RESUMO
Trypanosoma cruzi alpha-mannosidase has been purified to apparent homogeneity. It is a 240,000-Da tetramer composed of four identical subunits (58,000 Da). Each subunit contains one N-linked high-mannose oligosaccharide. Based on pH optimum and sensitivity to inhibition by swainsonine, we suggest it to be lysosomal, but this has yet to be demonstrated directly. The enzyme appears to be developmentally regulated and may be a key enzyme in the degradation of the lipopeptidophosphoglycan (LPPG) during transformation from epimastigote to trypomastigote. Preliminary experiments suggest T. cruzi does not utilize the mannose 6-phosphate recognition system for sorting alpha-mannosidase (or other acid hydrolases) to the lysosome. To clone the alpha-mannosidase from T. cruzi we have used the same approach that has been used for other alpha-mannosidases. The cDNA amplification product was subcloned and sequenced. Comparison of the T. cruzi alpha-mannosidase sequence with the alpha-mannosidases that were used in the original primer design demonstrated a greater similarity to murine lysosomal and Dictyostelium alpha-mannosidases than to Golgi alpha-mannosidases.
