Structural studies of the shortest extended synaptotagmin with only two C2 domains from Trypanosoma brucei

The transferrin receptor (TfR) of the bloodstream form (BSF) of Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site–associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting of three oligomannose and two unprocessed paucimannose structures. Its GPI anchor is modified by the addition of 4–6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, and previous studies have shown transport-dependent increases in E6 size consistent with post-glycan processing in the endoplasmic reticulum. Using pulse-chase radiolabeling, peptide-N-glycosidase F treatment, lectin pulldowns, and exoglycosidase treatment, we have now investigated TfR N-glycan and GPI processing. E6 increased ∼5 kDa during maturation, becoming reactive with both TL and Erythrina cristagalli lectin (ECL, terminal LacNAc), indicating synthesis of poly-LacNAc on paucimannose N-glycans. This processing was lost after exoglycosidase treatment and after RNAi-based silencing of TbSTT3A, the oligosaccharyltransferase that transfers paucimannose structures to nascent secretory polypeptides. These results contradict previous structural studies. Minor GPI processing was also observed, consistent with α-galactose addition. However, increasing the spacing between E6 protein and the GPI ω-site (aa 4–7) resulted in extensive post-translational processing of the GPI anchor to a form that was TL/ECL-reactive, suggesting the addition of LacNAc structures, confirmed by identical assays with BiPNHP, a non-N-glycosylated GPI-anchored reporter. We conclude that BSF trypanosomes can modify GPIs by generating structures reminiscent of those present in insect-stage trypanosomes and that steric constraints, not stage-specific expression of glycosyltransferases, regulate GPI processing.

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Structural Studies on the Glycophospholipid Membrane Anchor of Trypanosoma Brucei Variant Surface Glycoprotein

Host-Parasite Cellular and Molecular Interactions in Protozoal Infections ◽

10.1007/978-3-642-72840-2_3 ◽

1987 ◽

pp. 19-28 ◽

Cited By ~ 1

Author(s):

M. A. J. Ferguson ◽

R. A. Dwek ◽

S. W. Homans ◽

T. W. Rademacher

Keyword(s):

Trypanosoma Brucei ◽

Surface Glycoprotein ◽

Variant Surface Glycoprotein ◽

Structural Studies ◽

Membrane Anchor

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Antigenic variation in trypanosomes

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1978.0057 ◽

1978 ◽

Vol 202 (1146) ◽

pp. 55-72 ◽

Cited By ~ 45

Keyword(s):

Immune Response ◽

Trypanosoma Brucei ◽

Genetic Basis ◽

Antigenic Variation ◽

Structural Studies ◽

African Trypanosomes ◽

Domestic Livestock ◽

Polypeptide Backbone ◽

Single Polypeptide ◽

Serological Variant

The several species of African trypanosomes pathogenic to man and domestic livestock may evade the immune response through a process of antigenic variation. This phenomenon is the major obstacle to immunization. Variation may be recognized in the form of sequential changes in the serological characteristics of the trypanosome surface during the course of an infection. Each serological variant of Trypanosoma brucei possesses a unique glycoprotein covering the entire trypanosome surface. When an immune response occurs against the prevailing variant, the trypanosomes are able to express an alternative and immunologically distinct glycoprotein. The antigenic repertoire of a trypanosome clone or species is so far undetermined and the genetic basis of antigenic variation is unexplored. Structural studies indicate that the immunological uniqueness of each glycoprotein is due primarily to immense variations in amino acid sequence distributed throughout the single polypeptide backbone. The extent of glycosylation is also variable.

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Structural Studies of Trypanosoma brucei RNA Editing Ligases and Their Binding Partner Proteins

Biochemistry ◽

10.1021/acs.biochem.5b01257 ◽

2016 ◽

Vol 55 (16) ◽

pp. 2319-2331

Author(s):

Alireza Shaneh ◽

Enrico O. Purisima ◽

Reza Salavati ◽

Traian Sulea

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Structural Studies ◽

Binding Partner ◽

Partner Proteins

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Ribosome Structural Organization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051670 ◽

1974 ◽

Vol 32 ◽

pp. 332-333

Author(s):

James A. Lake

Keyword(s):

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Small Subunit ◽

Structural Studies ◽

X Ray Diffraction ◽

Specific Antibodies ◽

X Ray ◽

E Coli ◽

Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

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Structural Studies on Mitotic Spindle Fibres

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100070345 ◽

1978 ◽

Vol 36 (3) ◽

pp. 615-626

Author(s):

J.R. Mcintosh

Keyword(s):

Chemical Composition ◽

Mitotic Spindle ◽

Structural Studies ◽

Mitotic Apparatus ◽

Chemical Studies ◽

Medical Interest ◽

Spindle Fibres

The mitotic apparatus is a structure of obvious biological and medical interest, but it has proved to be a difficult cellular machine to understand. The chemical composition of the spindle is only slightly elucidated, largely because of the difficulties in preparing useful isolates of the structure. Chemical studies of the mitotic spindle have been reviewed elsewhere (Mcintosh, 1977), and will not be discussed further here. One would think that structural studies on the mitotic apparatus (MA) in situ would be straightforward, but even with this approach there is some disagreement in the results obtained with various methods and by different investigators. In this paper I will review briefly the approaches which have been used in structural studies of the MA, pointing out the strengths and problems of each approach. I will summarize the principal findings of the different methods, and identify what seem to be fruitful avenues for further work.

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Image registration with a computer driven S.T.E.M.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100107630 ◽

1978 ◽

Vol 36 (1) ◽

pp. 98-99

Author(s):

A.M.H. Schepman ◽

J.A.P. van der Voort ◽

J.E. Mellema

Keyword(s):

Spot Size ◽

Scanning Transmission Electron Microscope ◽

Small Computer ◽

Structural Studies ◽

Area Of Interest ◽

Transmission Electron ◽

Scanning Transmission ◽

Specimen Area ◽

On Line ◽

Minimum Distances

A Scanning Transmission Electron Microscope (STEM) was coupled to a small computer. The system (see Fig. 1) has been built using a Philips EM400, equipped with a scanning attachment and a DEC PDP11/34 computer with 34K memory. The gun (Fig. 2) consists of a continuously renewed tip of radius 0.2 to 0.4 μm of a tungsten wire heated just below its melting point by a focussed laser beam (1). On-line operation procedures were developped aiming at the reduction of the amount of radiation of the specimen area of interest, while selecting the various imaging parameters and upon registration of the information content. Whereas the theoretical limiting spot size is 0.75 nm (2), routine resolution checks showed minimum distances in the order 1.2 to 1.5 nm between corresponding intensity maxima in successive scans. This value is sufficient for structural studies of regular biological material to test the performance of STEM over high resolution CTEM.

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The Fine Structure of Phloem Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119867 ◽

1985 ◽

Vol 43 ◽

pp. 632-635

Author(s):

James Cronshaw

Keyword(s):

Structural Studies ◽

High Concentration ◽

Long Distance ◽

Phloem Tissue ◽

Sieve Elements ◽

Long Distance Transport ◽

P Protein ◽

Companion Cells ◽

Electron Microscopical ◽

Distance Transport

Long distance transport in plants takes place in phloem tissue which has characteristic cells, the sieve elements. At maturity these cells have sieve areas in their end walls with specialized perforations. They are associated with companion cells, parenchyma cells, and in some species, with transfer cells. The protoplast of the functioning sieve element contains a high concentration of sugar, and consequently a high hydrostatic pressure, which makes it extremely difficult to fix mature sieve elements for electron microscopical observation without the formation of surge artifacts. Despite many structural studies which have attempted to prevent surge artifacts, several features of mature sieve elements, such as the distribution of P-protein and the nature of the contents of the sieve area pores, remain controversial.

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