Structure of the D-Mannan and D-Arabino-D-Galactan in Crithidia fasciculata: Changes in Proportion with Age of Culture*

SYNOPSIS. Cells of the insect flagellate Crithidia fasciculata contained mannan and arabinogalactan components, whose porportion varied with culture age, the former predominating during early stages, and the latter during the later stages of exponential growth and the deceleration phase. The mannan was a β-D-(1→2)-linked D-mannopyranan. The arabinogalactan had a complex structure containing, in part, a β-D-(1→-3)-linked galactopyranose main-chain substituted in the 2 positions by single-unit D-arabinopyranose side-chains and with some unsubstituted units.     

Structural and immunochemical studies on D-arabino-D-mannans and D-mannans of Mycobacterium tuberculosis and other Mycobacterium species.

Serologically active D-arabino-D-mannas ([alpha]D, +82 degrees approximately 89 degrees; ratio of D-arabinose to D-mannose, 1-2:1) were isolated from the soluble fraction of disintegrated cells of M. tuberculosis, M. smegmatis, and several other Mycobacterium species. These arabinomannans had similar structures, consisting of alpha-(1 leads to 5)-linked D-arabinose residues and alpha-(1 leads to 6)-, and (1 leads to 2)-linked D-mannose residues. Methylation and enzymic degradation studies using Arthrobacter sp. alpha-D-mannosidase and M-2 enzyme (D-arabinan hydrolase) indicated that the arabinomannan of M. tuberculosis Aoyama B possesses short side chains built up from alpha-(1 leads to 2)-D-mannosidic linkages which are attached to an alpha-(1 leads to 6)-linked mannan back-bone chain. The alpha-(1 leads to 5)-linked D-arabinose residues located in the side chains were shown, by comparison of the immunochemical activities of the native and enzyme-degraded polysaccharides, to be the main immunodeterminants, as in the cell-wall arabinogalactan. There appeared to be variations in the ratio of arabinose and mannose residues, and also in the proportion of (1 leads to 2)-linked D-mannose units, depending on the individual strain; no (1 leads to 2)-mannosidic linkage was found in M. smegmatis arabinomannan. In addition to arabinomannan, a serologically inactive alpha-D-mannan ([alpha)D, +65 degrees approximately 68 degrees), whose structure may resemble that of the core mannan of the arabinomannan, was isolated as a copper hydroxide complex from the soluble fraction of disintegrated mycobacterial cells.     

Candida albicans cell wall comprises a branched beta-D-(1-->6)-glucan with beta-D-(1-->3)-side chains

The structure of immunogenic and immunomodulatory cell wall glucans of Candida albicans is commonly interpreted in terms of a basic polysaccharide consisting of a beta-D-(1-->3)-linked glucopyranosyl backbone possessing beta-D-(1-->6)-linked side chains of varying distribution and length. This proposed molecular architecture has been re-evaluated by the present study on the products of selective enzymolysis of insoluble C. albicans glucan particles (GG). High resolution 1H (400 and 700 MHz) and 13C (100 and 175 MHz) NMR analyses were performed on a soluble beta-glucan preparation (GG-Zym) obtained by GG digestion with endo-beta-D-(1-->3)-glucanase and on its high- (Pool 1) and low-molecular weight (Pool 2) sub-fractions. The resonances typical of uniformly beta-D-(1-->6)- and beta-D-(1-->3)-linked linear glucans, together with additional multiplets assigned to short-chain oligoglucosides, were detected in GG-Zym. Pool 1 (46.3+/-6.4% of GG-Zym content) consisted of beta-D-(1-->6)-linked glucopyranosyl polymers, with short beta-D-(1-->3)-branched side chains of 2.20+/-0.02 units (branching degree (DB)=0.14+/-0.03). Pool 2 was a mixture of glucose and linear short-chain beta-D-(1-->3)-oligoglucosides. Further digestion of Pool 1 by beta-D-(1-->6)-glucanase yielded a mixture of glucose and short beta-D-(1-->6)-linked, either linear or beta-D-(1-->3,6) branched, oligomers. These endoglucanase digestion patterns were consistent with the presence in C. albicans cell wall glucans of beta-D-(1-->6)-linked glucopyranosyl backbones possessing beta-D-(1-->3)-linked side chains, a structure very close to that of beta-D-(1-->6)-glucan from Saccharomyces cerevisiae yeast. This finding may provide the grounds for further elucidation of the cell wall structure and a better understanding of the biological properties of C. albicans beta-glucans.     

Structure-immunomodulating activity relationships of a pectic arabinogalactan from Vernonia kotschyana Sch. Bip. ex Walp

Structure and immunological characteristics of the pectic arabinogalactan Vk2a (previously reported as Vk100A2a) from the roots of Vernonia kotschyana Sch. Bip. ex Walp. were investigated after enzymatic digestion of the galacturonan moiety and the side chains of the rhamnogalacturonan structure of Vk2a. endo-alpha-D-(1-->4)-Polygalacturonase digestion released the high molecular weight 'hairy region' (Vk2a-HR) and oligogalacturonides. Vk2a-HR consisted of GalA (4-linked) and Rha (2- or 2,4-linked) in a 1:1 ratio, with 60% of Rha branched at C-4. The Rha located in the rhamnogalacturonan core was branched randomly by Gal units. Vk2a-HR was rich in neutral sugars such as Araf 5- (12.2%) and 3,5-substituted (12.8%) and terminally- (14.1%) linked and Gal 4- (13.0%), 3- (0.9%), 6- (2.2%) and 3,6- (1.1%) substituted. Arabinans with chain lengths up to 11 units were identified. Araf residues were attached to C-3 of alpha-L-(1-->5)-Araf chains and to C-4 of Gal residues. Single Gal units and chains of beta-D-(1-->6)-linked galacto di- to penta-saccharides were attached to a beta-D-(1-->3)-galactan core. All the enzyme resistant fractions expressed potent complement fixation and induction of B-cell mitogenic activity, and the present study indicates that there may be several and possibly structurally different active sites involved in the bioactivity of Vk2a. The bioactive sites may be located both in the more peripheral parts of the molecule but also in the inner core of the 'hairy region' or in larger enzyme-resistant chains.     

Different composition of the cell wall polysaccharides in Saccharomyces cerevisiae S288 and in an osmosis sensitive mutant]

Determination of the polysaccharide contents and structural studies on the mannan by acetolysis and permethylation analysis shows an altered polysaccharide biosynthesis of the osmotic-sensitive mutant VY 1160 of Saccharomyces cerevisiae S 288. The mutant contains more glucan, less mannan, and less alkali-soluble glycogen. Its mannan is characterized by more short side chains and less long side chains. Its main chain is 1 leads to 6-linked, but its side chains consist of more 1 leads to 3- than 1 leads to 2-linked mannose units.     

Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses

The peptidoglycan-bound arabinogalactan of a virulent strain of Mycobacterium tuberculosis was per-O-methylated, partially hydrolyzed with acid, and the resulting oligosaccharides reduced and O-pentadeute-rioethylated. The per-O-alkylated oligoglycosyl alditol fragments were separated by high pressure liquid chromatography and the structures of 43 of these constituents determined by 1H NMR and gas chromatography/mass spectrometry. The arabinogalactan was shown to consist of a galactan containing alternating 5-linked beta-D-galactofuranosyl (Galf) and 6-linked beta-D-Galf residues. The arabinan chains are attached to C-5 of some of the 6-linked Galf residues. The arabinan is comprised of at least three major structural domains. One is composed of linear 5-linked alpha-D-arabinofuranosyl (Araf) residues; a second consists of branched 3,5-linked alpha-D-Araf units substituted with 5-linked alpha-D-Araf residues at both branched positions. The non-reducing terminal region of the arabinan was characterized by a 3,5-linked alpha-D-Araf residue substituted at both branched positions with the disaccharide beta-D-Araf-(1----2)-alpha-D-Araf. 13C NMR of intact soluble arabinogalactan established the presence of both alpha- and beta-Araf residues in this domain. This non-reducing terminal motif apparently provides the structural basis of the dominant immunogenicity of arabinogalactan within mycobacteria. A rhamnosyl residue occupies the reducing terminus of the galactan core and may link the arabinogalactan to the peptidoglycan. Evidence is also presented for the presence of minor structural features involving terminal mannopyranosyl units. Models for most of the heteropolysaccharide are proposed which should increase our understanding of a molecule responsible for much of the immunogenicity, pathogenicity, and peculiar physical properties of the mycobacterial cell.     

Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.)

Extracts from pollen of timothy grass (Phleum pratense L.) contain up to 20% arabinogalactan proteins (AGPs). Separation of the AGP polysaccharide moieties by tryptic digestion, size exclusion chromatography (GPC), and reverse phase HPLC yielded arabinogalactan fractions AG-1 and AG-2 with molecular weights of approximately 15,000 and approximately 60,000Da, respectively. The backbones of both polysaccharides are composed of (1-->6)-linked beta-D-galactopyranosides with beta-D-GlcUAp or 4-O-Me-beta-D-GlcUAp at their terminal ends as revealed by chemical analysis, FT-IR, MALDI-MS, and NMR spectroscopy. AG-1 contains a small number of beta-l-Araf side chains while AG-2 possesses a variety of (1-->3)-linked units, which consist of beta-l-Araf-(1-->, alpha-l-Araf-(1-->3)-beta-l-Araf-(1-->, and alpha-l-Araf-(1-->5)-beta-l-Araf-(1--> as well as a small number of longer arabinogalactan side chains. In contrast to crude pollen extracts, the immunological properties of the arabinogalactan mixture reveal an IgG4 reactivity instead of IgE reactivity. Structural properties of timothy pollen arabinogalactan might thus influence the immune response.     

Characterization of a beta-D-(1-->3)-glucan from the marine diatom Chaetoceros mülleri by high-resolution magic-angle spinning NMR spectroscopy on whole algal cells

High-resolution magic-angle spinning (hr-MAS) NMR spectroscopy was used to record NMR spectra of a cell paste from the marine diatom Chaetoceros mülleri. This gave information on a cellular storage polysaccharide identified as a beta-D-(1-->3)-linked glucan, using hr-MAS one-dimensional 1H and 13C, two-dimensional 1H,1H-COSY and 13C,1H-correlation spectroscopy. The same structural information was deduced from the liquid state NMR data on the glucan extracted from C. mülleri. The extracted glucan proved to be a beta-D-(1-->3)-linked glucan with a degree of polymerization of 19 and a degree of beta-D-(1-->6) branching of 0.005. The hr-MAS spectrum of the diatom showed several nonglucan resonances in the carbohydrate region of the NMR spectrum (60-103 ppm) that were shown to be noncarbohydrate resonances by means of two-dimensional 13C,1H- and 1H,1H-correlated NMR data.     

Concise syntheses of arabinogalactans with beta-(1-->6)-linked galactopyranose backbones and alpha-(1-->3)- and alpha-(1-->2)-linked arabinofuranose side chains

4-methoxyphenyl glycosides of 2,3'-bis-alpha-L-arabinofuranosyl branched beta-D-(1-->6)-linked galactopyranosyl tetraose (16), 3',2'-bis-alpha-L-arabinofuranosyl branched beta-D-(1-->6)-linked galactopyranosyl hexaose (27), and a twentyose (42) consisting of beta-(1-->6)-linked D-galactopyranosyl pentadecaoligosaccharide backbone with alpha-L-arabinofuranosyl side chains alternately attached at C-2 and C-3 of the middle galactose residue of each consecutive beta-(1-->6)-linked galactotriose unit of the backbone, were synthesized with isopropyl 3-O-allyl-2,4-di-O-benzoyl-1-thio-beta-D-galactopyranoside (6), 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (7), 2,3,5-tri-O-benzoyl-alpha-L-arabinofuranosyl trichloroacetimidate (12), 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (17), 4-methoxyphenyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (19), and 2,6-di-O-acetyl-3,4-di-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (28) as the key synthons. Condensation of 6 with 7 gave the disaccharide donor 8, and subsequent condensation of 8 with 4-methoxyphenyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->6)-2-O-acetyl-3,4-di-O-benzoyl-beta-D-galactopyranoside (9) followed by selective deacetylation afforded the tetrasaccharide acceptor 11. Coupling of 11 with 12 gave the pentasaccharide 13, its deallylation followed by coupling with 12, and debenzoylation gave the hexasaccharide 16 with beta-(1-->6)-linked galactopyranose backbone and 2- and 3'-linked alpha-L-arabinofuranose side chains. The octasaccharide 27 was similarly synthesized, while the twentyoside 42 was synthesized with tetrasaccharides 33 or 24 as the donors and 23, 36, 38, and 40 as the acceptors by consecutive couplings followed by deacylation.     

Metabolism of 2-deoxy-d-glucose by baker's yeast. VI. A study on cell wall mannan

The incorporation of 2-deoxy-d-glucose into cell wall mannan of growing Saccharomyces cerevisiae proceeded continuously during culture growth and followed the cell multiplication. About 10% of mannan labelled with deoxyglucose was concurrently released into the medium. The distribution of deoxyglucose between the side-chains and the main chain of mannan has been established. Approximately 90% of deoxyglucose present in the polysaccharide was bound in the side-chains and only 10% was located in the (1 å 6)-linked main chain. This result suggested that deoxyglucose metabolites serving as glycosyl donors in mannan biosynthesis were much worse substrates for the enzyme(s) responsible for the formation of the main chain of the polysaccharide than for the mannosyl transferases involved in the formation of the mannan side-chains. Degradation of deoxyglucose-containing mannan by α-mannosidase of Arthrobacter GJM-1 stopped at the deoxyglucosyl residues.     

Chemical structure of the cell-wall mannan of Candida albicans serotype A and its difference in yeast and hyphal forms

The structure of the cell-wall mannan from the J-1012 (serotype A) strain of the polymorphic yeast Candida albicans was determined by acetolysis under mild conditions followed by HPLC and sequential NMR experiments. The serotype A mannan contained beta-1,2-linked mannose residues attached to alpha-1,3-linked mannose residues and alpha-1,6-linked branching mannose residues. Using a beta-1,2-mannosyltransferase, we synthesized a three-beta-1,2-linkage-containing mannoheptaose and used it as a reference oligosaccharide for 1H-NMR assignment. On the basis of the results obtained, we derived an additivity rule for the 1H-NMR chemical shifts of the beta-1,2-linked mannose residues. The morphological transformation of Candida cells from the yeast form to the hyphal form induced a significant decrease in the phosphodiesterified acid-labile beta-1,2-linked manno-oligosaccharides, whereas the amount of acid-stable beta-1,2 linkage-containing side chains did not change. These results suggest that the Candida mannan in candidiasis patients contains beta-1,2-linked mannose residues and that they behave as a target of the immune system.     

Structural characterisation of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa

An anti-complementary arabinogalactan (AGIIb-1), isolated from the roots of Angelica acutiloba Kitagawa, has been subjected to methylation analysis, digestion with alpha-L-arabinofuranosidase, controlled Smith-degradation, and partial acid hydrolysis. AGIIb-1 consisted of arabinose, galactose, rhamnose, galacturonic acid, and glucuronic acid in the molar ratios 1.8-2.2:1.0:0.2-0.3:0.2-0.4:0.1. AGIIb-1 contained mainly an arabino-3,6-galactan moiety, and most of the Ara was present as alpha-L-arabinofuranosyl residues in the non-reducing terminals and the highly polymerised and branched side-chains which were attached mainly to positions 3 and 6 of (1----6)- and (1----3)-linked Gal, respectively. Some Ara-containing chains were also attached to (1----4)-linked Gal residues. The 13C-n.m.r. data for AGIIb-1 showed that the Galp was beta. Mild acid hydrolysis of AGIIb-1 yielded several linear and highly branched arabino-oligosaccharides, a neutral arabinogalactan, and two acidic arabinogalactans. Some arabino-oligosaccharides contained a (1----4)-linked Arap at the reducing terminal. The neutral arabinogalactan contained (1----3)-, (1----4)-, and (1----6)-linked and 3,6-di-O-substituted Gal, whereas the acidic arabinogalactans contained, in addition, non-reducing terminal GlcA, (1----4)-linked GalA, and 2,4-di-O-substituted Rha. The anti-complementary activity was decreased when AGIIb-1 was partially hydrolysed with mild acid (10mM HCl, 100 degrees, 10 min), but treatment with exo-alpha-L-arabinofuranosidase markedly enhanced the activity.     

A (1-->6)-linked beta-mannopyrananan, pseudonigeran, and a (1-->4)-linked beta-xylan, isolated from the lichenised basidiomycete Dictyonema glabratum

Extraction of Dictyonema glabratum with hot 2% (w/v) aqueous KOH at 100 degrees C, followed by neutralisation and freeze-thawing, gave an insoluble glucan. The residue was further extracted by a similar process, but with hot 10% (w/v) aqueous KOH, furnishing a mixture of glucan, mannan and xylan. The mannan and xylan were obtained via precipitation of its copper complex with Fehling's solution, leaving the glucan in the supernatant. The insoluble complex was finally purified through gel permeation chromatography. Methylation analysis, one- and two-dimensional nuclear magnetic resonance examination showed the polysaccharides to be a (1-->3)-linked alpha-glucan (pseudonigeran) and a (1-->4)-linked beta-xylan, both not previously encountered in lichens, and a newly discovered (1-->6)-linked beta-mannan.     

An arabinogalactan from the culture medium of Rubus fruticosus cells in suspension

A water-soluble arabinogalactan, isolated from the extracellular medium of suspension-cultured cells of Rubus fruticosus, contained arabinose, rhamnose, galactose, and also protein (6.5%) and uronic acid (2.5%). Methylation analysis of the arabinogalactan and the arabinose-free product obtained by mild acid hydrolysis showed that the polysaccharide was a typical arabino-3,6-galactan in which rhamnose and glucuronic acid occupied non-reducing terminal positions. Successive Smith-degradations combined with methylation analysis and ¹³C-n.m.r. spectroscopy revealed that the arabinogalactan contained a main chain of (→3)-linked β-d-galactopyranosyl residues with a high degree of branching at positions 6 by (1→6)-linked d-galactopyranosyl side-chains of various lengths, in which several contiguous residues were substituted at positions 3. The polymer is thus an arabinogalactan-protein belonging to the galactans of Type II.     

An arabinogalactan from the skin of Opuntia ficus-indica prickly pear fruits

The cold-water extract from the skin of Opuntia ficus-indica fruits was fractionated by anion-exchange chromatography. The major fraction, which was purified by size exclusion chromatography, consisted of a polysaccharide composed of galactose and arabinose residues in the ratio 6.3:3.3, with traces of rhamnose, xylose and glucose, but no uronic acid. The results of methylation analysis, supported by (13)C NMR spectroscopy, indicated that this polysaccharide corresponded to an arabinogalactan having a backbone of (1-->4)-linked beta-D-galactopyranosyl residues with 39.5% of these units branched at O-3. The side-groups consisted either of single L-arabinofuranosyl units or L-arabinofuranosyl alpha-(1-->5)-linked disaccharides. This polysaccharide is thus an arabinogalactan that can be classified in the type I of the arabinogalactan family.     

Structural analysis of cell wall mannan of <Emphasis Type="Italic">Candida sojae</Emphasis>, a new yeast species isolated from defatted soybean flakes

We investigated the structural and immunochemical characteristics of cell wall mannan obtained from Candida sojae JCM 1644, which is a new yeast species isolated from defatted soybean flakes. The results of a slide-agglutination test and of an enzyme-linked immunosorbent assay using anti-factor sera to the pathogenic Candida species indicated that the cells and the C. sojae mannan were cross-reactive to the specific anti-factor sera against Candida albicans serotype A (FAb 6) and Candida guilliermondii (FAb 9). Two-dimensional homonuclear Hartmann–Hahn analysis indicated that the mannan consisted of various linked oligomannosyl side chains containing α-1,2-, α-1,3-, α-1,6- and β-1,2-linked mannose residues. However, although the determinants of antigenic factors 6 and 9 could be not found in this mannan, branched side chains, Manβ1-2Manα1-3[Manα1-6]Manα1-(2Manα1-)n2Man and a linear α-1,6-linked polymannosyl backbone, which are cross-reacted by FAbs 6 and 9, respectively, were identified. The mannan was subjected to acetolysis in order to determine the polymerization length of the α-1,2-linked oligomannosyl residue in the side chains. The result of ¹H-nuclear magnetic resonance analysis of the released oligosaccharides showed that the remarkable regularity in the length of α-1,2-linked oligomannosyl side chains, which were previously found in mannans of other Candida species, is not observed in this mannan.     

Water-soluble sulfated polysaccharides from the red seaweed Chaetangium fastigiatum. Analysis of the system and the structures of the α-d-(1 → 3)-linked mannans

The water-soluble polysaccharides from Chaetangium fastigiatum were fractionated with cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration and seven fractions were separated and analyzed. Two of the fractions were subjected to methylation and desulfation-methylation analyses. The results indicate that this seaweed contains a system of sulfated polysaccharides consisting in part of a galactan and an α-d-(1 → 3)-linked mannan, 2- and 6-sulfated, and having single stubs of β-(1 → 2)-linked d-xylose. Composition dispersity of the mannan is produced by variation of the amount and disposition of the sulfate groups and of the content of the xylose side-chains.     

The occurrence of internal (1 --> 5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances

CDTA-extractable soybean pectic substances were subjected to enzymatic digestion with arabinogalactan degrading enzymes yielding a resistant polymeric pectic backbone and arabino-, galacto-, and arabinogalacto-oligomers. The complex digest was fractionated using size-exclusion chromatography. Monosaccharide composition analysis, HPAEC fractionation and MALDI-TOF MS analysis of the resulting fractions showed that each contained a mixture of oligosaccharides of essentially the same degree of polymerisation, composed of only arabinose and galactose. MALDI-TOF MS analysis was used for molecular mass screening of oligosaccharides in underivatised HPAEC fractions. The monosaccharide sequence and the branching pattern of oligosaccharides (degree of polymerisation from 4 to 8) were determined using linkage analysis and ES-CID tandem MS analysis of the per-O-methylated oligosaccharides in each of the HPAEC fractions. These analyses indicated the presence of common linear (1 --> 4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1 --> 4)-linked galactose residues bearing an arabinopyranose residue as the non-reducing terminal residue, and a mixture of linear oligosaccharides constructed of (1 --> 4)-linked galactose residues interspersed with an internal (1 --> 5)-linked arabinofuranose residue. The consequences of these two new structural features of pectic arabinogalactan side chains are discussed.     

Changes in the Composition of Cell Wall Polysaccharides of Suspension-cultured Vinca rosea Cells during Culture

In a suspension culture of Vinca rosea L. three distinct growth phases were distinguished: a cell division phase, a cell expansion phase, and a stationary phase. At various stages of the growth cycle, extra-cellular polysaccharides (ECP) and cell wall were isolated and their compositions were investigated. ECP are mainly composed of xyloglucan, 3,6-linked arabinogalactan, and polyuronide. From the results of gas chromatographic analyses of sugars and partially methylated sugars in each fraction of cell wall polysaccharides, the following changes were observed in the composition of cell wall polysaccharides during culture: (1) a decrease in polyuronide content, (2) a decrease in non-cellulosic glucan content, (3) an increase in xyloglucan content, and (4) an increase in a-cellulose content. The significance of the changes is discussed in relation to the growth of cells.     

Distribution of antigenic oligomannosyl side chains in the cell wall mannans of several strains of<Emphasis Type="Italic"> Candida tropicalis</Emphasis>

In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall mannans of the pathogenic yeast Candida tropicalis, the chemical structure of mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the mannans. The linear side chain Manalpha1-3Manalpha1-(2Manalpha1-)(n)2Man [n> or =2] is present in the mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the mannans from IFO 0199 and IFO 1647. The mannan of IFO 0589 is the only mannan with the branched side chains, Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man and Manalpha1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man [n> or =2]. However, this mannan lacked the phosphate group and the beta-1,2-linked oligomannosyl side chain which are features of this group. The mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an alpha-1,3-linked mannose residue previously observed in Candida albicans.