Secretion, interaction and assembly of two O-glycosylated cell wall antigens from Candida albicans

• Published in: Microbiology (Society for General Microbiology) 2001-07, Vol.147 (7), p.1983-1991
• Main Authors: Pavia, Javier, Aguado, Carmen, Mormeneo, Salvador, Sentandreu, Rafael
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Fungal - biosynthesis; Antibodies, Fungal - immunology; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - immunology; Antigens, Fungal - metabolism; beta -1,3-Glucan; beta-Glucans; Biological and medical sciences; Candida albicans; Candida albicans - immunology; Candida albicans - metabolism; Cell Wall - immunology; Cell Wall - metabolism; Female; Fluorescent Antibody Technique, Indirect; Fundamental and applied biological sciences. Psychology; Glucans - metabolism; Glycosylation; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Membrane Glycoproteins - metabolism; Mice; Mice, Inbred BALB C; Microbiology; Mycology
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-147-7-1983

Secció Departamental de Microbiología, Facultat de Farmacia, Universitat de València, Vicent Andrés Estellés s/n, 46100 Burjassot, València, Spain 1 Author for correspondence: Rafael Sentandreu. Tel: [phone]. Fax: [phone]. e-mail: rafael.sentandreu{at}uv.es The mechanisms of incorporation of two antigens have been determined using a monoclonal antibody (3A10) raised against the material released from the mycelial cell wall by zymolyase digestion and retained on a concanavalin A column. One of the hybridomas secreted an IgG that reacted with two bands in Western blots. Indirect immunofluorescence showed that the antigens were located on the surfaces of mycelial cells, but within the cell walls of yeasts. These antigens were detected in a membrane preparation, in the SDS-soluble material and in the material released by a 1,3-ß-glucanase and chitinase from the cell walls of yeast and mycelial cells. In the latter three samples, an additional high-molecular-mass, highly polydispersed band was also detected. Beta-elimination of each fraction resulted in the disappearance of all antigen bands, suggesting that they are highly O -glycosylated. In addition, the electrophoretic mobility of the high-molecular-mass, highly polydispersed bands increased after digestion with endoglycosidase H, indicating that they are also N -glycosylated. New antigen bands were released when remnants of the cell walls extracted with 1,3-ß-glucanase or chitinase were digested with chitinase or 1,3-ß-glucanase. These results are consistent with the notion that, after secretion, parts of the O -glycosylated antigen molecules are transferred to an N -glycosylated protein(s). This molecular complex, as well as the remaining original 70 and 80 kDa antigen molecules, next bind to 1,3-ß-glucan or chitin, probably via 1,6-ß-glucan, and, in an additional step, to chitin or 1,3-ß-glucan. This process results in the final molecular product of each antigen, and their distribution in the cell walls. Keywords: monoclonal antibodies, cell wall biosynthesis, lysis of yeast wall, N -glycosylated proteins, cell wall architecture Abbreviations: Endo-H, N -acetylglucosaminidase H; GPI, glycosylphosphatidylinositol; IIF, indirect immunofluorescence