Characterization of α‐1,6‐Mannosyltransferase Responsible for the Synthesis of Branched Side Chains in Candida albicans Mannan

Abstract

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,

Identification of the antigenic determinants of factors 8, 9, and 34 of genus Candida

“…A 3,6-branched pentaose (M5-3,6) was synthesized from M4-3 by an enzymatic procedure using GDP-mannose and glabrata mannan (Fig. 3A), the side chain corresponding to an c~-l,6-mannosyltransferase of C. albicans serotype B [21]. The chethe antigenic determinant of factor 34 appears to be prefermical structures of the oligosaccharides were confirmed by 1H-NMR entially synthesized compared to that of antigenic factor 6.…”

“…downstream enzyme, e~-l,6-mannosyltransferase, which parsupport the findings of Ataoglu et al [25] who defined the ticipates in the synthesis of antigenic factor 4 [13] and requires determinant of FAb 9 as a linear c~-l,6-1inked mannose polya side chain possessing a non-reducing terminal c~-l,3-1inked mer. In addition to their findings, the results of ELISA-inhimannose residue in C. albicans mannan [21]. As a result of bition assay of Fr.…”

Characterization of β-1,2-Mannosyltransferase in Candida guilliermondii and Its Utilization in the Synthesis of Novel Oligosaccharides

“…However, the linkage of the penultimate mannose unit does not affect the substrate activity of the oligosaccharides. A similar substrate specificity has been found on the ␣-1,6-mannosyltransferase responsible for the synthesis of ␣-1,6-branched side chains (23) Man␣133Man␣133Man␣132Man␣132Man-pyridylamine 48 1 6 Man␣1 a The ␤-1,2-mannosyltransferase and ␣-1,6-mannosyltransferase activities were calculated from the amount of the jack bean ␣-mannosidaseresistant and susceptible enzyme reaction products, respectively.…”

“…Furthermore, the oligosaccharides synthesized by using enzymes of C. albicans and C. guilliermondii cells were labeled by the addition of small letters, a and g, respectively. Substrate oligosaccharides, Man␣132Man␣132Man␣132Man (AMan 4 ), and Man␣13 3Man␣132Man␣132Man␣132Man (AMan 5 ) were prepared from C. albicans mannan (24), Man␣133Man␣132Man␣132Man (SMan 4 ) and Man␣133Man␣133Man␣132Man␣132Man (SMan 5 ) were from S. cerevisiae serotype Ia mannan (27) 5 , and SMan 5 , respectively, using the ␣-1,6-mannosyltransferase of C. albicans NIH B-792 cells followed by the method described in a previous paper (23).…”

“…1 based on the results obtained by the structural analysis of C. guilliermondii mannan (18) and the substrate specificity of an ␣-1,6-mannosyltransferase of C. albicans (23) responsible for the formation of the branched oligosaccharides corresponding to factor 4 (24,25). Namely, the introduction of a ␤-1,2-linked mannose unit from GDP-mannose to the nonreducing terminal ␣-1,3-linked mannose unit of an ␣-linked mannopentaosyl side chain, Man␣133(Man␣136)Man␣132Man␣132Man, synthesized from Man␣133Man␣132Man␣132Man corresponding to factor 34 (26), takes place by participation of the ␤-1,2-mannosyltransferase IV, and then, the elongation of a ␤-1,2-linked one to a resulting product, Man␤132Man␣133-(Man␣136)Man␣132Man␣132Man, takes place by participation of the ␤-1,2-mannosyltransferase V in succession.…”