Biochemical Diversity in the Trypanosoma congolense Trans-sialidase Family

Trans-sialidases are key enzymes in the life cycle of African trypanosomes in both, mammalian host and insect vector and have been associated with the disease trypanosomiasis, namely sleeping sickness and nagana. Besides the previously reported TconTS1, we have identified three additional active trans-sialidases, TconTS2, TconTS3 and TconTS4, and three trans-sialidase like genes in Trypanosoma congolense. At least TconTS1, TconTS2 and TconTS4 are found in the bloodstream of infected animals. We have characterised the enzymatic properties of recombinant proteins expressed in eukaryotic fibroblasts using fetuin as model blood glycoprotein donor substrate. One of the recombinant trans-sialidases, TconTS2, had the highest specific activity reported thus far with very low sialidase activity. The active trans-sialidases share all the amino acids critical for the catalytic reaction with few variations in the predicted binding site for the leaving or acceptor glycan. However, these differences cannot explain the orders of magnitudes between their transfer activities, which must be due to other unidentified structural features of the proteins or substrates selectivity. Interestingly, the phylogenetic relationships between the lectin domains correlate with their specific trans-sialylation activities. This raises the question whether and how the lectin domains regulate the trans-sialidase reaction. The identification and enzymatic characterisation of the trans-sialidase family in T. congolense will contribute significantly towards the understanding of the roles of these enzymes in the pathogenesis of Animal African Trypanosomiasis.转唾液酸酶（trans-sialidases）是非洲锥虫（African trypanosomes）在哺乳动物宿主与昆虫媒介体内生命周期中的关键酶类，且与锥虫病——即昏睡病与牛锥虫病（nagana）——的发生密切相关。此前已有研究报道了TconTS1，本研究在刚果锥虫（Trypanosoma congolense）中额外鉴定出3种具有活性的转唾液酸酶，即TconTS2、TconTS3与TconTS4，以及3种转唾液酸酶样基因。至少TconTS1、TconTS2与TconTS4可在感染动物的血液中被检测到。我们以胎球蛋白（fetuin）作为模式血液糖蛋白供体底物，对在真核成纤维细胞中表达的重组蛋白的酶学特性进行了表征。其中重组转唾液酸酶TconTS2展现出目前已报道的最高比活性，且仅具有极低的唾液酸酶活性。具有活性的转唾液酸酶均保留了催化反应必需的全部氨基酸，仅在离去糖或受体糖聚糖的预测结合位点上存在少量差异。但上述差异无法解释其转唾液酸活性之间的数量级差异，该差异应源于蛋白质尚未明确的其他结构特征或底物选择性。值得注意的是，凝集素结构域（lectin domains）之间的系统发育关系与其特异性转唾液酸化活性存在相关性。这一发现引出了一个问题：凝集素结构域是否以及如何调控转唾液酸酶的催化反应。本研究对刚果锥虫中转唾液酸酶家族的鉴定与酶学表征，将为理解这些酶在非洲动物锥虫病（Animal African Trypanosomiasis）发病机制中的作用提供重要助力。