Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, 156-756, South Korea.
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, South Korea.
Sci Rep. 2023 Jan 20;13(1):1175. doi: 10.1038/s41598-023-27422-9.
Cryptococcus neoformans is an opportunistic human fungal pathogen causing lethal meningoencephalitis. It has several cell wall mannoproteins (MPs) identified as immunoreactive antigens. To investigate the structure and function of N-glycans assembled on cryptococcal cell wall MPs in host cell interactions, we purified MP98 (Cda2) and MP84 (Cda3) expressed in wild-type (WT) and N-glycosylation-defective alg3 mutant (alg3Δ) strains. HPLC and MALDI-TOF analysis of the MP proteins from the WT revealed protein-specific glycan structures with different extents of hypermannosylation and xylose/xylose phosphate addition. In alg3Δ, MP98 and MP84 had truncated core N-glycans, containing mostly five and seven mannoses (M5 and M7 forms), respectively. In vitro adhesion and uptake assays indicated that the altered core N-glycans did not affect adhesion affinities to host cells although the capacity to induce the immune response of bone-marrow derived dendritic cells (BMDCs) decreased. Intriguingly, the removal of all N-glycosylation sites on MP84 increased adhesion to host cells and enhanced the induction of cytokine secretion from BMDCs compared with that on MP84 carrying WT N-glycans. Therefore, the structure-dependent effects of N-glycans suggested their complex roles in modulating the interaction of MPs with host cells to avoid nonspecific adherence to host cells and host immune response hyperactivation.
新型隐球菌是一种机会性人类真菌病原体，可导致致命性脑膜脑炎。它有几种细胞壁甘露糖蛋白（MPs）被鉴定为免疫反应性抗原。为了研究在宿主细胞相互作用中组装在隐球菌细胞壁 MPs 上的 N-聚糖的结构和功能，我们纯化了在野生型（WT）和 N-糖基化缺陷型 alg3 突变体（alg3Δ）菌株中表达的 MP98（Cda2）和 MP84（Cda3）。WT 中 MP 蛋白的 HPLC 和 MALDI-TOF 分析显示，具有不同程度的高甘露糖化和木糖/木糖磷酸盐添加的蛋白质特异性聚糖结构。在 alg3Δ中，MP98 和 MP84 的核心 N-聚糖被截断，分别主要含有五个和七个甘露糖（M5 和 M7 形式）。体外粘附和摄取实验表明，改变的核心 N-聚糖不影响与宿主细胞的粘附亲和力，尽管诱导骨髓来源树突状细胞（BMDC）免疫反应的能力降低。有趣的是，与携带 WT N-聚糖的 MP84 相比，去除 MP84 上所有的 N-糖基化位点会增加对宿主细胞的粘附，并增强 BMDC 细胞因子分泌的诱导。因此，N-聚糖的结构依赖性效应表明它们在调节 MPs 与宿主细胞相互作用方面的复杂作用，以避免与宿主细胞的非特异性粘附和宿主免疫反应的过度激活。
