N-linked glycosylation sites affect secretion of cryptococcal phospholipase B1, irrespective of glycosylphosphatidylinositol anchoring

IP3-4kinase Arg1 regulates cell wall homeostasis and surface architecture to promoteCryptococcus neoformansinfection in a mouse model

“…We showed previously that approximately 30% of the molecular weight of Plb1 is attributable to N-linked mannosylation 35 , and that preventing the acquisition of N-linked mannosylation by site-directed mutagenesis prevents Plb1 transport to the cell surface in a heterologous expression system 30 . To determine whether the increase in Plb1 size in the arg1D mutant ( Figure 8A) is due to abnormal processing of Nlinked mannose sugars in the Golgi, we treated the secretions collected from WT and arg1D with N-glycosidase F (PNGase F) and assessed protein size by anti-Plb1 Western blotting ( Figure 9A).…”

“…One of these mannoproteins is the virulence factor Plb1 29,30 . Not only is Plb1 a fungal 'invasin' with a role in cell wall integrity 14 , it is also a marker of the classical secretion pathway in C. neoformans 31,32 .…”

Enhanced expression of heterologous proteins in yeast cells via the modification ofN-glycosylation sites

“…Furthermore, the effect of N-glycosylation on protein folding and secretion was site-specific: the attachment of N-glycan to a given protein at different sites exerted different effects on protein expression. 7,8,15,16,19 Determining the correlation between the position of an individual N-glycosylation site and its effect on protein folding and secretion is of great value, though it is also a challenging task. Even so, modifying Nglycosylation sites to stimulate protein expression is a feasible strategy for increasing protein expression that can be conveniently implemented by site-directed mutagenesis with ease and efficiency in a laboratory setting.…”

“…In 2006, Turner et al 16 reported the deletion of the N-glycosylation sites in wild-type PLB1 and its glycosylphosphatidylinositol (GPI) anchorless version (PLB1 GPI¡ ) expressed in S. cerevisiae by the conversion of Asn to Ala by site-directed mutagenesis. They found that 2 mutations, N56A and N550A, inhibited protein secretion completely, while N430A reduced the level of secretion by 60%.…”

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

“…However, only a small number of C. neoformans glycoproteins have been characterized, and detailed investigation on oligosaccharide structures has not been carried out except for carbohydrate composition (27)(28)(29). Previous work involving bioinformatics and radioactive N-glycan analyses indicated that the structure of the dolichol-linked N-glycosylation precursor in the ER of C. neoformans is Man 9 GlcNAc 2 -PP-Dol with no addition of glucose residues due to a lack of Alg glucosyltransferases (Alg6p, Alg8p, and Alg10p), implying different processing of cryptococcal N-glycans in the ER (30).…”