TRYPANOSOMA-RANGELI SIALIDASE - CLONING, EXPRESSION AND SIMILARITY TOTRYPANOSOMA-CRUZI TRANS-SIALIDASE

Sialidases are hydrolytic enzymes present from virus to higher eukaryo tes, catalyzing the removal of sialic acid from glycoconjugates. Some protozoa Trypanosomatidae secrete high levels of sialidase into the me dium, We have now purified the secreted sialidase from Trypanosoma ran geli, Its N-terminal sequence reveals 100% identity with the correspon ding region of the trans-sialidase from T.cruzi. Trans-sialidase, alth ough homologous to viral and bacterial sialidases, displays a novel si alyltransferase activity and is involved in host cell invasion, Severa l homologous trans-sialidase-like genes were cloned from genomic DNA o f T.rangeli, and grouped in three subfamilies. Active sialidase-encodi ng genes were found in one of them, The recombinant sialidase shows si milar properties to those of the native enzyme, including undetectable trans-sialidase activity, Nevertheless, it has an overall identity of 68.9% with the catalytic domain of T.cruzi trans-sialidase, increasin g to 86.7% admitting conservative substitutions, Only three other euka ryotic sialidases have been previously cloned, none of them showing si gnificant homology to trans-sialidase, The isolation of a highly simil ar sialidase is relevant to further identify the molecular determinant s allowing trans-sialidase activity, As a first approach, chimeric con structs between sialidase and trans-sialidase were generated, one of t hem rendering a sialidase with three times lower K-m than the natural enzyme.