A particulate insoluble fraction from Candida albicans J-1012 (serotype A) strain cells was obtained as the residue after extracting a 105,000 × g pellet of cell homogenate with 1% ~1 riton X-100. Incubation of this fraction with a mannopentaose, Man~l-->2Manotl-->(2Manal-Qz2Man (al~Mans), in the presence of GDP-mannose followed by high performance liquid chromatography showed the formation of a mannohexaose. Analysis of the product by ~H NMR indicates that a~Man5 was changed to Man~l--~ 2Manl~ll--~2Manal-~(2Manal--~)z 2Man (a~lMan6). This gl-l,2-mannosyltransferase (ManTase) II activity was completely inhibited by Zn 2÷ and was not restored by the addition of EDTA. The corresponding enzyme fraction from C. albieans NIH B-792 (serotype B) strain calls, the mannan of which does not possess both the otl3Mans and airMan6 side chains, also exhibited the same/3-1,2-ManTase II activity. /(ey words: Candida albicans; fl-1,2-Linked mannose; Mannosyltransferase ~. IntroductionCandidiasis is an infectious disease frequently seen in early hildhood and in adults with predisposing conditions such as ,tiabetes, cancer, AIDS, and immuno-suppressive therapy [1,2]. Candida albicans strains were divided into two serotypes, A and ~, by Hasenclever and Mitchell [3] from the antigenicity of cell vall mannans. A structural study of the mannans of the 2 albicans serotype A and B strains has been achieved during he last several years [4,5], and we have demonstrated the pres-'nce of phosphodiesterified fl-l,2-1inked oligomannosyl moieies as a group of common epitopes throughout the two seroype strains [6,7]. The major difference between the structural !eatures of mannans from C. albicans serotype A and B strain .',ells is the presence of a fl-1,2-1inked mannose unit attached to m ~-l,2-1inked one in the side chains of the former mannan ,train [8,9], but lacking in the latter ones. We also reported in 'ecent papers that the serotype A-specific fl-1,2-1inked mannose mit in the mannans of C. albicans disappeared by cultivation )fthe cells under acidic pH [10] dida and several other yeasts. Thus, the detection of the circulating mannan antigen containing fl-l,2-1inkages in patients' sera by immunological procedures is important for the diagnosis of invasive candidiasis. Furthermore, several workers reported that the fl-1,2-linkage-containing side chains participate in adherence of C albicans cells to mammalian cells in the initial step of Candida infection [14,15]. Therefore, we tried to detect and characterize a fl-l,2-mannosyltransferase (ManTase) which is responsible for the biosynthesis of the serotype A specific epitope of C. albicans mannan. The biosynthetic pathway of the serotype A-specific side chain can be depicted as shown in Fig. 1 based on the structural analysis findings of C. albicans mannans [4]. Namely, the first transfer of the fl-l,2-linked mannose unit from GDP-mannose to the non-reducing terminal site of the ~-l,2-1inked mannotetraosyl side chain takes place by participation of the fl-l,2-ManTase I. However,...

A particulate insoluble fraction from Candida albicans NIH B-792 (serotype B) strain cells was obtained as the residue after extracting a 105000Xg pellet of cell homogenate with 1% Triton X-100.Incubation of this fraction with a mannopentaose, Manal-3Manal-t2Manal-+Manal+2Man, in the presence of GDP-mannose and MnZ+ at pH 6.0 gave a branched mannohexaose, Manal+3Manal-2Manal-t2Manal-+2Man, f6 Manal the structure of which was identified by means of sequential NMR assignment, However, the enzyme fraction obtained from Candida parapsilosis gave Mana1+2Manal-3Manal+2Manal-2Manal-2-Man under the same conditions. These results demonstrate the finding that the structural difference in the mannans of these two species is due to the presence of a-1,6-linked branching mannose units in the C. albicans mannan [Shibata, N., Ikuta, K., Imai, T., Satoh, Y., Satoh, R., Suzuki, A,, Kojima, C., Kobayashi, H., Hisamichi, K. & Suzuki, S. (1995) J. Biol. Chem. 270, 1113-11221. The substrate-specificity study of the enzyme indicated that the structural requirement of the a-l,6-mannosyltransferase is Manal-3-Manal-. The a-l,6-mannosyltransferase also transferred the a-l,6-linked branching mannose unit to the mannan of Saccharomyces cerevisiue. The transformation of the mannan was detected by the appearance of antigenic factor 4 using an enzyme-linked immunosorbent assay and two-dimensional homonuclear Hartmann-Hahn spectroscopy.Keywords: Candida ulbicans; mannosyltransferase ; mannan ; NMR; biosynthesis.The pathogenic yeast Candidu species is the leading cause of disseminated fungal infection in the immunocompromised host, the diabetic, the neonate, and the post-operative patient. An outburst of severe infections caused by Candida species, especially in patients with impaired immune defense systems, led us to investigate these microorganisms in detail including the study of their cell wall components. Candida albicans is the most virulent strain and has been divided into two serotypes, A and B, based on the antigenicity of the cells [I]. Tsuchiya et al.[2] determined the antigenic patterns of medically important yeasts by cross-adsorption experiments and proposed antigenic formulas for their serologic classification. Namely, C. albicans serotype A has antigenic factors 1, 4, 5 , and 6, while serotype B shows antigenic factors 1, 4, 5, and 13b. In a previous study [3], we detected and characterized the /I-1 ,Zmannosyltransferase I1 which participates in the biosynthesis of the serotype A specific epitope [4, 51, factor 6 [6]. This enzyme transfers a mannose unit from GDP-mannose to Manpl-2Manal-2 Manal-+2Manal-+2Man and synthesizes Manpl-+2Man~I--2Manal-+2Manal+2Manal+2Man. found the presence of a-1,6-branched mannohexaose and mannoheptaose side chains,