N-linked glycosylation sites affect secretion of cryptococcal phospholipase B1, irrespective of glycosylphosphatidylinositol anchoring.
Secreted phospholipase B enzymes (PLB1) with high levels of N-linked glycosylation are proven fungal virulence determinants. We demonstrated that removal of N-linked glycans from secreted cryptococcal PLB1 leads to loss of enzyme activity. To determine if individual N-glycan attachment sites affect secretion of active enzyme, we altered three along the entire length of the protein, by site-directed mutagenesis, namely Asn56, Asn430 and Asn550 to Ala, in wild-type PLB1 (full length) and a glycosylphosphatidylinositol (GPI) anchorless version (PLB1(GPI-)) that is hypersecreted due to lack of membrane association. Alteration of Asn56 and Asn550 in both PLB1 and PLB1(GPI-) abolished enzyme secretion while alteration of Asn430 reduced secretion by 60%, following expression in Saccharomyces cerevisiae. Reduced secretion coincided with reduced enzyme in membranes and cell walls confirming a reduction in the rate of PLB1 transport to the cell surface. Deglycosylation of cryptococcal PLB1 increased its susceptibility to proteolysis suggesting that the absence of full glycosylation status leads to degradation of unstable PLB1, resulting in reduced traffic through the secretory pathway. We conclude that individual N-linked glycans are required for optimal transport of PLB1 to the cell surface and optimal secretion of both PLB1 and PLB1(GPI-).
Turner
Kylie M
KM
Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute and University of Sydney, Level 3, ICPMR Building, Westmead Hospital, Darcy Rd., Westmead, NSW 2145, Australia.
Wright
Lesley C
LC
Sorrell
Tania C
TC
Djordjevic
Julianne T
JT
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D002473:Cell Wall / Q000201:enzymology; D003455:Cryptococcus neoformans / Q000201:enzymology*; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D008245:Lysophospholipase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; D011994:Recombinant Proteins; D008245:Lysophospholipase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase