An alpha-1,3-mannosyltransferase of Cryptococcus neoformans.
Cryptococcus neoformans is a pathogenic fungus, distinguished by an elaborate polysaccharide capsule that is essential for its virulence. As part of an effort to understand the biosynthesis of this important structure, we initiated purification of an alpha-1,3-mannosyltransferase with appropriate specificity for a role in building the main capsule polysaccharide, glucuronoxylomannan. A pool of proteins that was 5,000-fold enriched in this activity included several polypeptides, which acted potentially as the catalytic protein. These were analyzed using sequence information and double-stranded RNA interference. Interference that targeted a sequence corresponding to part of a 46 kDa protein in the enriched fraction abolished the activity of interest and reduced the capsule on the affected cells. This gene was cloned and expressed in active form in Saccharomyces cerevisiae to confirm function, and was termed CMT1, for cryptococcal mannosyltransferase 1. CMT1 has no confirmed homologs in GenBank other than CAP59, a cryptococcal gene encoding a protein of unknown function that is required for capsule synthesis and virulence. The Cmt1p protein also co-purifies with a homolog of CAP64, a gene whose product has similarly been implicated in capsule synthesis and virulence. A strain disrupted in CMT1 was generated in C. neoformans; this had no effect on virulence in an animal model of cryptococcosis.
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D003001:Cloning, Molecular; D003208:Concanavalin A / Q000737:chemistry; D003453:Cryptococcosis / Q000378:metabolism; D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000472:pathogenicity*; D017886:Durapatite / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007150:Immunohistochemistry; D008364:Mannosyltransferases / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D034622:RNA Interference; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D010455:Peptides; D011134:Polysaccharides; D003208:Concanavalin A; C027478:glucuronoxylomannan; D017886:Durapatite; D008364:Mannosyltransferases; C121563:alpha 1,3-mannosyltransferase