T lymphoblasts and the human T leukaemic cell line Jurkat 6. The type 1 phosphorylase phosphatase activity is present in a latent form which can be stimulated synergistically by deinhibitor and p-nitrophenyl phosphate. The PCSH form of the type 2A phosphatase appears to be the predominant oligomer in the plasma-membrane fraction. All three phosphatases can be extracted from membranes with Nonidet P40, but whereas the type 1c and type 2Ac phosphatases separate into the detergent-poor phase of Triton X-114, calcineurin separates into both detergent-rich and -poor phases. It is probable that one or more of these three plasma-membrane-associated phosphatases play regulatory roles in determining the phosphorylation state of membrane-bound proteins involved in human T-cell activation.

Calcineurin↗
PubMed1988

Evidence that a GTP binding protein regulates phosphorylation of the CD3 antigen in human T lymphocytes.

The role of guanine nucleotide binding regulatory proteins (G proteins) in the regulation of phosphorylation of the gamma subunit of the CD3 antigen has been examined. CD3 gamma chain phosphorylation in isolated T cell microsomes was stimulated by the G protein activator guanosine 5'-0 thiotriphosphate (GTP gamma S), but cyclic adenosine monophosphate and guanosine 5'-diphosphate were ineffective at inducing gamma chain phosphorylation. The effect of GTP gamma S was rapid and transient; a half maximal effect was observed with 50 microM of the nucleotide. gamma polypeptide phosphorylated in vitro in GTP gamma S stimulated microsomes incorporated phosphate on Serines 123 and 126. These data are consistent with the involvement of a G protein in the signalling mechanisms that regulate the phosphorylation of the CD3 gamma chain.

Amino Acid Sequence↗
PubMed1987

The human T3 gamma chain is phosphorylated at serine 126 in response to T lymphocyte activation.

The gamma subunit of the human T lymphocyte T3 antigen is rapidly phosphorylated on serine residues in vivo during the initiation of T cell activation by a polyclonal mitogen (Phaseolus vulgaris phytohemagglutinin), an activator of protein kinase C (phorbol 12,13-dibutyrate), and an elevator of intracellular calcium (ionomycin). The sites of phosphorylation were identified by comparing tryptic peptide analyses of T3 gamma chains labeled in vivo with various synthetic peptides, corresponding to portions of the cytoplasmic domain of the gamma chain that had been labeled in vitro using purified protein kinase C. Two sites, serines 123 and 126, were phosphorylated in response to ionomycin, whereas a single site, serine 126, was phosphorylated when T lymphocytes were stimulated by P. vulgaris phytohemagglutinin or when protein kinase C was directly activated by phorbol 12,13-dibutyrate. Immune activation of T cells via the protein kinase C pathway thus induces phosphorylation of a single site on the T3 gamma chain, namely serine 126.

Amino Acid Sequence↗
PubMedDate not supplied

A consensus amino-acid sequence repeat in Torpedo and mammalian Ca2+-dependent membrane-binding proteins.

A group of calcium-binding proteins which bind to biomembranes has recently been identified in widely different cells and tissues (refs 1-7, reviewed in ref. 8). Three of these proteins (p70, p36 and p32.5) cross-react with antiserum to calelectrin, a Ca2+-binding protein (relative molecular mass 34,000 (34K] from the ray Torpedo marmorata, giving rise to their designation as calelectrin-related proteins. We now report that calelectrin, p36 and p32.5 contain a 17-amino-acid consensus sequence which is conserved and present in multiple copies. We suggest that this sequence may be common to other members of this new group of Ca2+-binding proteins and may underlie their unusual mode of combination with biomembranes.

Amino Acid Sequence↗