Trypanosoma brucei: Effect of inhibition of N-linked glycosylation on the nearest neighbor analysis of the major variable surface coat glycoprotein

Abstract

As an assay for the surface deposition of newly synthesized major variable surface coat glycoprotein (VSCG) we have treated intact Trypanosoma brucei cells with the cleavable cross-linking reagent dithiobis-(succinimidyl propionate). Under appropriate conditions, surface VSCG is converted to oligomers of n not less than 8. The oligomeric protein, apparent molecular weight greater than 4 × 10 5, does not migrate more than 1 to 2 mm into a 3–15% linear polyacrylamide gradient gel containing 0.1% sodium dodecyl sulfate, hence the appearance of newly synthesized radiolabeled protein in the top 2 mm of the gel indicates the translocation of VSCG from the site of synthesis to the surface and the gross establishment of normal interactions among the molecules. In addition, purified VSCG treated with the cross-linking reagent yielded a dimeric product on gel electrophoresis. To examine the role of N-linked carbohydrate in the translocation of the protein and in intermolecular interactions we have allowed trypanosomes to incorporate L-[ 14C]serine into protein in the presence of the antibiotic tunicamycin. Our results show that N-linked carbohydrate is not essential to the transfer of VSCG to the cell surface nor does its absence interfere with gross intermolecular interactions in the short term. On the other hand N-linked carbohydrate does appear to play an essential role in dimer formation.