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 One. 2011;6(12):e29216. doi: 10.1371/journal.pone.0029216. Epub 2011 Dec 21.
The fungus Paracoccidioides brasiliensis is a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The cell wall of P. brasiliensis is a network of glycoproteins and polysaccharides, such as chitin, that perform several functions. N-linked glycans are involved in glycoprotein folding, intracellular transport, secretion, and protection from proteolytic degradation. Here, we report the effects of tunicamycin (TM)-mediated inhibition of N-linked glycosylation on P. brasiliensis yeast cells. The underglycosylated yeasts were smaller than their fully glycosylated counterparts and exhibited a drastic reduction of cell budding, reflecting impairment of growth and morphogenesis by TM treatment. The intracellular distribution in TM-treated yeasts of the P. brasiliensis glycoprotein paracoccin was investigated using highly specific antibodies. Paracoccin was observed to accumulate at intracellular locations, far from the yeast wall. Paracoccin derived from TM-treated yeasts retained the ability to bind to laminin despite their underglycosylation. As paracoccin has N-acetyl-β-d-glucosaminidase (NAGase) activity and induces the production of TNF-α and nitric oxide (NO) by macrophages, we compared these properties between glycosylated and underglycosylated yeast proteins. Paracoccin demonstrated lower NAGase activity when underglycosylated, although no difference was detected between the pH and temperature optimums of the two forms. Murine macrophages stimulated with underglycosylated yeast proteins produced significantly lower levels of TNF-α and NO. Taken together, the impaired growth and morphogenesis of tunicamycin-treated yeasts and the decreased biological activities of underglycosylated fungal components suggest that N-glycans play important roles in P. brasiliensis yeast biology.
巴西副球孢子菌是一种人类病原体，可引起副球孢子菌病，这是拉丁美洲最常见的系统性真菌病。巴西副球孢子菌的细胞壁是糖蛋白和多糖（如几丁质）的网络，具有多种功能。N-连接糖基化参与糖蛋白折叠、细胞内运输、分泌和保护免受蛋白水解降解。在这里，我们报告了衣霉素（TM）介导的 N-连接糖基化抑制对巴西副球孢子菌酵母细胞的影响。糖基化不足的酵母比完全糖基化的酵母小，并且细胞出芽明显减少，这反映了 TM 处理对生长和形态发生的损害。使用高度特异性抗体研究了 TM 处理酵母中巴西副球孢子菌糖蛋白 paracoccin 的细胞内分布。观察到 paracoccin 在细胞内的位置积累，远离酵母壁。尽管 TM 处理的酵母糖基化不足，但源自 TM 处理的酵母的 paracoccin 仍然保留与层粘连蛋白结合的能力。由于 paracoccin 具有 N-乙酰-β-d-氨基葡萄糖苷酶（NAGase）活性，并诱导巨噬细胞产生 TNF-α和一氧化氮（NO），我们比较了糖基化和糖基化不足的酵母蛋白之间的这些特性。当糖基化不足时，paracoccin 的 NAGase 活性较低，尽管两种形式的 pH 和最适温度没有差异。用糖基化不足的酵母蛋白刺激的鼠巨噬细胞产生的 TNF-α和 NO 水平显著降低。综上所述，衣霉素处理的酵母生长和形态发生受损以及糖基化不足的真菌成分的生物活性降低表明 N-聚糖在巴西副球孢子菌酵母生物学中发挥重要作用。
