Reporters for the analysis of N-glycosylation in Candida albicans

Authors

Shahida Shahana, University of Aberdeen
Hector M. Mora-Montes, Universidad de Guanajuato
Luis Castillo, Universidad de La Serena
Iryna Bohovych, University of Nebraska-LincolnFollow
Chirag C. Sheth, University of Nebraska-Lincoln
Frank C. Odds, University of Aberdeen
Neil A.R. Gow, University of Aberdeen
Alistair J.P. Brown, University of AberdeenFollow

Document Type

Article

Date of this Version

2013

Citation

Fungal Genetics and Biology 56 (2013) 107–115

Comments

Copyright 2013 The Authors. Published by Elsevier Inc.

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.