PubMed Health⌕ Search

Biomedical subjects

P T Englund

Publications and source records attributed to P T Englund.

At least 91 records · Page 5Linked to original sources
PubMed1987

A glycan-phosphatidylinositol-specific phospholipase D in human serum.

A group of proteins anchored to the cell by phosphatidylinositol (PI) has recently been identified. The significance of this new class of membrane anchor is unknown; one possibility is that it facilitates release of the molecule by phospholipases. In fact, phospholipase C enzymes specific for the complex carboxyl-terminal glycolipids of these proteins have been isolated from African trypanosomes and from hepatocyte plasma membranes. This study reports the discovery of a glycan-PI-specific phospholipase D in human serum that cleaves both the membrane form of the variant surface glycoprotein of African trypanosomes and its glycolipid precursor, but not phosphatidylethanolamine, phosphatidylcholine, or phosphatidylinositol. Decay-accelerating factor, another PI-anchored molecule, is also cleaved by the enzyme and converted from a hydrophobic to a soluble protein. The enzyme is Ca2+-dependent, heat labile, and not affected by the inhibitor of serine proteases, phenylmethylsulfonylfluoride. Its function is not known, but the present findings indicate that it participates in the metabolism of glycolipid-anchored membrane proteins.

CD55 Antigens↗
PubMed1986

A phospholipase C from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein.

The surface coat of Trypanosoma brucei is composed of 10(7) molecules of the variant surface glycoprotein (VSG). Each VSG molecule is tethered to the cell membrane by a glycolipid moiety which contains 1,2-dimyristoyl-sn-phosphatidylinositol (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Following cell lysis, an endogenous phospholipase C cleaves dimyristoyl glycerol from the glycolipid, releasing soluble VSG. We have purified this enzyme, which we designate VSG lipase, by detergent extraction, (NH4)2SO4 fractionation, hydrophobic chromatography, and cation exchange chromatography. It is purified 2600-fold and is virtually homogeneous. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass is 37 kDa. In solutions containing the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), the Stokes radius (2.6 nm), S20,w (3.7 S), and v (0.77 cm3/g) of VSG lipase suggest a molecular mass for the native enzyme of about 47 kDa, part of which may be due to bound CHAPS. Therefore, it is probably monomeric. VSG lipase does not require Ca2+; it is stimulated by chelating agents or dithiothreitol, and it is inhibited by some sulfhydryl reagents. The purified enzyme appears to be highly specific. Under the conditions of our assay, it cleaves the VSG glycolipid, a biosynthetic precursor of the VSG glycolipid, and, to a much lesser extent, 1,2-dimyristoyl-sn-phosphatidylinositol. There was no apparent cleavage of other myristate-containing lipids of trypanosomes or 1-stearoyl-2-arachidonoyl-sn-phosphatidylinositol.

Animals↗
PubMed1986

Identification of a glycolipid precursor of the Trypanosoma brucei variant surface glycoprotein.

The variant surface glycoprotein (VSG) of Trypanosoma brucei has a glycolipid covalently attached to its C terminus. This glycolipid, which anchors the protein to the cell membrane, is attached to the VSG polypeptide within 1 min after translation (Bangs, J. D. Hereld, D., Krakow, J.L., Hart, G. W., and Englund, P. T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3207-3211). This rapid processing suggests that, prior to incorporation, the glycolipid may exist in the cell as a preformed precursor which is transferred to the VSG polypeptide en bloc. We have isolated a molecule which has properties consistent with it being a VSG glycolipid precursor. It is highly polar and can be labeled by [3H] myristate but not by [3H]palmitate. It reaches steady state during continuous labeling with [3H]myristate and shows rapid turnover in pulse-chase experiments, suggesting that it is a metabolic intermediate rather than an end product. When treated with HNO2 it liberates phosphatidylinositol, as does VSG (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Also, like VSG, it releases a compound which co-migrates on thin layer chromatography with dimyristylglycerol when treated with the purified endogenous phospholipase C from trypanosomes. After treatment with this lipase, the putative precursor can be immunoprecipitated by antibodies directed against the C-terminal cross-reactive antigenic determinant of the VSG. These data provide strong evidence that this glycolipid is a VSG precursor.

Animals↗
PubMed1986

Ribonucleotides associated with a gap in newly replicated kinetoplast DNA minicircles from Trypanosoma equiperdum.

In Trypanosoma equiperdum, some newly replicated kinetoplast DNA minicircles contain a single gap at a unique location in their newly synthesized strand (Ntambi, J. M., and Englund, P. T. (1985) J. Biol. Chem. 260, 5574-5579). We now report that ribonucleotides are associated with this gap, with one or two covalently attached to the 5' terminus of the newly synthesized strand. There appear to be two possible RNA/DNA junctions at adjacent positions in the sequence. The ribonucleotides may be remnants of a replication primer, and their presence strongly implies that the gap is at the site of a replication origin.

Animals↗
PubMed1986

Visualization of the bent helix in kinetoplast DNA by electron microscopy.

Kinetoplast DNA minicircles from the trypanosomatid Crithidia fasciculata contain a segment of approximately 200 bp which is probably more highly bent than any other DNA previously studied. Electron microscopy (EM) of relaxed minicircles (2.5 kb) revealed 200-300 bp loops within the larger circles, and the loops could also be detected on full-length linear molecules. Examination by EM of a 219 bp cloned fragment which contains the bent helix revealed that up to 70% of the molecules appeared circular whether or not the ends were cohesive. In contrast, a 207 bp fragment from pBR322 showed no circles and the fragments in general appeared much straighter than the kinetoplast fragments. Treatment of the 219 bp bent kinetoplast fragment with the drug distamycin caused a striking reduction in curvature.

Animals↗
PubMed1986

A highly bent fragment of Crithidia fasciculata kinetoplast DNA.

Kinetoplast DNA minicircles from Crithidia fasciculata contain a single major region of bent helix. Restriction fragments containing this bent helix have electrophoretic behavior on polyacrylamide gels which is much more anomalous than that of previously studied bent fragments. Therefore, the C. fasciculata fragments probably have a more extreme curvature. Sequencing part of a cloned minicircle revealed an unusual structure for the bent region. In a sequence of 200 bases, the bent region contains 18 runs of 4-6 As with 16 of these runs in the same strand. In some parts of this sequence the A runs are regularly spaced with a periodicity of about 10 base pairs. This spacing is nearly in phase with the twist of the DNA helix. This same sequence arrangement has been observed in other bent fragments, but the number of A runs is much greater in this C. fasciculata sequence. It is likely that there are small bends associated with each A run which, because of their periodic spacing, add up to produce substantial curvature in this molecule. In addition to having highly anomalous electrophoretic behavior, the fragment has unusual circular dichroism spectra. Its spectrum in the absence of ethanol is that of B DNA, but ethanol in the concentration range of 51-71% (w/w) induces changes to forms which are different from those of any well characterized DNA structure. The C. fasciculata bent helix is neither cleaved by S1 nuclease nor modified by bromoacetaldehyde under conditions in which other unusual DNA structures (such as cruciforms or B-Z junctions) are susceptible to attack by these reagents. Finally, a two-dimensional agarose gel analysis of a family of topoisomers of a plasmid containing the bent helix revealed no supercoil-induced relaxation.

Animals↗
PubMed1986

Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein.

After synthesis on membrane-bound ribosomes, the variant surface glycoprotein (VSG) of Trypanosoma brucei is modified by: (a) removal of an N-terminal signal sequence, (b) addition of N-linked oligosaccharides, and (c) replacement of a C-terminal hydrophobic peptide with a complex glycolipid that serves as a membrane anchor. Based on pulse-chase experiments with the variant ILTat-1.3, we now report the kinetics of three subsequent processing reactions. These are: (a) conversion of newly synthesized 56/58-kD polypeptides to mature 59-kD VSG, (b) transport to the cell surface, and (c) transport to a site where VSG is susceptible to endogenous membrane-bound phospholipase C. We found that the t 1/2 of all three of these processes is approximately 15 min. The comparable kinetics of these processes is compatible with the hypotheses that transport of VSG from the site of maturation to the cell surface is rapid and that VSG may not reach a phospholipase C-containing membrane until it arrives on the cell surface. Neither tunicamycin nor monensin blocks transport of VSG, but monensin completely inhibits conversion of 58-kD VSG to the mature 59-kD form. In the presence of tunicamycin, VSG is synthesized as a 54-kD polypeptide that is subsequently processed to a form with a slightly higher Mr. This tunicamycin-resistant processing suggests that modifications unrelated to N-linked oligosaccharides occur. Surprisingly, the rate of VSG transport is reduced, but not abolished, by dropping the chase temperature to as low as 10 degrees C.

Animals↗
PubMed1985

A gap at a unique location in newly replicated kinetoplast DNA minicircles from Trypanosoma equiperdum.

Kinetoplast DNA is a network containing thousands of interlocked minicircles. The minicircles replicate as free molecules after release from the network, and their progeny are then reattached (Englund, P. T., (1979) J. Biol. Chem. 254, 4895-4900). In Trypanosoma equiperdum, some of the newly replicated minicircles which have recatenated to the network contain a single gap. This gap is about 10 nucleotides in size and it is in the newly synthesized strand. Based on several criteria (S1 nuclease digestion, denaturing polyacrylamide gel analysis and DNA sequencing), the gap residues at a unique site on the molecule. This site overlaps the sequence GGGGTTGGTGTAA, which is the only common sequence found in all minicircles, from several different species, which have been examined.

Animals↗
PubMed1985

Intermediates in the replication of kinetoplast DNA minicircles.

Kinetoplast DNA of Crithidia fasciculata and other trypanosomatids is in the form of a network of thousands of minicircles and a few dozen maxicircles. Minicircles replicate as free molecules after release from the network, and their progeny subsequently reattach to the network (Englund, P. T. (1979) J. Biol. Chem. 254, 4895-4900). The minicircles just released from the network are covalently closed and apparently completely relaxed. After Cairns-type (theta) replication, the two minicircle progeny have different structures. One has a nascent H (heavy) strand which initially is in the form of 20-110 nucleotide fragments that are separated by gaps (Kitchin, P. A., Klein, V. A., Fein, B. I., and Englund, P. T. (1984) J. Biol. Chem. 259, 15532-15539). The other initially has a full-size (2.5 kilobase) nascent L (light) strand. During the time between formation of these progeny molecules and network reattachment, the nascent L strand is nicked (or gapped) and nascent H strand is partially repaired. Therefore, both progeny, at the time of reattachment, have several nicks (or gaps) in their nascent strand. Minicircle progeny with a nascent L strand reattach to the network quickly, whereas those with a nascent H strand reattach more slowly. Once reattached to the network, the nicks or gaps in the minicircles are repaired until finally covalent closure occurs.

Animals↗
PubMed1985

Rapid processing of the carboxyl terminus of a trypanosome variant surface glycoprotein.

The variant surface glycoprotein of the parasite Trypanosoma brucei contains a glycolipid of unknown structure covalently attached to its COOH terminus. We have shown, by using metabolic labeling with [35S]methionine or [3H]myristic acid, precipitation with specific antibodies, and NaDodSO4/polyacrylamide gel electrophoresis, that this glycolipid is attached to the variant surface glycoprotein polypeptide within 1 min after its translation.

Amino Acid Sequence↗
PubMed1984

Gapped Minicircles. A novel replication intermediate of kinetoplast DNA.

Kinetoplast DNA, the mitochondrial DNA in trypanosomatids, is a network of thousands of interlocked circles. Most of these circles are minicircles and a few are maxicircles. Minicircles replicate, after decatenation from the network, by a Cairns-type mechanism. The minicircle progeny then reattach to the network (Englund, P. T. (1979) J. Biol. Chem. 254, 4895-4900). We have now discovered a novel intermediate in Crithidia fasciculata minicircle replication. It is a highly gapped 2.5-kilobase free minicircle with nascent fragments of only 20 to 110 nucleotides. These fragments are nonligasable, and some remain nonligasable even after gap filling with DNA polymerase. Solution hybridization studies show that the nascent fragments are predominantly, if not exclusively, heavy strand.

Animals↗
PubMed1984

Physical characterization of a kinetoplast DNA fragment with unusual properties.

A 414-base pair fragment from a Leishmania tarentolae kinetoplast DNA minicircle has unusual physical properties. We reported previously that in comparison to phi X174 and pBR322 control fragments, the kinetoplast fragment behaves in gel electrophoresis, gel filtration, and electric dichroism experiments as if it has an unusually compact conformation. We accounted for these unusual properties by proposing that the fragment is a systematically bent helix (Marini, J.C., Levene, S.D., Crothers, D.M., and Englund, P.T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7664-7668). In this paper, we further explore the properties of the kinetoplast fragment. Because of its compact conformation, the kinetoplast fragment has difficulty in snaking through polyacrylamide gels and therefore migrates unusually slowly in electrophoresis experiments. Warming (53 degrees C) and ethanol (5-20%) partially normalize gel migration; glyoxal treatment results in denatured strands with electrophoretic mobility close to that expected for their size. In vivo modification does not appear to be responsible for the fragment's properties; its anomalous electrophoretic behavior persists after proteinase K treatment, phenol extraction, or after cloning into pBR322 and reisolation. Velocity sedimentation experiments rule out fragment aggregation. Secondary structure, such as a cruciform, is not detectable by S1 or mung bean nuclease digestion. The kinetoplast fragment has circular dichroism spectra characteristic of a B-type helix. With increasing temperature, there is an increase in the 270/280 ellipticity ratio. Circular dichroism spectra taken in the presence of ethanol show a B to A helix transition at unusually low ethanol concentrations (between 44 and 54% (w/w]. Thermal denaturation reveals a triphasic melting curve.

Animals↗
PubMed1984

Replication of kinetoplast DNA maxicircles.

The kinetoplast DNA of Crithidia fasciculata is a massive network composed of thousands of topologically interlocked circles. Most of these circles are minicircles (2.5 kb), and about 50 are maxicircles (37 kb). Previous studies showed that minicircles replicate, after release from the network, via Cairns (theta) intermediates. Here we show that maxicircles replicate, while attached to the network, by an entirely different mechanism involving rolling circle intermediates. After the network-bound maxicircle has finished replication, the branch of the rolling circle is apparently cleaved off to form a linear free maxicircle. A restriction map of the linearized free maxicircles shows that these molecules have unique termini, one of which presumably corresponds to the replication origin.

Animals↗
PubMed1984

Presence of a bent helix in fragments of kinetoplast DNA minicircles from several trypanosomatid species.

Some restriction fragments of kinetoplast minicircles from several trypanosomatid species (Leishmania tarentolae, Trypanosoma brucei, T. equiperdum, Herpetomonas muscarum, Crithidia fasciculata, but not T. cruzi) migrate anomalously on polyacrylamide gels. This behavior is probably due to a natural curvature of the helix. Bent helices appear to be a common property of kinetoplast minicircles, and may be important for minicircle function. In the case of T. equiperdum, we present evidence that each minicircle has a single bent region which resides in or near the 'conserved sequence.'

Animals↗
PubMed1982

Bent helical structure in kinetoplast DNA.

We have investigated the unusual physical properties of a restriction fragment of Leishmania tarentolae kinetoplast DNA. A gel-purified fragment comprising slightly more than half of a minicircle was determined by Maxam-Gilbert sequence determination to be 490 base pairs (bp) in length. This fragment has dramatically anomalous electrophoretic behavior; it has an apparent size of 450 bp on a 1% agarose gel but migrates as 1,380 bp on a 12% polyacrylamide gel. However, in gel filtration on Sephacryl S-500, the fragment elutes with an apparent size of 375 bp. Finally, it behaves anomalously in electric dichroism experiments. Field-free rotational relaxation times from transient electric dichroism studies are highly sensitive to effective molecular dimensions. The rotational relaxation time of the kinetoplast fragment is smaller than that of a 309-bp control fragment from pBR322. Because rigorous control experiments rule out the possibility that this fragment is modified, these anomalous properties must be dictated by the sequence itself. Fragment behavior indicates that it has an unusually compact configuration; we propose that this molecule contains a region of systematically bent B-DNA. This model accounts for the fragment's difficulty in snaking through the pores of a polyacrylamide gel, its ease in diffusing into Sephacryl beads, and its smaller rotational relaxation time. Bending of this molecule may be caused by periodicities in the DNA sequence.

Journal Article↗