Centre for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University of Bremen, Germany.
BMC Biochem. 2011 Jul 30;12:39. doi: 10.1186/1471-2091-12-39.
Animal African trypanosomiasis, sleeping sickness in humans and Nagana in cattle, is a resurgent disease in Africa caused by Trypanosoma parasites. Trans-sialidases expressed by trypanosomes play an important role in the infection cycle of insects and mammals. Whereas trans-sialidases of other trypanosomes like the American T. cruzi are well investigated, relatively little research has been done on these enzymes of T. congolense.
Based on a partial sequence and an open reading frame in the WTSI database, DNA sequences encoding for eleven T. congolense trans-sialidase 1 variants with 96.3% overall amino acid identity were amplified. Trans-sialidase 1 variants were expressed as recombinant proteins, isolated and assayed for trans-sialylation activity. The purified proteins produced α2,3-sialyllactose from lactose by desialylating fetuin, clearly demonstrating their trans-sialidase activity. Using an HPLC-based assay, substrate specificities and kinetic parameters of two variants were characterized in detail indicating differences in substrate specificities for lactose, fetuin and synthetic substrates. Both enzymes were able to sialylate asialofetuin to an extent, which was sufficient to reconstitute binding sites for Siglec-4. A mass spectrometric analysis of the sialylation pattern of glycopeptides from fetuin revealed clear but generally similar changes in the sialylation pattern of the N-glycans on fetuin catalyzed by the trans-sialidases investigated.
The identification and characterization of a trans-sialidase gene family of the African parasite T. congolense has opened new perspectives for investigating the biological role of these enzymes in Nagana and sleeping sickness. Based on this study it will be interesting to address the expression pattern of these genes and their activities in the different stages of the parasite in its infection cycle. Furthermore, these trans-sialidases have the biotechnological potential to be used for enzymatic modification of sialylated glycoconjugates.
动物非洲锥虫病，人类昏睡病和牛纳格那病，是由锥虫寄生虫引起的非洲复发性疾病。锥虫表达的转涎酸酶在昆虫和哺乳动物的感染周期中发挥重要作用。虽然其他锥虫如美洲 T. cruzi 的转涎酸酶得到了很好的研究，但对 T. congolense 的这些酶的研究相对较少。
根据 WTSI 数据库中的部分序列和开放阅读框，扩增了编码 11 种 T. congolense 转涎酸酶 1 变体的 DNA 序列，这些变体的总体氨基酸同一性为 96.3%。转涎酸酶 1 变体作为重组蛋白表达，分离并测定转涎酰基活性。从乳糖中通过去唾液酸化胎球蛋白产生α2,3-唾液乳糖的纯化蛋白，清楚地证明了它们的转涎酰基酶活性。使用基于 HPLC 的测定法，详细表征了两种变体的底物特异性和动力学参数，表明对乳糖、胎球蛋白和合成底物的底物特异性存在差异。两种酶都能够将无唾液酸胎球蛋白唾液酸化到足以重新形成 Siglec-4 结合位点的程度。胎球蛋白糖肽的唾液酸化模式的质谱分析显示，在所研究的转涎酸酶催化下，胎球蛋白的 N-糖链的唾液酸化模式发生了明显但通常相似的变化。
鉴定和表征非洲寄生虫 T. congolense 的转涎酸酶基因家族为研究这些酶在纳格那病和昏睡病中的生物学作用开辟了新的视角。基于这项研究，研究这些基因在寄生虫感染周期的不同阶段的表达模式及其活性将非常有趣。此外，这些转涎酸酶具有用于酶促修饰唾液酸化糖缀合物的生物技术潜力。
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