title: Reporters for the analysis of N-glycosylation in Candida albicans.
abstract: A large proportion of Candida albicans cell surface proteins are decorated post-translationally by glycosylation. Indeed N-glycosylation is critical for cell wall biogenesis in this major fungal pathogen and for its interactions with host cells. A detailed understanding of N-glycosylation will yield deeper insights into host-pathogen interactions. However, the analysis of N-glycosylation is extremely challenging because of the complexity and heterogeneity of these structures. Therefore, in an attempt to reduce this complexity and facilitate the analysis of N-glycosylation, we have developed new synthetic C. albicans reporters that carry a single N-linked glycosylation site derived from Saccharomyces cerevisiae Suc2. These glycosylation reporters, which carry C.albicans Hex1 or Sap2 signal sequences plus carboxy-terminal FLAG₃ and His₆ tags, were expressed in C.albicans from the ACT1 promoter. The reporter proteins were successfully secreted and hyperglycosylated by C.albicans cells, and their outer chain glycosylation was dependent on Och1 and Pmr1, which are required for N-mannan synthesis, but not on Mnt1 and Mnt2 which are only required for O-mannosylation. These reporters are useful tools for the experimental dissection of N-glycosylation and other related processes in C.albicans, such as secretion.
authors.affiliation: Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK.
authors.forename: Shahida
authors.initials: S
authors.lastname: Shahana
authors.forename: Hector M
authors.initials: HM
authors.lastname: Mora-Montes
authors.forename: Luis
authors.initials: L
authors.lastname: Castillo
authors.forename: Iryna
authors.initials: I
authors.lastname: Bohovych
authors.forename: Chirag C
authors.initials: CC
authors.lastname: Sheth
authors.forename: Frank C
authors.initials: FC
authors.lastname: Odds
authors.forename: Neil A R
authors.initials: NA
authors.lastname: Gow
authors.forename: Alistair J P
authors.initials: AJ
authors.lastname: Brown
mesh_terms: D002176:Candida albicans / Q000235:genetics* / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry; D015870:Gene Expression; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D006031:Glycosylation; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D043324:beta-Fructofuranosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C468437:SUC2 protein, S cerevisiae; D043324:beta-Fructofuranosidase