Use of chimeric recombinant polypeptides to analyse conformational, surface epitopes on trypanosome variant surface glycoproteins.
Identification of surface-exposed epitopes on the variant surface glycoproteins (VSGs) of African trypanosomes has been complicated by the observation that most such epitopes are highly conformational. As a result, whenever the molecule is broken down for analysis, the epitope is generally lost. We have exploited the existence of closely related gene families to create chimeric molecules in which particular segments of one VSG are placed in the analogous position of a related but antigenically distinct VSG. The process is used in both a positive and negative manner, so that the epitope can be specifically added or destroyed in a given chimera. As an example, we have used this approach to identify the regions involved in reactivity to a monoclonal antibody specific for VSG117 on the surface of live trypanosomes. We show that while deletion of almost any region of VSG117 results in loss of reactivity to this monoclonal antibody, substituting particular regions with the corresponding segment of the structurally related but antigenically distinct VSG FM8.5 restores reactivity in most but not all cases, thereby delimiting the antigenically key regions. Likewise, substituting key regions from VSG117 into FM8.5 confers reactivity on the resulting chimeras. This approach circumvents some of the problems that result from the highly conformational nature of VSG and should allow further elucidation of the biologically relevant antigenic topology of VSGs.
Hsia
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Department of Microbiology and Immunology, Stanford University School of Medicine, California 94305-5402, USA.
Beals
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T
Boothroyd
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JC
D000349:Africa; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D002874:Chromosome Mapping; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D005455:Fluorescent Antibody Technique; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma