Structural study of a cell‐wall mannan of Saccharomyces kluyveri IFO 1685 strain

Abstract

Acetolysis of the cell-wall mannan of Saccharomyces kluyveri under mild conditions, gave fragments with 1-6 mannose residues. The structures of mannopentaose and mannohexaose were determined to be Manal+3Manal-+2Manal+2Man 1 6 Manal and Manfil+2Manal+3Mannl+2Manal+2Man, FManal respectively, by two-dimensional homonuclear Hartmann-Hahn spectroscopy and a sequential NMR assignment method that combines 'H-I3C correlated spectroscopy, relayed coherence transfer spectroscopy, 'H-detected heteronuclear multiple-bond connectivity and methylation analysis. The HI proton chemical shift of a neighboring a-1,2-linked mannose unit of the 3-0-substituted structure was shifted upfield by the addition of a mannose unit to the adjacent 3-0-substituted unit by an a-1,6 linkage. The characteristic H1 -H2-correlated cross-peak of the a-1,3-linked mannose unit substituted by a B1,2 linkage, pl+2Mana1+3, in the mannan of S. kluyveri, as also found by twodimensional homonuclear Hartmann-Hahn spectroscopy in the rnannan of Cundidu guilliermondii, a pathogenic yeast in man.In a previous study, it was found that mannans of the pathogenic yeast, Candida albicans, strains NIH A-207 (serotype A), NIH B-792 (serotype B) and J-1012 (serotype A) all contained /3-1,2-linked mannose units [l -81. Furthermore, we defined that a P-1,2-linked mannose unit is linked to an a-1,2-linked unit, Manfil+2Manal+2Manal+ andManpl-+2Manpl+2Manal-+2Manal-+, and that these specific structures are present in serotype-A epitopes corresponding to antigenic factor 6 in C. albicans species [9]. The phosphodiesterified p-1,2-linked oligomannosyl residues, Man,!ll+(2Manpl+),2Man (n = 0-5), are common feaCorrespondence to S. Suzuh, The Second Department of Hygienic Chemistry, Tohoku College of Pharmacy, 4-4-1 Komatsushima, Sendai Aoba-ku, Miyagi, Japan 981 Abbreviations. 2D, two-dimensional; HOHAHA, homonuclear Hartmann-Hahn spectroscopy ; COSY, correlated spectroscopy ; relayed COSY, relayed coherence transfer spectroscopy; HMBC, 'Hdetected heteronuclear multiple-bond connectivity; Fr. S, alkalimodified mannan: SM,, mannooligosaccharide obtained after mild acetolysis, where x indicates number of mannose residues; SM,-e, a-rnannosidase-resistant hexaose; SM,-eH, NaBH,-reduction product of SM,-e. tures present in the mannans of strains of both serotypes A and €3, and correspond to antigenic factor 5 [lo].However, Zhang and Ballou [111 reported the presence of a fi-1,2-linked mannose unit, in the oligomannosyl moiety which can be /? eliminated, for the 0-glycosylated form. This unit is linked to serine and/or threonine in the peptide moiety of a mannoprotein of Saccharomyces kluyveri. In this study [l I], the structure of the oligomannosyl residue containing the p-1,2 linkage was determined to be Manpl+2Mana 1 +3Manal+2Manal+2Manal +Ser/Thr I6Manal .

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

“…Therefore, we can also assign through the H-1-H-2-correlated cross-peak C2 and the NOE cross-peak C2Ј between the H-2 of Man-D and the H-1 of Man-C. Additionally, we could find the NOE cross-peak D2Ј between the H-2 of Man-D and the H-1 of Man-E, of which the signal, 4.764 ppm, corresponds to the ␤-1,2-linked mannose unit (10,11,17). Using this procedure, we could sequentially assign the H-1 signal from Man-A to Man-E as A2-A2Ј-B2-B2Ј-C3-C3Ј-(or -C2-C2Ј-)-D2-D2Ј-E2.…”

“…The H-1 and H-2 signals of SaggMan 7 were sequentially assigned from Man-A to Man-F, A2-A2Ј-B2-B2Ј-C3-C3Ј-(or -C2-C2Ј-)-D2-D2Ј-E2-E2Ј-F2. It has been shown that the H-1 signal of an ␣-linked mannose unit substituted by two consecutive ␤-1,2-linked units causes an upfield shift (⌬␦ ϭ 0.02 ppm) (10,11,17,18). The H-1 proton of the Man-D of SaggMan 7 at 5.216 ppm appeared at about 0.02 ppm upfield from that of SagMan 6 at 5.239 ppm.…”

“…Although KaMan 4 , AaMan 6 , and SaMan 6 can also possibly act as the acceptors of both the ␤-1,2-mannosyltransferase IV and an ␣-1,2-mannosyltransferase judging from the structure of C. guilliermondii mannan, their enzyme reaction products, designated as KagMan 5 , AagMan 7 , and SagMan 7 , exhibited a signal at about 4.76 ppm in the 1 H NMR spectra (data not shown). Therefore, it is apparent that the transferred mannose unit is attached to each substrate with a ␤-1,2 linkage (10,11,17). Since these ␤-1,2 linkagecontaining branched oligosaccharides have not been found in the acetolysate of yeast mannans, it is reasonable to say that we could prepare novel oligosaccharides using the ␤-1,2-mannosyltransferase IV of C. guilliermondii.…”

“…Therefore, we assumed that Man-D of SagMan 6 is substituted by a ␤-1,2-linked mannose unit, Man-E. It has been shown that the H-1 signal of an ␣-linked mannose unit substituted by a single ␤-1,2-linked unit causes a downfield shift (⌬␦ ϭ 0.09 -0.12 ppm) (10,11,17) and that the ␤-1,2-linked mannose units gave H-1-H-5-correlated cross-peaks in a characteristic region (14,17,18). In the spectrum of SagMan 6 , the H-1 proton of Man-D at 5.239 ppm appeared at 0.1 ppm more downfield than that of SaMan 5 at 5.129 ppm, and there is a ␤-mannose specific H-1-H-5-correlated cross-peak.…”

“…The third type of ␤-1,2 linkage-containing side chains can be observed in the mannan of Candida guilliermondii. This type of oligosaccharide contains ␤-1,2-linked mannose units attached to an ␣-1,3-linked mannose unit, the presence of which has been demonstrated in the cell wall mannans of Saccharomyces kluyveri (16,17), C. guilliermondii (18), and Candida saitoana (19) and have been identified to correspond to antigenic factor 9 (18).…”