Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins.
Covalently linked cell wall proteins (CWPs) of the dimorphic fungus Candida albicans are implicated in virulence. We have carried out a comprehensive proteomic analysis of the covalently linked CWPs in exponential-phase yeast cells. Proteins were liberated from sodium dodecyl sulfate (SDS)-extracted cell walls and analyzed using immunological and advanced protein sequencing (liquid chromatography-tandem mass spectrometry [LC/MS/MS]) methods. HF-pyridine and NaOH were used to chemically release glycosylphosphatidylinositol-dependent proteins (GPI proteins) and mild alkali-sensitive proteins, respectively. In addition, to release both classes of CWPs simultaneously, cell walls were digested enzymatically with a recombinant beta-1,3-glucanase. Using LC/MS/MS, we identified 14 proteins, of which only 1 protein, Cht2p, has been previously identified in cell wall extracts by using protein sequencing methods. The 14 identified CWPs include 12 GPI proteins and 2 mild alkali-sensitive proteins. Nonsecretory proteins were absent in our cell wall preparations. The proteins identified included several functional categories: (i) five CWPs are predicted carbohydrate-active enzymes (Cht2p, Crh11p, Pga4p, Phr1p, and Scw1p); (ii) Als1p and Als4p are believed to be adhesion proteins. In addition, Pga24p shows similarity to the flocculins of baker's yeast. (iii) Sod4p/Pga2p is a putative superoxide dismutase and is possibly involved in counteracting host defense reactions. The precise roles of the other CWPs (Ecm33.3p, Pir1p, Pga29p, Rbt5p, and Ssr1p) are unknown. These results indicate that a substantial number of the covalently linked CWPs of C. albicans are actively involved in cell wall remodeling and expansion and in host-pathogen interactions.
Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. pgroot@science.uva.nl
Piet W J
PW
de Groot
Albert D
AD
de Boer
Jeff
J
Cunningham
Henk L
HL
Dekker
Luitzen
L
de Jong
Klaas J
KJ
Hellingwerf
Chris
C
de Koster
Frans M
FM
Klis
D000595:Amino Acid Sequence; D002176:Candida albicans / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D002473:Cell Wall / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism; D016415:Sequence Alignment
C109455:ALA1 protein, Candida albicans; D015815:Cell Adhesion Molecules; D005656:Fungal Proteins; D017261:Glycosylphosphatidylinositols; D020543:Proteome