Analysis of Wall Glucans from Yeast, Hyphal and Germ-tube Forming Cells of Candida albicans

Gopal, Pramod K.; Shepherd, Maxwell G.; Sullivan, Patrick A.

doi:10.1099/00221287-130-12-3295

Cited by 44 publications

(36 citation statements)

References 21 publications

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“…Presumably, Quantazyme had difficulty in passing ␤-1,6 branch points in the ␤-1,3-glucan polymers present in the cell wall, thereby leaving some undigested ␤-1,3-glucan attached to the ␤-1,6-glucosylated mannoproteins. It is tempting to speculate that the protein-bound ␤-1,6-glucan-␤-1,3-glucan demonstrated here is related to the ␤-1,6-glucan-␤-1,3-glucan heteropolymer isolated from the alkali-soluble wall fraction of C. albicans (1,11,25). Montijn et al (19) have shown that the ␤-1,6-glucose-containing side chains of glucanase-extracted cell wall proteins of S. cerevisiae are not modified or extended N chains and are also not O linked.…”

Section: Discussionmentioning

confidence: 99%

Covalent association of beta-1,3-glucan with beta-1,6-glucosylated mannoproteins in cell walls of Candida albicans

et al. 1995

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Yeast and hyphal walls of Candida albicans were extracted with sodium dodecyl sulfate (SDS). Some of the extracted proteins reacted with a specific ␤-1,6-glucan antiserum but not with a ␤-1,3-glucan antiserum. They lost their ␤-1,6-glucan epitope after treatment with ice-cold aqueous hydrofluoric acid, suggesting that ␤-1,6-glucan was linked to the protein through a phosphodiester bridge. When yeast and hyphal walls extracted with SDS were subsequently extracted with a pure ␤-1,3-glucanase, several mannoproteins that were recognized by both the ␤-1,6-glucan antiserum and the ␤-1,3-glucan antiserum were released. Both epitopes were sensitive to aqueous hydrofluoric acid treatment, suggesting that ␤-1,3-glucan and ␤-1,6-glucan are linked to proteins by phosphodiester linkages. The possible role of ␤-glucans in the retention of cell wall proteins is discussed.Candida albicans is an opportunistic, dimorphic fungal pathogen that grows in either yeast or hyphal form, depending on the environmental conditions. It is generally assumed that the morphological transition from the yeast to the hyphal form is a prerequisite for successful tissue invasion by C. albicans. This transition is correlated with changes in the composition of cell wall mannoproteins (5,8,9). Cell wall mannoproteins are also involved in other pathogenicity-related aspects, such as adhesion of Candida cells to mammalian tissues and immunogenicity (3, 4). It is, therefore, of interest to obtain more knowledge of the mechanisms by which mannoproteins are incorporated into the fungal cell wall. Some of the cell wall-associated proteins are extractable with hot sodium dodecyl sulfate (SDS). These proteins are mainly O mannosylated and have relatively low molecular masses (8, 9). The remaining mannoproteins, which might have both N-and O-linked carbohydrate chains, can be extracted by ␤-1,3-glucanase digestion, suggesting that these proteins are linked to the ␤-1,3-glucan skeleton of the cell wall (8,14,17,23). However, the exact nature of the bonds between these mannoproteins and the ␤-1,3-glucan skeleton is unknown. Recently, it was shown that the glucanaseextractable mannoproteins of C. albicans and Saccharomyces cerevisiae contain a novel type of side chain, carrying ␤-1,6-linked glucose residues, that was proposed to be involved in cell wall anchorage (13,16,19,22,23). Evidence that the ␤-1,6-glucan side chains of C. albicans are phosphodiester linked to the mannoproteins was presented (13). Interestingly, it has been shown that the alkali-soluble glucan of C. albicans cell walls consists of a ␤-1,6-glucan-␤-1,3-glucan heteropolymer (1,25). This raises the question whether the proteinbound ␤-1,6-glucan might also be part of such a heteropolymer in vivo.Here, by immunological means, we show that the ␤-1,6-glucosylated cell wall proteins contain a ␤-1,3-glucan epitope. Both the ␤-1,6-glucan and ␤-1,3-glucan epitopes are presumably phosphodiester linked to the protein. We propose that the incorporation of proteins into the cell wall is accomplished by ext...

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Section: Discussionmentioning

confidence: 99%

Covalent association of beta-1,3-glucan with beta-1,6-glucosylated mannoproteins in cell walls of Candida albicans

et al. 1995

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“…Since only linear polymers of 8-1,3 glucan and chitin are synthesized in vitro [I, 21, while branched glucans [19] and linkages to chitin [20-221 are found in vivo, some form of assembly reactions most likely occur post synthetically for the major structural polymers of fungal cell walls. The most likely candidate enzyme activity would be glycosyltransferases capable of assembling nascent glucan and chitin into the final wall structure.…”

Section: Discussionmentioning

confidence: 99%

Kinetics of β‐1,3 Glucan Interaction at the Donor and Acceptor Sites of the Fungal Glucosyltransferase Encoded by the BGL2 Gene

Goldman

Sullivan

Zakula

et al. 1995

European Journal of Biochemistry

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Formation of branched glucan, glucan-glucan cross links, and glucan-chitin cross links most likely involves the action of fungal wall glucanases and transglycosylases. We developed an HPLC assay using radiolabeled substrates in order to study the kinetics of interaction of donor and acceptor molecules with a glucosyltransferase present in the cell walls of both Saccharomyces cerevisiae and Candida albicans. Purified transferase first forms an activated intermediate from a donor p-1,3 glucan, releasing free disaccharide. The activated intermediate is transferred, in the presence of an appropriate acceptor /3-1,3 glucan, yielding a linear glucan containing a p-1,6 linkage at the transfer site [Yu, L., Goldman, R., Sullivan, P., Walker, G. & Fesik, S. W. (1993) J. Biomol. NMR 3, 429-4411, An apparent K,, of 0.41 mM for the acceptor site was determined using laminaritetraose as the acceptor. An apparent K, of 31 mM for the donor site was determined using increasing concentrations of laminaripentaose, and monitoring formation of laminaribiose. The enzyme functioned as a glucanase at low concentrations of acceptor molecules, with excess H,O competing for reaction at the activated donor site, thus resulting in hydrolysis.However, as the concentration of acceptor increased, the reaction shifted from hydrolysis to glucosyltransfer. The reaction appeared specific for p-1,3 glucan as acceptor, in as much as no transfer was detected when either hexa-N-acetyl-chitohexaose or maltooligosaccharides were used as acceptors. The roles of such an enzymic activity in cell wall metabolism is discussed in terms of repair, cross linking and incorporation of newly synthesized chains of p-1,3 glucan into the previously existing cell wall structure.Keywords. Glucosyltransferase ; fungal wall ; BGL2 protein ; wall assembly ; Saccharomyces.Synthesis and assembly of the fungal cell wall is a complex process about which we know much less compared to the analogous events occurring in bacteria. We do know that linear polymers of chitin, p-1,4 N-acetylglucosamine, and glucan, p-1,3 glucose, are synthesized by microsomal fractions prepared from fungal cells when iincubated in the presence of their appropriate UDP-sugars [l, 21. The process appears to be vectorial in that UDP-sugar presenf in the cytoplasm is polymerized, with concurrent translocation through the membrane of the growing polymeric chain. However, little if anything is known about the following aspects of p-1,3 polymer synthesis: (a) initiation of chain synthesis, (b) regulation of chain initiation, (c) translocation through the membrane, (d) mechanism and regulation of termination of chain synthesis, and (e) localization of chain synthesis during different phases of wall expansion. Furthermore, glucan found in the mature cell wall consists of highly branched molecules containing both p-1,3 and p-1,6 linkages.

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“…It is also of interest that second site suppressors containing no (1-*6)-P-D-glucan can at least partially bypass the KRES defect, although in all cases examined, these suppressors grew considerably more slowly than did wild-type cells. The demonstration here that (1-*6)- (14), and as (1--6)-P-D-glucans are apparently absent from mammalian cells, the KRE5 gene product is a new target for antifungals that should show specificity of action.…”

Section: S V N N T a L E S V C S E R I G Y Y T K N E E Y N L L H T I mentioning

confidence: 99%

The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1----6)-beta-D-glucan synthesis and normal cell growth.

Meaden¹,

Hill²,

Wagner³

et al. 1990

Mol. Cell. Biol.

126

120

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Yeast kre mutants define a pathway of cell wall (1-*6)-jp-D-glucan synthesis, and mutants in genes KRES and KRE6 appear to interact early in such a pathway. We have cloned KRES, and the sequence predicts the product to be a large, hydrophilic, secretory glycoprotein which contains the COOH-terminal endoplasmic reticulum retention signal, HDEL (1->3)-P-D-glucan and a (1->6)-P-D-glucan (12). The (1->3)-,B-D-glucan is the most abundant polysaccharide, contains some 1,500 glucose residues, and is composed of repeating units of linear (1->3)-13-linked residues, with some 3% of these branched through (1->6)-p3-linkages (20). The (1->6)-,-D-glucan is a smaller, highly branched molecule containing approximately 140 glucose residues. This glucan is largely connected through linear (1--6)-,-linked units, though it contains some linear (1->3)-1 linkages and a high proportion, some 14%, of (1->3, 1->6)-linked branched residues (21).(1-+6)-P-D-Glucan serves as a cell wall receptor for the yeast Kl killer toxin protein, and the binding of the toxin to the cell wall appears essential for toxin action (1, 6). Mutants have been isolated which are resistant to killer toxin (killer resistant) and which have reduced levels of (1->6)-P-Dglucan (15; Boone et al., in press). Such mutants define three genes, KREJ, KRE5, and KRE6, whose components are required for the sequential assembly of (1-+6)-P-D-glucan.The KREI gene codes for a secreted threonine-and serinerich agglutinin-like protein necessary for the addition of (1--6)-,-linked outer chains to a mixed linked core glucan structure (Boone et al., in press). Mutants

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Analysis of Wall Glucans from Yeast, Hyphal and Germ-tube Forming Cells of Candida albicans

Cited by 44 publications

References 21 publications

Covalent association of beta-1,3-glucan with beta-1,6-glucosylated mannoproteins in cell walls of Candida albicans

Covalent association of beta-1,3-glucan with beta-1,6-glucosylated mannoproteins in cell walls of Candida albicans

Kinetics of β‐1,3 Glucan Interaction at the Donor and Acceptor Sites of the Fungal Glucosyltransferase Encoded by the BGL2 Gene

The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1----6)-beta-D-glucan synthesis and normal cell growth.

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