Essential Role of Yl MPO1 , a Novel Yarrowia lipolytica Homologue of Saccharomyces cerevisiae MNN4 , in Mannosylphosphorylation of N- and O-Linked Glycans

Abstract

Yeast species are important hosts for the production of therapeutic secretory proteins owing to their ability to secrete and glycosylate proteins, their rapid growth to high cell density, and the ease with which they can be genetically manipulated. However, the glycosylation pathway of yeast cells is known to be different from that of mammalian cells, and yeast glycans could induce immunological responses in humans (5). In the traditional yeast Saccharomyces cerevisiae, the typical characteristics of the Asn (N)-linked glycan include hypermannosylation (Man 50-150 GlcNAc 2 ), mannosylphosphorylation in the core and outer regions, and termination with the ␣1,3-linked mannose residue. These yeast-specific sugar moieties could prove problematic in the production of therapeutic glycoproteins; hypermannosylation can impair protein activity, and mannosylphosphate residues and terminal ␣1,3-linked mannoses may elicit an antigenic response (14).Several nonconventional yeast species, including the methylotrophic species Pichia pastoris and Hansenula polymorpha, have emerged as alternative systems for the production of recombinant therapeutic proteins. Advantages over S. cerevisiae include reduced hypermannosylation GlcNAc 2 ), no terminal ␣1,3-linked mannose of N-linked oligosaccharides, and efficient heterologous protein secretion (18,20,21,22). As another alternative expression system for the production of human-derived therapeutic glycoproteins, the dimorphic yeast Yarrowia lipolytica has recently drawn attention since this yeast naturally secretes several enzymes, including proteases, lipases, esterases, and RNases, at elevated levels and its posttranslational modification ability is similar to that of mammalian systems (3,26,29).Recently, we reported that the N-linked glycans of Y. lipolytica are composed of neutral and acidic sugars lacking a terminal ␣1,3-linked mannose (38). The neutral sugars of Nlinked glycans are high-mannose oligosaccharides, principally Man 7-12 GlcNAc 2 , and the acidic sugars of N-linked glycans are composed of monomannosylphosphorylated Man 7-9 GlcNAc 2 sugars. In the case of S. cerevisiae, at least four mannosylphosphorylation loci have been detected in the core and outer chain regions of N-linked glycans, and the mannosylphosphorylation of N-linked glycans in the Golgi apparatus requires both S. cerevisiae Mnn4p (ScMnn4p) and ScMnn6p (19). Deletion of ScMNN4 or ScMNN6 induced a significant reduction in alcian blue intensity, which reflects the presence of negatively

Phosphomannosylation and the Functional Analysis of the Extended Candida albicans MNN4-Like Gene Family

“…Although the functional role of this LicD domain in the phosphomannosylation mechanism remains unknown, we conclude this is a signature domain for the identification of proteins involved in the cell wall phosphomannosylation pathway in fungi. In Ogataea minuta (Akeboshi et al, 2009 ), Yarrowia lipolytica (Park et al, 2011 ), Pichia pastoris (Miura et al, 2004 ), Candida parapsilosis , and Candida tropicalis (our unpublished observations), the Mnn4-like proteins involved in the modification of glycoproteins with phosphomannan all contain the LiCD domain, providing additional support for this conclusion.…”

Unraveling Unique Structure and Biosynthesis Pathway of N-Linked Glycans in Human Fungal Pathogen Cryptococcus neoformans by Glycomics Analysis

“…N-Glycans of several yeast species also contain acidic sugars, which are composed of mannosyl phosphorylated sugars in most yeast species such as S. cerevisiae, C. albicans, P. pastoris, and Y. lipolytica. However, in S. pombe, the addition of pyruvate generates acidic glycans (11,43).…”

Unraveling the Novel Structure and Biosynthetic Pathway of O-Linked Glycans in the Golgi Apparatus of the Human Pathogenic Yeast Cryptococcus neoformans

“…Preparation of O-Glycans for HPLC Analysis-The O-linked oligosaccharides were released from the cryptococcal cwMPs by modified hydrazinolysis as described previously (34). Briefly, dried cwMPs (100 g) were resuspended in 100 l of hydrazine monohydrate (Tokyo Chemical Industry Co.), and the mixture was incubated at 60°C for 4 h. After cooling and desiccation of the reactant without heating, the pellets were dissolved in 100 l of saturated NaHCO 3 , mixed with 10 l of (CH 3 CO) 2 O, and incubated on ice for 30 min.…”