Conversion of α1,2-mannosidase E-I from Candida albicans to α1,2-mannosidase E-II by limited proteolysis

• Published in: Antonie van Leeuwenhoek 2008-02, Vol.93 (1-2), p.61-69
• Main Authors: Mora-Montes, Héctor Manuel, López-Romero, Everardo, Zinker, Samuel, Ponce-Noyola, Patricia, Flores-Carreón, Arturo
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Enzyme engineering; Fundamental and applied biological sciences. Psychology; Improved methods for extraction and purification of enzymes; Life Sciences; Medical Microbiology; Methods. Procedures. Technologies; Microbiology; Original Paper; Plant Sciences; Soil Science & Conservation
• ISSN: 0003-6072; 1572-9699
• DOI: 10.1007/s10482-007-9179-x

Previous studies demonstrated the presence in Candida albicans ATCC 26555 of two soluble α1,2-mannosidases: E-I and E-II. In contrast, in the C. albicans CAI-4 mutant only E-I was detected and it could be processed by a membrane-bound proteolytic activity from the ATCC 26555 strain, generating an active 43 kDa polypeptide. Here, α1,2-mannosidase E-I from strain ATCC 26555 was purified by conventional methods of protein isolation and affinity chromatography in Concanavalin A-Sepharose 4B. Analytical electrophoresis of the purified enzyme revealed two polypeptides of 52 and 23 kDa, the former being responsible for enzyme activity as revealed by zymogram analysis. Time course proteolysis with an aspartyl protease from Aspergillus saitoi, converted α1,2-mannosidase E-I into an active polypeptide of 43 kDa which trimmed Man₉GlcNAc₂, generating Man₈GlcNAc₂ isomer B and mannose. Trimming was inhibited preferentially by 1-deoxymannojirimycin. Both, the molecular mass and the enzyme properties of the proteolytic product were identical to those described for α1,2-mannosidase E-II therefore supporting the notion that E-I is the precursor of E-II.