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G A Cross

Publications and source records attributed to G A Cross.

At least 73 records · Page 4Linked to original sources
PubMed1991

Characterization of a candidate gene for GP72, an insect stage-specific antigen of Trypanosoma cruzi.

The Trypanosoma cruzi insect stage-specific antigen GP72 was purified from epimastigotes and the amino acid sequences of peptide fragments determined. Oligonucleotides derived from these data were used to amplify and clone a cDNA sequence, which was used to isolate a full-length gene. All the sequenced peptides were encoded within the gene. The characteristics of the encoded 62,600-Da protein, including a potential amino-terminal signal sequence, a hydrophobic carboxy-terminus, and a large number of potential O-glycosylation sites, are consistent with the properties of GP72. No sequence homologies were found in searches of DNA and protein data banks. GP72 is encoded by a single pair of non-telomeric allelic genes.

Amino Acid Sequence↗
PubMed1991

Transfer of glycosyl-phosphatidylinositol membrane anchors to polypeptide acceptors in a cell-free system.

Glycosylinositol phospholipid (GPI) membrane anchors are the sole means of membrane attachment of a large number of cell surface proteins, including the variant surface glycoproteins (VSGs) of the parasitic protozoan, Trypanosoma brucei. Biosynthetic data suggest that GPI-anchored proteins are synthesized with carboxy-terminal extensions that are immediately replaced by GPI, suggesting the existence of preformed GPI species available for transfer to the nascent protein in the ER. Candidate precursor glycolipids having a linear sequence indistinguishable from the conserved core structure found on all GPI anchors, have been characterized in T. brucei. In this paper we describe the transfer of three GPI variants to endogenous VSG in vitro. GPI addition is not reduced by inhibitors of protein synthesis and does not require ATP or GTP, consistent with a transpeptidation mechanism.

Adenosine Triphosphate↗
PubMed1990

Cell-free synthesis of glycosyl-phosphatidylinositol precursors for the glycolipid membrane anchor of Trypanosoma brucei variant surface glycoproteins. Structural characterization of putative biosynthetic intermediates.

Trypanosome variant surface glycoproteins exemplify a class of eukaryotic cell surface glycoproteins that rely on a carboxyl-terminal covalently-attached inositol-containing glycophospholipid for membrane attachment. The glycolipid anchor is acquired soon after translation of the polypeptide, apparently by replacement of a short carboxyl-terminal peptide sequence with a prefabricated glycolipid. A candidate glycolipid precursor (referred to as P2), and a related glycolipid (P3) have been identified recently in polar lipid extracts from trypanosomes. In this paper we describe the synthesis of P2 and P3 by trypanosome membranes. Analyses of organic solvent extracts from membranes incubated with radioactive sugar nucleotides (GDP-[3H]mannose or UDP-[3H]GlcNAc) showed a spectrum of labelled lipids, ranging from partially glycosylated species to the final products, P2 and P3. Structural analyses of these putative biosynthetic intermediates suggest that glycolipid assembly occurs via the sequential glycosylation of phosphatidylinositol.

Animals↗
PubMed1990

Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. I. Can structure of the phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids.

A number of eukaryotic surface glycoproteins, including the variant surface glycoproteins of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosyl-phosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated precursor glycolipid that can be transferred en bloc to the polypeptide. We have reported the purification and partial characterization of a candidate precursor glycolipid (P2) and of a compositionally similar glycolipid (P3) from T. brucei (Menon, A. K., Mayor, S., Ferguson, M. A. J., Duszenko, M., and Cross, G. A. M. (1988) J. Biol. Chem. 263, 1970-1977). The primary structure of the glycan portions of P2 and P3 have now been analyzed by a combination of selective chemical fragmentation and enzymatic glycan sequencing at the subnanomolar level. The glycans were generated by deamination, NaB3H4 reduction, and dephosphorylation of glycolipids purified from different trypanosome variants. Glycan fragments derived from biosynthetically labeled glycolipids were also analyzed. The cumulative data strongly suggest that P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The structural similarities suggest that GPI membrane anchors are derived from common precursor glycolipids that become variably modified during or after addition to newly synthesized proteins.

Animals↗
PubMed1990

Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. II. Lipid structures of phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids.

A common diagnostic feature of glycosylinositol phospholipid (GPI)-anchored proteins is their release from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). However, some GPI-anchored proteins are resistant to this enzyme. The best characterized example of this subclass is the human erythrocyte acetylcholinesterase, where the structural basis of PI-PLC resistance has been shown to be the acylation of an inositol hydroxyl group(s) (Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G., and Rosenberry, T. L. (1988) J. Biol. Chem. 263, 18766-18775). Both PI-PLC-sensitive and resistant GPI-anchor precursors (P2 and P3, respectively) have been found in Trypanosoma brucei, where the major surface glycoprotein is anchored by a PI-PLC-sensitive glycolipid anchor. The accompanying paper (Mayor, S., Menon, A. K., Cross, G. A. M., Ferguson, M. A. J., Dwek, R. A., and Rademacher, T. W. (1990) J. Biol. Chem. 265, 6164-6173) shows that P2 and P3 have identical glycans, indistinguishable from the common core glycan found on all the characterized GPI protein anchors. This paper shows that the single difference between P2 and P3, and the basis for the PI-PLC insusceptibility of P3, is a fatty acid, ester-linked to the inositol residue in P3. The inositol-linked fatty acid can be removed by treatment with mild base to restore PI-PLC sensitivity. Biosynthetic labeling experiments with [3H]palmitic acid and [3H]myristic acid show that [3H]palmitic acid specifically labels the inositol residue in P3 while [3H]myristic acid labels the diacylglycerol portion. Possible models to account for the simultaneous presence of PI-PLC-resistant and sensitive glycolipids are discussed in the context of available information on the biosynthesis of GPI-anchors.

Animals↗
PubMed1990

Leishmania mexicana mexicana gp63 is a site-specific neutral endopeptidase.

Leishmania mexicana, like other species of the genus, has a major 63-kDa surface glycoprotein (gp63) that is an active protease. Reports differ as to whether gp63 is a neutral or an acidic protease. Using three radiolabeled synthetic peptide substrates, gp63 purified from L. m. mexicana is most active at pH 6.5-7.5, in three different buffer systems, and appears to be a sequence-specific endopeptidase. The full extent of sequence specificity is undetermined, but these experiments suggest a strong preference for cleavage at serine or threonine residues. In common with other metalloproteases, the cleavage is on the amino side of the recognition residue.

Amino Acid Sequence↗
PubMed1989

Expression of a bacterial gene in a trypanosomatid protozoan.

A simple and reproducible assay for DNA-mediated transfection in the trypanosomatid protozoan Leptomonas seymouri has been developed. The assay is based on expression of the Escherichia coli chloramphenicol acetyl transferase (CAT) gene flanked by Leptomonas DNA fragments that are likely to contain necessary elements for gene expression in trypanosomes. After electroporation of cells in the presence of plasmid DNA, CAT activity was detected in crude cell lysates. No activity was detected when the orientation of the L. seymouri mini-exon sequence (placed upstream of the CAT gene) was reversed, or in additional control experiments. This system provides a method for defining transcriptional control elements in trypanosomes.

Animals↗
PubMed1989

Evaluation of evolutionary divergence in the genus Naegleria by analysis of ribosomal DNA plasmid restriction patterns.

Ribosomal DNA (rDNA) plasmid restriction maps of 10 strains and rDNA hybridisation patterns of 61 additional strains have been used to assess inter- and intra-specific diversity and phylogenetic relationships in the genus Naegleria. The results obtained by this method largely confirm those of previous studies based on a variety of other criteria. They indicate that very little variation exists within the pathogenic species Naegleria fowleri despite its worldwide distribution and that it is closely related to the nonpathogenic Naegleria lovaniensis. Naegleria gruberi is most likely a polyphyletic grouping and care should be taken when using one strain as a reference point for this species. In addition, the two subspecies of the pathogenic Naegleria australiensis arose separately from within the range of variability encompassed by N. gruberi, as did the species Adelphamoeba galeacystis which should probably be assigned to the genus Naegleria. The species Naegleria jadini and Naegleria andersoni are not closely related to any other in the genus based on their rDNA patterns.

Animals↗
PubMed1989

Transport of fluorescent phospholipid analogues from the erythrocyte membrane to the parasite in Plasmodium falciparum-infected cells.

The asexual development of the human malaria parasite Plasmodium falciparum is largely intraerythrocytic. When 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazole-4-yl)amino]caproyl] phosphatidylcholine (NBD-PC) was incorporated into infected and uninfected erythrocyte membranes at 0 degrees C, it remained at the cell surface. At 10 degrees C, the lipid was rapidly internalized in infected erythrocytes at all stages of parasite growth. Our results indicate that the internalization of NDB-PC was not because of endocytosis but rapid transbilayer lipid flip-flop at the infected erythrocyte membrane, followed by monomer diffusion to the parasite. Internalization of the lipid was inhibited by (a) depleting cellular ATP levels; (b) pretreating the cells with N-ethyl maleimide or diethylpyrocarbonate; and (c) 10 mM L-alpha-glycerophosphorylcholine. The evidence suggests protein-mediated and energy dependent transmembrane movement of the PC analogue. The conditions for the internalization of another phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) were distinct from that of NBD-PC and suggest the presence of additional mechanism(s) of parasite-mediated lipid transport in the infected host membrane. In spite of the lack of bulk, constitutive endocytosis at the red cell membrane, the uptake of Lucifer yellow by mature infected cells suggests that microdomains of pinocytotic activity are induced by the intracellular parasite. The results indicate the presence of parasite-induced mechanisms of lipid transport in infected erythrocyte membranes that modify host membrane properties and may have important implications on phospholipid asymmetry in these membranes.

Animals↗
PubMed1988

Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5' cap structure, m7GpppA*A*C(2'-O)mU*A.

Trypanosoma brucei mRNA is discontinuously synthesized via the 5' addition of a "mini-exon" sequence. The mini-exon-specific cap structure was purified from a complete RNase T2 and phosphatase digest of in vivo 32P-labeled poly(A)+RNA. The purified cap structure was sequenced by a series of partial and complete enzymatic digests by nuclease P1 and venom phosphodiesterase. This approach demonstrated that the T. brucei mini-exon cap structure consists of N7-methylguanosine linked in a conventional 5'-5' triphosphate bond to five nucleotides, in the sequence A*A*C(2'-O)mU*A (asterisks denote modifications that were not fully characterized in this work). 2'-O-methylations and other modifications appear to be present in this novel cap structure, which could have a functional role in the metabolism of the mini-exon.

Animals↗
PubMed1988

Discontinuous transcription in Leptomonas seymouri: presence of intact and interrupted mini-exon gene families.

Mature mRNAs of trypanosomatid protozoa result from the joining of at least two exons, which are initially transcribed as separate RNAs. In all trypanosomatids examined to date, the first exon (mini-exon) is encoded by approximately 200 tandemly reiterated genes. In characterizing the mini-exon genes of Leptomonas seymouri, we identified two predominant size classes of repetitive sequences that hybridized strongly to the L. seymouri mini-exon sequence. These two sequences are arranged as interspersed clusters. DNA sequence analysis of a clone representing the smaller size class demonstrated that these sequences have the capacity to encode a mini-exon donor (med)RNA corresponding to the 86 nt component seen in Northern blots of L. seymouri RNA. The larger size class comprises a family of related sequences, some of which contain DNA inserted into the mini-exon portion of the medRNA gene. The specific insert identified here (LINS 1) is exclusively associated with medRNA sequences, and is present in approximately 20% of the larger size class of L. seymouri medRNA genes. Disregarding the insertion, the sequences of the smaller bona fide mini-exon genes and the gene copy containing the insert were almost identical. The insert sequence is transcribed in the same direction as medRNA to yield at least four small non-polyadenylated RNAs, which appeared not to be linked to medRNA sequences.

Animals↗
PubMed1988

Characterization of RNA transcripts from the alpha tubulin gene cluster of Leptomonas seymouri.

A tandem cluster of alpha tubulin genes was identified in the trypanosomatid protozoan Leptomonas seymouri. One repeat unit and the first gene of the cluster, with its upstream flanking region, were cloned and analyzed for their transcriptional and coding capacities. The 12-15 copies per cell of the 4 kb repeat encode a stable 2 kb transcript, which contains a mini-exon at its 5' end and two closely spaced polyadenylation sites. Transcription of the alpha tubulin gene cluster in isolated nuclei was unidirectional. Intergenic and coding regions were transcribed at the same rate, and nascent intergenic and coding region transcripts were quantitatively linked. These results are consistent with the possibility that the primary transcripts are polycistronic, or that there is a single small intercistronic gap.

Amino Acid Sequence↗
PubMed1988

Candidate glycophospholipid precursor for the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoproteins.

Trypanosoma brucei variant surface glycoproteins are apparently synthesized with a hydrophobic carboxyl-terminal peptide that is cleaved and replaced by a complex glycosylphosphatidylinositol membrane anchor within 1 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated core glycolipid that would be transferred en bloc to the variant surface glycoprotein polypeptide. We report the purification and chemical characterization of a glycolipid from T. brucei that has properties consistent with a role as a variant surface glycoprotein glycolipid donor. This candidate glycolipid precursor has been defined by thin-layer chromatography of extracts of trypanosomes metabolically labeled with radioactive myristic acid, ethanolamine, glucosamine, mannose, and phosphate and by enzymatic, chemical, and gas chromatographic-mass spectrometric analysis. Mild alkali released 100% of the myristic acid, and reaction with phospholipase A2 released 50%. Nitrous acid deamination generated dimyristylphosphatidylinositol, and periodate oxidation released phosphatidic acid. Treatment of purified glycolipid with phosphatidylinositol-specific phospholipase C released dimyristylglycerol and a water-soluble glycan that was sized on Bio-Gel P-4 columns. The candidate precursor contained mannose, myristic acid, phosphate, and ethanolamine with an unsubstituted amino group, but not galactose.

Animals↗