Biomedical subjects

W Harnett

Publications and source records attributed to W Harnett.

At least 37 records · Page 2Linked to original sources

PubMed2000

Presence of phosphorylcholine on a filarial nematode protein influences immunoglobulin G subclass response to the molecule by an interleukin-10-dependent mechanism.

The filarial nematode product ES-62 contains phosphorylcholine (PC) covalently attached to N-linked glycans. ES-62 induced high levels of immunoglobulin G1 (IgG1) antibodies, but no IgG2a, to non-PC epitopes of the molecule following subcutaneous injection into BALB/c mice. Conversely, mice given ES-62 lacking PC demonstrated significant production of both IgG subclasses. Thus, PC appears to block production of IgG2a antibodies to other epitopes on the parasite molecule. A role for interleukin-10 (IL-10) in this effect was shown by the ability of IL-10(-/-) mice to make an IgG2a antibody response to non-PC epitopes of ES-62.

Animals↗

PubMed1999

Molecular cloning and demonstration of an aminopeptidase activity in a filarial nematode glycoprotein.

ES-62 is an abundant phosphorylcholine-containing secreted glycoprotein of the filarial nematode Acanthocheilonema viteae. Using an antiserum directed against the parasite molecule, 3 cDNAs of size, approximately 1.5-1.6 kbp were isolated from an A. viteae expression library. Sequence analysis in combination with N-terminal amino acid sequencing of purified ES-62 revealed that each clone contained a full-length cDNA for ES-62 corresponding to 474 amino acid residues but differed in their 5' and 3' untranslated regions. Characterisation of the 5' end of ES-62 mRNA using 5' rapid amplification of cDNA ends showed that it coded for a signal sequence. Several tryptic peptides were independently sequenced using quadruple-time-of-flight mass spectrometry and used to confirm the cDNA sequence. The mature protein was found to contain three potential N-linked glycosylation sites. Comparison of the derived amino acid sequence of ES-62 with the SwissProt database identified a sequence (between amino acid residues approximately 250 and 350 of mature ES-62) with significant similarity to several bacterial/fungal aminopeptidases. Incubation of ES-62 with leucine-7-amino-4-methylcoumarin as substrate confirmed that ES-62 possessed aminopeptidase activity.

Amino Acid Sequence↗

PubMed1999

Structural studies of N-glycans of filarial parasites. Conservation of phosphorylcholine-substituted glycans among species and discovery of novel chito-oligomers.

N-Type glycans containing phosphorylcholine (PC-glycans), unusual structures found in the important human pathogens filarial nematodes, represent a novel target for chemotherapy. Previous work in our laboratories produced compositional information on the PC-glycan of ES-62, a secreted protein of the rodent parasite Acanthocheilonema viteae. In particular, we established using fast atom bombardment mass spectrometry (MS) analysis that PC was attached to a glycan with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. In the present study, we demonstrate that this structure is conserved among filarial nematodes, including the parasite of humans, Onchocerca volvulus, for which new drugs are most urgently sought. Furthermore, by employing a variety of procedures, including collision-activated dissociation MS-MS analysis and matrix-assisted laser desorption MS analysis, we reveal that surprisingly, filarial nematodes also contain N-linked glycans, the antennae of which are composed of chito-oligomers. To our knowledge, this is the first report of such structures in a eukaryotic glycoprotein.

Animals↗

PubMed1999

Phosphorylcholine: friend or foe of the immune system?

Phosphorylcholine (PC) is a structural component of a variety of prokaryotic and eukaryotic pathogens. In some cases, PC in infectious agents can benefit the infected host due to its targeting by both the innate and adaptive immune responses. However, as discussed here, PC exhibits a surprising range of immunomodulatory properties that might be to the detriment of the host.

Animals↗

PubMed1999

Mechanisms underlying the transfer of phosphorylcholine to filarial nematode glycoproteins--a possible role for choline kinase.

Phosphorylcholine (PC) is a common constituent of proteins secreted by filarial nematodes. As this substance has been shown to interfere with immune responses, we are interested in designing strategies for blocking its attachment. Towards this end, we are investigating the mechanism of incorporation of PC into filarial molecules and in the present manuscript we describe experiments relating to elucidating the source of PC for attachment. Synthesis of phosphatidylcholine in eukaryotic organisms can occur by a mechanism involving the transfer of PC from CDP-choline to diacylglycerol (the Kennedy pathway). By (i) measuring transfer of radio-isotope labelled PC from CDP-choline to parasite molecules and (ii) employing inhibitors of CDP-choline synthesis, we have investigated whether CDP-choline can act as a source of PC for transfer to ES-62, a major secreted glycoprotein of the rodent filarial nematode Acanthocheilonema viteae. Although we can find no evidence of this, we show that attachment of PC is blocked by hemicholinium-3, an inhibitor of choline kinase, the first enzyme in the Kennedy pathway. Thus, at least the first step in this pathway--phosphorylation of choline, would appear to be necessary for attachment of PC to ES-62.

Animals↗

PubMed1999

Immunomodulatory properties of a phosphorylcholine-containing secreted filarial glycoprotein.

ES-62 is a phosphorylcholine (PC)-containing glycoprotein which is secreted by the rodent filarial nematode Acanthocheilonema viteae. A homologue exists in the human filarial nematode Brugia malayi and indeed PC is found attached to glycoproteins of many, if not all, filarial species. At concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitized humans, ES-62 is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal-transduction elements in B and T lymphocytes following in-vitro exposure. Although this interaction is insufficient to cause lymphocyte proliferation per se, it serves to desensitize the cells to subsequent activation of the phosphoinositide-3-kinase, protein kinase C and Ras mitogen-activating protein kinase pathways and hence also to proliferation via the antigen receptors. The active component of ES-62 appears to be PC, as the results obtained with ES-62 are broadly mimicked by PC conjugated to BSA or PC alone. Although PC can also be shown to desensitize B cells following in-vivo administration, not all cells are affected, as it is still possible to generate an antibody response. Dissection of this response indicates that it is of the Th2 type.

Animals↗

PubMed1998

A phosphorylcholine-containing filarial nematode-secreted product disrupts B lymphocyte activation by targeting key proliferative signaling pathways.

Filarial nematodes infect more than 100 million people in the tropics, causing elephantiasis, chronic skin lesions, and blindness. The parasites are long-lived as a consequence of being able to evade the host immune system, but an understanding of the molecular mechanisms underlying this evasion remains elusive. In this study, we demonstrate that ES-62 (2 microg/ml), a phosphorylcholine (PC)-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal-transduction elements in B lymphocytes. Although this interaction is insufficient to cause B lymphocyte proliferation per se, it serves to desensitize the cells to subsequent activation of the phosphoinositide-3-kinase and Ras mitogen-activating protein kinase pathways, and hence also to proliferation, via the Ag receptor. The active component of ES-62 appears to be PC, a molecule recently shown to act as an intracellular signal transducer, as the results obtained with ES-62 are broadly mimicked by PC alone. As PC-containing secreted products (PC-ES) are also released by human filarial parasites, our data suggest that PC-ES, by interfering with B cell function, could play a role in prolonging filarial infection in parasitized individuals.

Amino Acid Sequence↗

PubMed1998

Heterotrimeric Guanine Nucleotide-binding Proteins in Eukaryotic Parasites.

Parasites, in order to survive, must be able to recognize and respond to signals in their environment. This is particularly so when parasite development is associated with moving from one environment to another as it is likely that a rapid process of adaptation that requires changes in gene expression will have to be initiated. In general, interaction with host signals takes place at the parasite surface, with the resulting need for information to be trasmitted to the interior. Many such signalling events in mammalian cells are regulated by a family of molecules referred to as guanine nucleotide-binding proteins (G proteins). The structure and function of these molecules, and evidence obtained to date of the roles that they may play in parasitic organisms, are discussed here by Bill and Maggie Harnett.

Journal Article↗

PubMed1998

Molecular and immunodiagnosis of human filarial nematode infections.

The filarial nematodes Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus represent major public health problems in the Tropics. Effective diagnosis of infection with these parasites is required both for administration of drugs to infected individuals and for monitoring of control programs. However parasitological diagnosis is associated with a number of problems including frequently inadequate sensitivity, long pre-patency of infection and inconvenience for patients. For these reasons there has been considerable effort expended in developing other forms of diagnosis, in particular immunoassays for measuring antibody and circulating parasite antigen as well as molecular-biology-based assays for detecting parasite DNA. This article reviews the progress and achievements obtained to date. The latter include the development of ELISAs employing recombinant antigen for detection of antibody to O. volvulus which have both high sensitivity and specificity, the commercial availability of immunoassays to measure circulating antigen in W. bancrofti infection and the generation of specific DNA-based detection systems for all three parasites.

Animals↗

PubMed1998

MAPkinase: a second site of G-protein regulation of B-cell activation via the antigen receptors.

Ligation of the antigen receptors on B cells transduces transmembrane signals leading to the induction of DNA synthesis. We now show that a pertussis toxin-sensitive heterotrimeric G-protein(s) of the Gi class plays a key role in the regulation of surface immunoglobulin (sIg)-mediated DNA synthesis in B cells. This site of G-protein regulation is distinct from that we have previously reported to govern the coupling of the antigen receptors on B cells to the phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate. We have, moreover, identified a candidate target for this new G-protein regulation by showing that mitogen-activating protein kinase (MAPkinase) activity, which plays a key role in the transduction of sIg-mediated proliferative signals in B cells, is abrogated by pre-exposure to pertussis toxin that covalently modifies and inactivates heterotrimeric G-proteins of the Gi class. Furthermore, our data suggest that this pertussis toxin-sensitive G-protein couples the antigen receptors to MAPkinase activation, at least in part, by regulating sIg-coupling to Lyn, Syk and perhaps Blk and Fyn activity, results consistent with studies in other systems which show that classical G-protein-coupled receptors recruit such protein tyrosine kinases to tranduce MAPkinase activation. Interestingly, however, this G-protein plays no apparent role in the control of up-regulation of major histocompatibility complex class II expression on B cells, suggesting that such G-protein-regulated-tyrosine kinase and MAPkinase activation is not required for the induction of this biological response following antigen receptor ligation.

Animals↗

PubMed1998

Induction of signalling anergy via the T-cell receptor in cultured Jurkat T cells by pre-exposure to a filarial nematode secreted product.

Filarial nematodes constitute major causes of morbidity in the Tropics. The worms have a life-span exceeding five years, a longevity which is considered to reflect at least in part, their ability to interfere with host lymphocyte responsiveness. To date the molecular mechanisms underlying this ability have not been defined but we now demonstrate that ES-62, a phosphorylcholine (PC)-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, is able to render Jurkat T cells anergic to intracellular signalling via the antigen receptor (TCR). In particular, ES-62 acts by modulating activation of the tyrosine kinases Fyn, Lck and ZAP-70 leading to selective disruption of TCR coupling to the phospholipase D, protein kinase C, phosphoinositide-3-kinase and RasMAPkinase signalling cascades. These cascades are key elements in the transduction of transcriptional and proliferative signals following ligation of TCR. As PC-containing secreted products (PC-ES) are also released by human filarial parasites, our data suggest that PC-ES may play a role in the induction of T lymphocyte hyporesponsiveness observed during filarial infections.

Amino Acid Sequence↗

PubMed1997

A filarial nematode secreted product differentially modulates expression and activation of protein kinase C isoforms in B lymphocytes.

Filarial nematodes, parasitic worms that cause elephantiasis, chronic skin lesions, and blindness in the tropics, release a number of molecules, some of which appear to be immunomodulatory/suppressive, into the host environment. Here we demonstrate that ES-62, a phosphorylcholine-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, interferes with activation of B lymphocytes by differential modulation of protein kinase C isoform expression. Indeed, while ES-62 selectively down-regulates expression of the alpha, beta, iota/lambda, delta, and zeta isoforms of PKC, it up-regulates expression of PKC-gamma and -epsilon in B cells. Inhibitor studies suggest that ES-62 appears to promote down-regulation of PKC isoforms mainly by stimulating proteolytic degradation. ES-62 also disrupts the normal activation and nuclear translocation patterns of the alpha and iota/lambda isoforms of PKC following ligation of the Ag receptor. The effects of ES-62 on certain PKC isoforms were found to be modified by coculture with IL-4. Of particular interest was the observation that IL-4 prevented down-regulation of PKC alpha and iota/lambda, isotypes considered to be active in transducing mitogenic signals. Phosphorylcholine-containing secreted products (phosphorylcholine-ES) are also released by human filarial parasites; hence we discuss how these findings may relate to the nature of the human B cell response during filarial infections.

Animals↗

PubMed1997

Studies on the site and mechanism of attachment of phosphorylcholine to a filarial nematode secreted glycoprotein.

We have recently shown that the immunomodulatory substance phosphorylcholine (PC) is covalently attached to ES-62, a major secreted protein of the filarial nematode parasite Acanthocheilonema viteae, via an N-linked glycan. Linkage of PC to N-glycans is previously unreported, and hence we have investigated the biochemical events underlying it. PC addition was found by pulse-chase experiments to be a fairly early event during intracellular transport, occurring within 40-60 min of protein synthesis. Biosynthetic labeling/immunoprecipitation experiments revealed that addition of PC to ES-62 was blocked by (i) brefeldin A, an inhibitor of trafficking of newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi, (ii) 1-deoxynorijirimycin, an inhibitor of glucosidase activity in the ER, and (iii) 1-deoxymannojirimycin, an inhibitor of mannosidase I in the cis Golgi. Swainsonine, an inhibitor of mannosidase II in the medial Golgi, did not affect PC addition. Taken together these data indicate that PC attachment is a post-ER event which is dependent on generation of an appropriate substrate during oligosaccharide processing. Furthermore, they strongly suggest that PC addition takes place in the medial Golgi and that the substrate for addition is the 3-linked branch of Man5GlcNAc3 or Man3GLcNAc3.

Animals↗

PubMed1997

Acanthocheilonema viteae: stage-specific expression of G-protein alpha-subunits.

We have previously demonstrated by Western blot analysis that the adult stage of the filarial nematode Acanthocheilonema viteae expresses the alpha-subunits of heterotrimeric G-proteins corresponding to GS and Gq. We now show, using the same technique, that these two alpha-subunits are not detectable in the microfilaria stage of the parasite. Conversely, microfilariae contain Go, an alpha-subunit not expressed by the adult worm. No other G-protein alpha-subunits were found in microfilariae by Western blotting. However, reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate G-protein oligonucleotide primers, followed by hybridisation analysis, using oligonucleotides specific for individual G-protein alpha-subunits, not only confirmed expression of Go, but also detected Gi1 and G11 alpha-subunits. G-protein expression in infective larvae was also investigated by RT-PCR analysis: this stage of the organism was found to resemble the adult more than the microfilaria but differed from the adult in that GS was absent and Gi3 was present. The significance of these stage-specific differences in G-protein expression is discussed with respect to their possible role in parasite development and survival.

Animals↗

PubMed1997

The use of inhibitors of N-linked glycosylation and oligosaccharide processing to produce monoclonal antibodies against non-phosphorylcholine epitopes of Brugia pahangi excretory-secretory products.

Adult Brugia pahangi were cultured with [3H]-choline in both the absence and the presence of either tunicamycin or 1-deoxymannojirimycin (dMM), inhibitors of N-linked glycosylation and N-linked oligosaccharide processing, respectively. Excretory-secretory products (ES) were recovered from the spent medium and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography. Both inhibitors were found to prevent radiolabeling of ES, a result consistent with blockage of the addition of the highly immunodominant phosphorylcholine (PC) group. Tunicamycin/dMM-treated ES were subsequently employed to immunise a mouse in an attempt to produce monoclonal antibodies (mAbs) against non-PC epitopes of ES. Three mAbs were isolated, each of which reacted with ES but not with PC.

1-Deoxynojirimycin↗

PubMed1997

Characterisation of the phosphorylcholine-containing N-linked oligosaccharides in the excretory-secretory 62 kDa glycoprotein of Acanthocheilonema viteae.

The major excretory-secretory product of the rodent filarial nematode Acanthocheilonema viteae is a 62 kDa glycoprotein (ES-62), which has phosphorylcholine, attached to the N-linked carbohydrates. In this paper, we describe structural studies of N-glycans released from ES-62 by peptide N-glycosidase F. Three major classes of N-glycan structures were observed: high mannose type structures; those which had been fully trimmed to the trimannosyl core and were sub-stoichiometrically fucosylated; and those with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine resides. Of the three classes of glycans, only the last was found to be substituted with detectable levels of phosphorylcholine. The implications of these results with respect to the probable glycosylation pathways operating in A. viteae are discussed.

Animals↗

PubMed1997

Variation in the nature of attachment of phosphorylcholine to excretory-secretory products of adult Brugia pahangi.

The mechanism of linkage of phosphorylcholine (PC) to excretory-secretory products (ES) of adult Brugia pahangi has been investigated. Biosynthetic radio-isotope labelling of ES with [3H]choline followed by SDS-PAGE/fluorography revealed a smear of molecular weight approximately 40-100 kDa which loses its radiolabel following exposure to N-glycosidase F, but not mild alkali. PC is thus attached to this smear of molecules via N-type glycans, a mechanism of linkage previously observed with respect to PC-ES of Acanthocheilonema viteae. Western blotting analysis of non-radiolabelled ES demonstrated the existence of additional PC-ES which were insensitive to N-glycosidase F, but not to alkali. This second group of molecules is therefore likely to contain PC linked to O-glycans. Filarial nematodes may thus utilize 2 classes of glycan for attachment of PC. Examination of B. pahangi and A. viteae whole worm extracts by Western blotting indicated that their PC content could not be cleaved by N-glycosidase F and hence the use of N-type glycans may be restricted to a subset of ES products. The implications of these findings with respect to developing inhibitors of PC attachment for use as anti-filarial drugs are discussed.

Amidohydrolases↗

PubMed1997

Differential G-protein expression during B- and T-cell development.

The molecular mechanisms underlying B- and T-cell development are, as yet, poorly understood. However, as G proteins regulate a diverse range of biological responses including growth, proliferation and differentiation, we have investigated differential expression of G proteins during B- and T-cell development with the aim of identifying key signals involved in lymphocyte maturation. Differential expression of beta 1/2 and alpha-subunits of the Gs-, i- and q-families was found throughout lymphoid development. Most strikingly, G alpha i1 and G alpha i1 were very weakly, or not expressed in pre-, immature and mature B cells, thymocytes or mature T cells, but strongly induced in mature B-lymphoblastoid cell lines, some of which have been used as models of germinal centre B cells, suggesting that expression of these G proteins may correlate with the later stages of B-cell development. In contrast, G alpha 16 expression was highest in T cells and pre-B cells and progressively declined with B-cell maturation. These findings suggest that G proteins, and the signals they regulate, such as ion channels and/or adenylate cyclase (G alpha s/i) and phospholipase C (G beta gamma and G alpha 11/16) are differentially regulated in lymphoid cells in a maturation-and lineage-dependent manner.

Animals↗