Rapid purification of the membrane-bound and released forms of the variant surface glycoprotein from Trypanosoma brucei

1985 ◽  
Vol 13 (2) ◽  
pp. 493-494
Author(s):  
DAVID G. JACKSON ◽  
H. PAUL VOORHEIS
Keyword(s):  
Trypanosoma Brucei ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Membrane Bound ◽  
Rapid Purification

scholarly journals A new method for the rapid purification of both the membrane-bound and released forms of the variant surface glycoprotein from Trypanosoma brucei

1985 ◽  
Vol 230 (1) ◽  
pp. 195-202 ◽  
Author(s):  
D G Jackson ◽  
M J Owen ◽  
H P Voorheis
Keyword(s):  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Aqueous Salt Solution ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
High Yield ◽  
Dependent Manner ◽  
Wide Range ◽  
Membrane Bound ◽  
Rapid Purification

A simple new technique was developed for the rapid purification of either the membrane-bound or the released forms of the variant surface glycoprotein of Trypanosoma brucei in high yield. Whole cells were used as the source of the membrane-bound form, and the supernatant of benzyl alcohol-treated cells was used as the source of the released form. The technique was based on extraction of the acid-treated protein into chloroform/methanol, followed by selective re-partition into aqueous salt solution. The yield of purified protein was found to be dependent critically on a low pH during the extraction/re-partition stages. This finding and the ability to cycle the protein repeatedly through organic and aqueous phases in a strictly pH-dependent manner suggested that the protein could undergo fully reversible denaturation/renaturation only while in an extensively protonated form. The yield was independent of the polarity of the organic phase and the protein concentration over a wide range. After purification, both forms retain their ability to react with specific antibody raised against the authentic native protein purified by conventional means. The amino acid composition and the identity of the N-terminal amino acid was the same for both forms of the protein. In addition, both forms had blocked C-terminal residues. There were determined to be 1.13 × 10(7) copies of the variant surface glycoprotein per cell.

scholarly journals Expression of a variant surface glycoprotein of Trypanosoma gambiense in procyclic forms of Trypanosoma brucei shows that the cell type dictates the nature of the glycosylphosphatidylinositol membrane anchor attached to the glycoprotein

1997 ◽  
Vol 324 (3) ◽  
pp. 885-895 ◽  
Author(s):  
Françoise PATURIAUX-HANOCQ ◽  
Nicole ZITZMANN ◽  
Jacqueline HANOCQ-QUERTIER ◽  
Luc VANHAMME ◽  
Sylvie ROLIN ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Degradation Products ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Mrna Levels ◽  
Membrane Anchor ◽  
Gpi Anchor ◽  
Intact Membrane ◽  
Membrane Bound ◽  
Trypanosoma Gambiense

Procyclic forms of Trypanosoma brucei have been genetically modified to express the major metacyclic variant surface glycoprotein (VSG variant AnTat 11.17) of Trypanosoma gambiense. The VSG is expressed in an intact membrane-bound form that can be detected over the entire plasma membrane, together with procyclin, and as a series of lower-molecular-mass fragments that are mostly soluble degradation products. The presence of degraded VSG in the cells and the culture medium suggests that VSG is not efficiently processed and/or efficiently folded when expressed in procyclic cells. The level of procyclin expressed on the surface of these cells is slightly reduced, although there is no difference in procyclin mRNA levels. The intact membrane-bound form of the VSG is N-glycosylated with oligomannose structures and contains a glycosylphosphatidylinositol (GPI) membrane anchor that can be biosynthetically labelled with [3H]ethanolamine. The anchor is sensitive to mammalian GPI-specific phospholipase D but, like the anchor of procyclin, it is resistant to the action of bacterial phosphatidylinositol-specific phospholipase C. This pattern of phospholipase sensitivity suggests that the GPI anchor acquired by VSG when expressed in procyclics is acylated on the inositol ring and therefore resembles a procyclic procyclin-type anchor rather than a trypomastigote VSG-type anchor with respect to the lipid structure. The VSG expressed in procyclics was sensitive to the action of a mixture of sialidase, β-galactosidase and β-hexosaminidase, suggesting that the VSG GPI anchor also contains a sialylated polylactosamine side-chain modification similar to that described for procyclin. These results indicate that the nature of the protein expressed has little influence on the post-translational modifications performed in the secretory pathway of procyclic trypanosomes.

scholarly journals Synchronous expression of individual metacyclic variant surface glycoprotein genes in Trypanosoma brucei

2015 ◽  
Vol 200 (1-2) ◽  
pp. 1-4 ◽  
Author(s):  
Kiantra Ramey-Butler ◽  
Elisabetta Ullu ◽  
Nikolay G. Kolev ◽  
Christian Tschudi
Keyword(s):  
Trypanosoma Brucei ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein

Stability and reconstitution of the soluble variant surface glycoprotein (sVSG) from Trypanosoma brucei

Biochemistry ◽  
1990 ◽  
Vol 29 (36) ◽  
pp. 8217-8223 ◽  
Author(s):  
Paul Rehaber ◽  
Norbert Staudacher ◽  
Robert Seckler ◽  
Roland Buelow ◽  
Peter Overath ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein

Frequent independent duplicative transpositions activate a single VSG gene

1987 ◽  
Vol 7 (1) ◽  
pp. 357-364
Author(s):  
M G Lee ◽  
L H Van der Ploeg
Keyword(s):  
Gene Conversion ◽  
Trypanosoma Brucei ◽  
Surface Antigen ◽  
Strong Evidence ◽  
Base Pair ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Gene Variants ◽  
Repeat Array ◽  
Expression Site

The expression of several surface antigen genes in Trypanosoma brucei is mediated by the duplicative transposition of a basic-copy variant surface glycoprotein (VSG) gene into an expression site. We determined that the appearance of variant 118, in a parasitemia, resulted from at least four independent duplicative transpositions of the same VSG 118 gene. Variants 117 and 118 both appeared at specific periods but resulted from multiple independent activations. Antigenic variants thus occur in an ordered manner. We show that in the duplicative transpositions of VSG genes, the ends of the transposed segments were homologous between the basic copy and the expression site. Sequences other than the previously reported 70-base-pair (bp) repeats could be involved. In one variant, 118 clone 1, the homology was between a sequence previously transposed into the expression site and a sequence located 6 kilobases upstream of the VSG 118 gene. In variant 118b the homology was presumably in 70-bp repeat arrays, while in a third 118 variant yet another sequence was involved. The possibility that the 70-bp repeats are important in the initial steps of the recombinational events was illustrated by a rearrangement involving a 70-bp repeat array. The data provide strong evidence for the notion that gene conversion mediates the duplicative transposition of VSG genes. We discuss a model that explains how the process of duplicative transposition can occur at random and still produce an ordered appearance of variants.

Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level

1991 ◽  
Vol 11 (1) ◽  
pp. 338-343
Author(s):  
D Jefferies ◽  
P Tebabi ◽  
E Pays
Keyword(s):  
Trypanosoma Brucei ◽  
Gene Promoter ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Gene Promoters ◽  
Control Of Gene Expression ◽  
Transient Activity ◽  
Expression Site ◽  
Cat Gene ◽  
Cat Expression

The putative promoter of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei was cloned into a plasmid containing the chloramphenicol acetyltransferase (CAT) gene. After electroporation into trypanosomes, this construct directed the expression of the CAT reporter gene. The essential region for promoter activity was found to reside within 88 bp upstream of the putative transcription start site. Transcription of the CAT construct occurred at approximately the same level in both bloodstream and procyclic forms and was resistant to alpha-amanitin. However, CAT expression appeared to be modulated in the two forms of the parasite. Sequences 3' to the gene seemed to be important in this respect, as CAT activity in bloodstream forms was readily detectable only when the 3' region of a VSG cDNA was placed downstream of the CAT gene. Two separate VSG gene promoter sequences, both cloned from T. brucei AnTat 1.3A, were equally able to direct CAT expression, which suggests that there are a number of potential VSG gene promoters in the genome, although usually only one expression site is fully active at any one time.

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