Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
PLoS Negl Trop Dis. 2014 Apr 17;8(4):e2788. doi: 10.1371/journal.pntd.0002788. eCollection 2014 Apr.
Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains.
METHODOLOGY/PRINCIPAL FINDINGS: The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347), was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis.
CONCLUSIONS/SIGNIFICANCE: Our results showed that the recombinant protein reproduced the biological properties described for the native protein-including binding to laminin in a manner that is dependent on carbohydrate recognition-showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-α and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls), mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-γ. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated inflammation. The results indicated that paracoccin is the protein encoded by PADG-3347, and we propose that this gene and homologous proteins in other P. brasiliensis strains be called paracoccin. We also concluded that recombinant paracoccin confers resistance to murine P. brasiliensis infection by exerting immunomodulatory effects.
巴西副球孢子菌的一种双功能蛋白，即副球菌素，具有凝集素特性和 N-乙酰氨基葡萄糖苷酶活性。对巴西副球孢子菌副球菌素制剂的蛋白质组学分析表明，其序列与 Pb-18 株假定蛋白 PADG-3347 编码的序列相匹配，其多肽序列与家族 18 内切几丁质酶相似。这些内切几丁质酶是多功能蛋白，具有不同的凝集素和酶结构域。
方法/主要发现：预测的 ORF（PADG-3347）的多外显子组装和最大外显子被克隆并在大肠杆菌细胞中表达，并将重组蛋白的特性与天然副球菌素进行了比较。多外显子蛋白还用于巴西副球孢子菌感染的小鼠模型的保护试验。
结论/意义：我们的结果表明，重组蛋白再现了天然蛋白所描述的生物学特性，包括依赖碳水化合物识别与层粘连蛋白结合的能力、具有 N-乙酰氨基葡萄糖苷酶活性，并刺激小鼠腹腔巨噬细胞产生高水平的 TNF-α 和一氧化氮。考虑到糖结合蛋白的免疫调节潜力，我们还研究了预防性给予重组副球菌素是否会影响小鼠实验性巴西副球孢子菌病的病程。与注射载体（对照）的动物相比，用重组副球菌素治疗的小鼠肺部真菌负荷降低，肺部肉芽肿减少。这些保护作用与肺内 IL-12 和 IFN-γ 水平增加有关。我们还观察到，在挑战前三天注射副球菌素是最有效的给药方案，因为诱导的 Th1 免疫被高水平的肺内 IL-10 平衡，这可能防止由炎症加剧引起的组织损伤。结果表明，副球菌素是 PADG-3347 编码的蛋白，我们建议将该基因和其他巴西副球孢子菌株中的同源蛋白称为副球菌素。我们还得出结论，重组副球菌素通过发挥免疫调节作用赋予小鼠巴西副球孢子菌感染的抗性。
