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Structural analysis of N-/O-glycans assembled on proteins in yeasts
Thak, Eun Jung,Kim, Jungho,Lee, Dong-Jik,Kim, Jeong Yoon,Kang, Hyun Ah MICROBIOLOGICAL SOCIETY OF KOREA 2018 JOURNAL OF MICROBIOLOGY -SEOUL- Vol.56 No.1
Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species- and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N- and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.
Eun Jung Thak,Dong-Jik Lee,Hyunah Kim,Ye Ji Son,Jung Ho Kim,Su-Bin Lee,Yong-Sun Bahn,Hyun Ah Kang 한국당과학회 2021 한국당과학회 학술대회 Vol.2021 No.01
The human fungal pathogen Cryptococcus neoformans assembles N-/O-linked glycans on its proteins in two types with and without xylose (1, 2). In this study, the CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to the minor O-glycans with xylose, was identified and functionally analyzed. The CAP6 deletion in the ktr3Δ strain, in which the α1,2-mannose addition to the major O-glycans is defected, resulted in almost complete blockage of O-glycan extension. Notably, the putative two cell surface sensor proteins, Wml (Wsc/Mid2-Like)1p and Wml2p, were shown to be subjected to minor and major O-mannosylation by Cap6 and Ktr3, respectively. The proteins levels of Wml1 and Wml2 were remarkably decreased in the ktr3Δ cap6Δ mutant, indicating that proper O-mannosylation is essential for their stability. The phosphorylation of Mpk1, induced by tunicamycin, was greatly decreased in the ktr3Δcap6Δ and the wml1Δwml2Δ, supporting an essential role of O-glycans on cell surface sensors in cell wall integrity signaling. As reflecting its defective growth under several stress conditions, the ktr3Δcap6Δ strain showed fully attenuated virulence in a mouse model of cryptococcosis. The delineation of the roles of protein glycosylation in fungal pathogenesis will not only provide a deep insight into the glycan-based fungal infection mechanism but also aid in the development of novel antifungal agents.
Eun Jung Thak,Dong-jik Lee,Hyun Ah Kang 한국당과학회 2017 한국당과학회 학술대회 Vol.2017 No.01
We have identified an ALG3 homolog (CnALG3), coding for a dolichyl-phosphate-mannose dependent α-1,3-mannosyltransferase, in the human pathogenic yeast Cryptococcus neoformans. The CnALG3 gene encodes a protein of 447 amino acids, showing approximately 33% sequence identity to the homologs of other yeast. For functional analysis of CnALG3, we constructed a null mutant strain and analyzed the N-glycan structures by HPLC and exoglycosidase treatment. CnALG3 was shown to be responsible for the conversion of Man5GlcNAc2-Dol-PP to Man6GlcNAc2-Dol-PP, the earliest step to attach a mannose to the lipid-linked oligosaccharide in the ER. The Cnalg3Δ mutant strain displayed a moderate defect in capsule biosynthesis, and exhibited more increased sensitivity to oxidative and cell wall stresses compared to the wild-type. The C. neoformans phospholipase PLB1, which is a glycoprotein aiding fungal traversal across the blood-brain barrier, was shown to have truncated N-glycans in the alg3Δ, which showed more apparent decrease than in the och1Δ and mnn2Δ. Moreover, the Cnalg3Δ showed fully attenuated virulence in a mouse model of cryptococcosis, suggesting that the alteration of N-glycan assembly affect considerably pathogenicity of C. neoformans. However, the non-opsonic phagocytosis of Cnalg3Δ was shown to be comparable to that of Cncap59Δ during cryptococcal pathogenesis, indicating that the truncated N-linked glycans may not affect the early steps in interaction with macrophages.