Functional analysis of the Candida albicans ALS1 gene product.
ALS1 encodes a cell surface protein that mediates adherence of Candida albicans to endothelial cells. The predicted Als1p has an N-terminal region, which contains a signal peptide; a middle region, which contains 20 36-amino acid tandem repeats; and a C-terminal region, which contains a glycosylphosphotidylinositol-anchorage sequence. We used site-directed mutagenesis to delineate the regions in Als1p required for endothelial cell adherence and cell surface expression of the protein. Mutant alleles of ALS1 containing either deletions or insertions were expressed in the normally non-adherent Saccharomyces cerevisiae. These transformants were analysed for endothelial cell adherence and cell surface expression of Als1p. We found that mutations centred around amino acid 285 in the N-terminus completely abolished adherence, but had no effect on cell surface expression of Als1p. Deletion of 15 of the tandem repeats reduced adherence by 50%, whereas deletion of all abolished adherence completely, even though cell surface expression of the N-terminus of Als1p was maintained. Insertions into the C-terminus at amino acids 413 and 254 upstream of the stop codon resulted in a modest loss of adherence, while cell surface expression of Als1p was maintained. An insertion at amino acid 249 in the C-terminus caused complete loss of both adherence and cell surface expression, even though the glycosylphosphotidylinositol-anchorage sequence remained intact. These data suggest a model of Als1p in which the endothelial cell binding region is localized within its N-terminus, the tandem repeats are essential for the proper presentation of the binding site, and the C-terminus is required for localizing Als1p to the cell surface.
Loza
Lucio
L
Division of Infectious Diseases, St. John's Cardiovascular Research Center, Harbor-UCLA Research and Education Institute, 1124 West Carson Street, Torrance, CA 90502, USA.
Fu
Yue
Y
Ibrahim
Ashraf S
AS
Sheppard
Donald C
DC
Filler
Scott G
SG
Edwards
John E
JE
D019138:Antibodies, Blocking / Q000502:physiology; D002176:Candida albicans / Q000235:genetics* / Q000502:physiology; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D042783:Endothelial Cells; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020080:Tandem Repeat Sequences / Q000235:genetics / Q000502:physiology; D014170:Transformation, Genetic
C093538:ALS1 protein, Candida albicans; D019138:Antibodies, Blocking; D004271:DNA, Fungal; D005656:Fungal Proteins