Isolation and characterization of a polysaccharide containing 3-O-methyl-D-mannose from Mycobacterium phlei.

Abstract A polysaccharide composed of 3-O-methyl-d-mannose and d-mannose in the molar proportion of 10:2 has been isolated from Mycobacterium phlei. This methylmannose polysaccharide (MMP) did not contain acidic or basic groups, acyl esters, or a reducing end. MMP had a molecular weight of 2084 ± 35 as determined by sedimentation equilibrium and had a partial specific volume of 0.663 cc per g as found both by sedimentation equilibrium and summation of the molal volumes of the hexose residues. The structure of MMP was investigated by methylation analysis, Smith degradation, and immunochemistry. In order to distinguish between mannose and 3-O-methylmannose residues in the polysaccharide after exhaustive methylation, MMP was isolated from cells grown in the presence of l-[methyl-14C]methionine, under conditions which resulted in the incorporation of radioactivity exclusively into the methyl groups of the 3-O-methylmannose residues. Exhaustive methylation of [methyl-14C]MMP with nonradioactive methyl iodide and hydrolysis of the fully methylated polysaccharide yielded radioactive 2,3,6-tri-O-methylmannose as the major component. Radioactive 2,3-di-O-methylmannose, and nonradioactive 3,4,6-tri-O-methylmannose and 2,3,4,6-tetra-O-methylmannose were obtained as minor components. These findings showed that all of the 3-O-methylmannose residues were (1 → 4)-linked with 1 residue containing a substituent at position 6 as well. One of the two mannose residues was (1 → 2)-linked and the other occupied a terminal position in the polysaccharide. Smith degradation of MMP converted the 2 mannose residues to glycerol and ethylene glycol, yielding a nonreducing polysaccharide containing only 3-O-methylmannose. Exhaustive methylation of Smith-degraded [methyl-14C]MMP and hydrolysis of the fully methylated polysaccharide yielded radioactive 2,3,6-tri-O-methylmannose as the major component, although a small quantity of radioactive 2,3-di-O-methylmannose and a trace of radioactive 2,3,4,6-tetra-O-methylmannose were still detected. With some slight discrepancies, these results indicate that the ten 3-O-methylmannose residues are (1 → 4)-linked and occur in a homologous chain which probably forms a macrocyclic ring. The 2 mannose residues are attached to each other in a (1 → 2)-linkage and occur as a "nonreducing" end of the polysaccharide, or as a side chain on the postulated macrocyclic ring. Further evidence in support of a (1 → 2)-linked mannobiose side chain was provided by testing MMP as an inhibitor of the precipitin reaction between Kloeckera brevis phosphomonoester mannan and antisera obtained against whole cells of the yeast. MMP and α-(1 → 2)-linked mannotriose were equally effective as inhibitors of this precipitin reaction, which is specific for α-(1 → 2)-linked mannose residues at a nonreducing terminus. Smith-degraded MMP, which had the mannobiose side chain removed, failed to inhibit the precipitin reaction. These results confirm the presence of at least 2 α-(1 → 2)-linked mannose residues at a terminal position of MMP.

This methyhnannose polysaccharide (MMP) did not contain acidic or basic groups, acyl esters, or a reducing end. MMP had a molecular weight of 2084 rt 35 as determined by sedimentation equilibrium and had a partial specific volume of 0.663 cc per g as found both by sedimentation equilibrium and summation of the molal volumes of the hexose residues.
One of the two mannose residues was (1 -+ 2)-linked and the other occupied a terminal position in the polysaccharide.
Smith degradation of MMP converted the 2 mannose residues to glycerol and ethylene glycol, yielding a nonreducing polysaccharide containing only 3-0-methylmannose. Exhaustive methylation of Smith-degraded [methyZ-14C]MMP and hydrolysis of the fully methylated polysaccharide yielded radioactive 2,3,6-tri-0-methylmannose as the major component, although a small quantity of radioactive 2,3-d&0methylmannose and a trace of radioactive 2,3,4,6-tetra-Omethylmannose were still detected. With some slight discrepancies, these results indicate that the ten S-O-methylmannose residues are (1 + 4)-linked and occur in a homologous chain which probably forms a macrocyclic ring.
The 2 mannose residues are attached to each other in a (1 -+ 2)-* This work was supported by Grant, AM884 from the United States Public Health Service and by United States Public Health Service Postdoctoral

Fellowship
No. 1 F02 AI42452-01 to G. R. G. linkage and occur as a "nonreducing" end of the polysaccharide, or as a side chain on the postulated macrocyclic ring.
Further evidence in support of a (1 + 2)-linked mannobiose side chain was provided by testing MMP as an inhibitor of the precipitin reaction between Kloeckera brevis phosphomonoester mannan and antisera obtained against whole cells of the yeast.
MMP and a-(1 + 2)-linked mannotriose were equally effective as inhibitors of this precipitin reaction, which is specific for a-(1 + 2)-linked mannose residues at a nonreducing terminus. Smith-degraded MMP, which had the mannobiose side chain removed, failed to inhibit the precipitin reaction.
These results confirm the presence of at least 2 oc-(1 + 2)-linked mannose residues at a terminal position of MMP.
Although rare in nature, several methylated sugars are found in Mycobacterium species as components of glycolipids, glycopeptides, or lipopolysaccharides.
In this paper we report our studies on the structure of this "methylmannose polysaccharide (MMP)".' In the course of this work, a paper appeared which described the presence of Y-0-methylmannose in a polysaccharide from Mycobactertim phlei (8), and it was reported that the polysaccharide had the unusual property of stimulating the fatty acid synthetase of d4. phlei by a large factor.
These authors found that MMP acted in a similar fashion.
Since this stimulatory effect of the methylmannose polysaccharides on the fatty acid synthetase may result from interaction of the polysaccharide with the protein complex, any information of the structure of the polysaccharide would be particularly important in understanding the basis of this interaction.
As reported herein, our studies reveal that the polysaccharide does have a very unusual structure and possesses properties that may enhance its ability to form specific complexes with other macromolecules. ,&Amylase (sweet potato, specific activity 600 units per mg) was a product of Worthington.
a-O-Methyln-mannose was prepared by a slight modification of the procedure of Candy and Baddiley (9).
Growth of Bacteria--M. phlei (ATCC 346) was grown to stationary phase (40 hours) on a complex medium (10) in a New Brunswick Fermacell Fermentor, and the cells were harvested by centrifugation and stored at -10". Two hundred liters of medium yielded 6.35 kg of M. phlei cell paste.
f7il. phlei cells were also grown in the presence of L-[methyl-i4C]methionine in a medium previously described (II). A 10% inoculum from a 24.hour culture and 50 ,.&I of L-[methyl-W]methionine were added per liter of fresh growth medium, and the incubation was continued for 24 hours at 37". Cells were harvested by centrifugation and stored frozen. Analytical Procedures-Total carbohydrate was measured by the phenolsulfuric acid method with n-mannose as the standard (la), reducing sugar by the method of Park and Johnson (13), and acyl esters were determined by a modification of the hydroxamic acid method (14). Column effluents were assayed for radioactivity by counting aliquots of the fractions in 10 ml of Bray's solution (15) in a Nuclear Chicago scintillation counter. Paper chromatograms were scanned for radioactivity by cutting the isolated 2-cm wide strip into l-cm horizontal bands which were counted in 10 ml of Bray's solution.
Sugars were detected on chromatograms with the aniline hydrogen phthalate reagent (16), or with alkaline silver nitrate (17). The latter reagent detects sugars and polyols, but it was necessary to hold the chromatogram over a steam bath to detect 3-0-methylmannose and polyols. Gas-liquid chromatography was performed on an Aerograph Hy-Fi, model 600-B, equipped with a flame ionization detector. The following stainless steel columns were used: Column 1, 3% OV-225 on Gas Chrom Q, 2 feet x 0.125 inch (100 to 120 mesh), nitrogen flow rate of 30 ml per min at 180"; Column 2, 10% Carbowax 20M on Aeropak 30, 4 feet x 0.125 inch (100 to 120 mesh), nitrogen flow rate of 30 ml per min at 140' for silyl derivatives or 200" for methyl glycosides of methylated sugars. Combined gas-liquid chromatography-mass spectrometry was carried out on Du Pont model 21-491 and Finnigan model 1015SL mass spectrometers at an ionizing potential of 70 volts.

AND DISCUSSION
Isolation of il[MP-Frozen M. phlei cells (250 g) were estracted twice with 5 volumes of acetone and air dried, and the residue was extracted twice with 15 volumes (based on the volume of wet cells) of refluxing 70% ethanol.
After ev-aporation of solvents, the ethanol extract was partitioned between the two layers of a mixture of chloroform-methanol-water (8:4:3, 750 ml). The upper aqueous layer was removed and centrifuged at 10,000 x g to remove the remaining chloroform layer, was evaporated under vacuum to remove methanol and traces of chloroform, and then was lyophilized.
The lyophilized water layer gave 21.75 g of solid material which was combined with [14C]MMP prepared from the cells described above, and then was fractionated on a Bio-Gel P-2 column (4 x 180 cm). Elution with water (containing 0.02$~& sodium aside as a preservative) gave the pattern shown in Fig. 1A. The appearance of MMP near the void volume resulted in its separation from trehalose, the major carbohydrate component of the aqueous extract.
The radioactive fractions were combined (2.1 g) and reapplied to the same column giving the result. shown in Fig. 1B. Fractions from this second column, containing radioactivity, were combined (1.18 g) and fractionated on a column (1 x 10 cm) of DEAE Sephadex A-25 (bicarbonate form).
Elution with water afforded neutral material (950 mg) which contained MMP as well as a large amount of glucan.
The latter was removed by digestion with 16 mg of p-amylase in 20 ml of 0.1 M sodium acetate buffer at pH 4.0. After 48 hours, the digest was heated and centrifuged to remove protein, reduced in volume, and then was fractionated on a Sephadex G-25 column (2.5 x 95 cm) (Fig. 2). Peak A (125 mg) gave mostly 3-0-methylmannose on hydrolysis, with a smaller amount of mannose; but, in some experiments it also contained traces of glucose and arabinose.
Identity of Unknown Sugar-Paper chromatography of the hydrolyzed polysaccharide indicated the presence of mannose and an unknown sugar with RmitnnOse values of 1.55 (Solvent A), 1.89 (Solvent B), and 1.48 (Solvent C). These values are those expected for a monomethylhexose.
The unknown sugar was isolated by preparative paper chromatography on Whatman No. 3MM paper in Solvent A, demethylated (18), and the product was rechromatographed in Solvents A and C. Mannose was the only sugar present.
The identity of the monomethylmannose was established by combined gas chromatography-mass spectrometry.
The purified polysaccharide was hydrolyzed and the monosaccharides were converted to alditol acetates (19). Gas chromatography (Column 1) indicated the presence of mannitol hexaacetate and the unknown acetate with a slightly slower retention time. The mass spectrum of the acetyl derivative of the unknown monomethylmannitol gave primary fragments of m/e 43, 85, 87, 99, 129, 189, and 261, those expected for the acetyl derivative of a 3-O-methylhexitol (20). The identity of the unknown sugar was confirmed by comparison with authentic 3-O-methyl-n-mannose.
Both had identical RmannDSe values in Solvents A, B, and C, were indistinguishable by gas chromatography of their silyl derivatives (Column 2), and had identica1 proton magnetic resonance spectra. Both sugars had the same equilibrium anomeric composition as mannose (69 % (Y-, 31 y0 P-pyranose) as determined by proton magnetic resonance Issue of November 25,1971 G. R. Gray and C. E. Ballou 6837 - proportion of reducing end was suggested by sodium borotritide reduction (21). MMP and a standard tetrasaccharide of mannose (MJ were reduced with the same sodium borotritide-sodium borohydride mixture, and the radioactive products were purified by gel filtration on Sephadex G-25. The specific activities of the isolated oligosaccharides were 166,000 cpm per pmole for reduced M, and 8,400 cpm per pmole for MMP, corresponding to 0.05 reducing end per mole of MMP.
This radioactivity appeared to be in mannitol following acid hydrolysis of the reduced MMP, and may have been derived from a minor contaminant.
The specific optical rotation of lyophilized MMP was [cx]$~ +128" (c, 0.4 water), or [CY] i,", +105" when hexose concentration was determined by the phenolsulfuric assay with mannose as standard, values which are characteristic of n-mannose in the o( configuration.
The relative proportions of mannose and 3-0-methylmannose in the polysaccharide were determined, after hydrolysis, by gasliquid chromatography of the trimethylsilyl derivatives (Column 2). As shown in Table I, this column separates the (Y and /3 anomers of glucose, mannose, and 3-0-methylmannose; and the conditions of silylation reflect their equilibrium anomeric composition in aqueous solution. Two different preparations of MMP gave 3-O-methylmannose to mannose ratios of 6.35 and 6.40, but one preparation gave a lower value (4.1). It is not clear whether the lower value represented variation because of growth conditions or the presence of a mannan impurity, but all further studies were carried out on the preparation with a ratio of 6.4.
The above properties are in general agreement with those of PSI isolated by Ilton et al. (8). PSr contained mannose and 3-0-methylmannose, was neutral, did not contain acyl groups and was nonreducing.
However, PSr had a lower content of mannose (4.5y0), and appeared to be larger than MMP since it was eluted from Bio-Gel P-6 with a lower elution volume than the methylglucose lipopolysaccharide of Saier and Ballou (6). In contrast, our MMP had a higher elution volume on Bio-Gel P-2 ( Fig. 1) than the methylglucose lipopolysa,ccharide, indicative of a smaller molecular weight.
The basis of these differences is not known.
The proton magnetic resonance spectrum of MMP is presented in Fig. 3. The anomeric hydrogens appear at 74.8, indicative of mannose and 3-O-methylmannose units linked in the (Y configuration. Protons with a chemical shift (75.06) corresponding to HI of the /3 form of mannose or 3-0-methylmannose were absent. The methyl hydrogens of the 3-0-methylmannose residues gave a singlet at 76.56, and the ring hydrogens of the hexose residues occurred in the region 75.7 to 6.5. The multiplet at 75.8, which is unusually deshielded for a hexose ring hydrogen, was also ob- Fractions were 4 ml each. Peak A is MMP and Peak B is maltose. spectroscopy and gas chromatography.
Neither was cleaved by periodate when its anomeric center was in a glycosidic linkage.
General Properties of Afethylmannose Polysaccharide-MMP contained mannose and 3-0-methylmannose as the only reducing sugars, the hydroxamic acid test showed that it did not contain ester groups, and it had no reducing end as measured by a reducing sugar titration (13). However, the possibility of a small Vol. 246, L-o. 22 served in the spectra of 3-O-methyl-n-mannose, 3,4-di-O-methyln-mannose, and 3,4,6-tri-O-methyl-n-mannose; but it was absent in the spectrum of methyl 2,3-di-0-methyl-a-n-mannoside. Proton decoupling established that the multiplet was spin coupled to the anomeric hydrogen.
These results show that the multiplet at 75.8 comes from the equatorial hydrogen at C-2, and that it is present only when the C-2 hydroxyl is unmethylated.
The same observations and conclusions have been made for methylated 6 I  I  I  I  I  I  I  I derivatives of myoinositol in which an equatorial methoxyl group is vicinal to an axial hydroxyl group (22). Molecular Weight of IWdirP-The molecular weight of AIMP was determined by sedimentation equilibrium with interference optics (23). In order to determine both the molecular weight and the partial specific volume (ti), the method of Edelstein and Schachman (24) was used, which is based on the change in the concentration distribution at equilibrium produced by increasing the density of the solvent with DzO. Solutions of MMP (6 mg per ml) in 0.1 M NaCl in both I-I,0 and DzO were centrifuged to equilibrium (24 hours) in a Yphantis cell. A plot of the logarithm of the concentration (c) across the cell against the square of the distance (r) from the center of rotation for each solution is shown in Fig. 4. The partial specific volume (6) of MMP was calculated from the slopes of these lines and the known densities of the solvents, knowing the ratio of the molecular weight in DzO to the molecular weight in HzO. The latter value was calculated to be 1.0130 from the increase in molecular weight because of replacement of the hydroxyl hydrogens with deuterium.
From these data a fi of 0.663 was found.
To verify this value, the 0 of MMP was calculated in the following manner.
The partial specific volume of mannose was determined by pycnometry to be 0.610 cc per g, in excellent agreement with a value (0.613 cc per g) obtained by addition of the molal volumes of the atoms (25). The partial specific volume of mannose was converted to a molal volume (109.8 cc per mole) from which was subtracted the molal volume of Hz0 (8.5 cc per mole), since a molecule of water is lost for each hexose linkage in a polysaccharide.
This gave the molal volume of a mannose residue in a polysaccharide, which, when divided by the residue weight, yielded the partial specific volume The same calculations were performed for 3-0-methylmannose, with a value of 16.2 cc per mole for the molal volume of the added methyl group (25). The partial specific volume for a 3-0-methylmannose residue in a polysaccharide was calculated to be 0.668 cc per g, and the part-ial specific volume of the polysaccharide, containing 13.5% mannose and 86.5y0 3-0-methylmannose, was 0.662 cc per g (0.668 X 0.865 + 0.625 X 0.135). With a value of 0.663 for 6, the molecular weight of MMP was calculated to be 2084 =t 35, corresponding to a dodecamer containing ten 3-0-methylmannose and 2 mannose residues.
Calculated for Cs2H140060 is 2086. From this structural formula, a fi of 0.663 was found by adding the molal volumes of the atoms and dividing by the molecular weight.
Methylation Analysis of MJ1P-MMP, isolated from 24. phlei grown in the presence of L-[methyl-14C]mebhiorline, contained radioactivity which was associated only with the methyl group of 3-0-methylmannose.
These results indicated that the 3-O-methylmannose residues were (1 ---f 4)-linked and that one was substituted on position 6 as well. They also suggest that 1 mannose residue was (1 + 2)-linked, while the other formed the nonreducing terminus of the polysaccharide.
Gas chromatography of the methylated sugars as t)heir methyl glycosides (Column 2) or as their alditol acetates (Column 1) confirmed the above assignments.
The ratio of 2,3,4,6-tetra-Omethylmannose to 2,3,6-tri-0-methyhnannose varied from 1:9.5 to 1: 12, the variability probably resulting from some loss of the volatile tetramethyl derivative during sample preparation. The amount of material with the retention time of 2,3-di-Omethylmannose was also variable, perhaps owing to incomplete methylation or to degradation of the free sugars during sodium borohydride reduction.
A control experiment showed that di-Omethylmannose was not produced from 2,3,6-tri-O-methylmannose under the conditions of acid hydrolysis used above.
Smith Degradation of MMP-MMP (10.7 mg, 5.13 pmoles) was oxidized with 0.05 M sodium metaperiodate in 0.033 M sodium acetate buffer at pH 4.0. The consumption of periodate, followed spectrophotometrically, was 15.9 qnoles in 36 hours, or 3.1 pmoles per pmole of MMP.
After destruction of excess periodate, the oxidized polysaccharide was purified by passage over Sephadex G-25, and then was reduced with sodium borotritide. Tritium-labeled, Smith-degraded MMP, again purified by passage over Sephadex G-25, emerged as a single peak with the same elution volume as intact MMP.
Total acid hydrolysis of Smith- Complete methylation of the unhydrolyzed Smith-degraded MMP followed by analysis of the methyl glycoside fraction by gas chromatography (Column 2) revealed the absence of 'methyl 2,3,4,6-tetra-0-methylmannose, as expected if mannose were originally at the nonreducing end of the intact MMP, thus confirming the results obtained by the methylation studies on [methyl-14C]MMP.
Partial acid hydrolysis of Smith-degraded MMP (0.25 N HCI, 25" for 24 hours) resulted in breaking only those linkages in which oxidized sugar residues were glycosidically linked to unoxidized sugar residues (28). After removal of HCl by evaporation under vacuum, sodium borohydride was added to reduce the newly formed aldehyde fragments.
Excess sodium borohydride was Polysaccharide Vol. 246, ATo. 22 removed by treatment with a cation exchange resin and was followed by repeated evaporation of methanol from the mixture to remove boric acid. Separation of the oxidized and unoxidized fragments was accomplished by gel filtration on Sephadex G-25 (Fig. 6). 2211 carbohydrate appeared in a single peak with the same elution volume as undegraded MMP.
A small amount of radioactivity (27J was associated with this fraction, which probably represented incomplete partial acid hydrolysis. The major radioactive peak was identified by paper radiochromatography as a mixture of glycerol (90%) and ethylene glycol (1070).
These results showed that the 2 mannose residues occurred together at the nonreducing end of the polysaccharide chain. If they were located in the interior of the chain, smaller molecular weight oligomers of 3-0-methylmannose would be formed after Smith degradation.
The fact that the mannose residues were converted to glycerol and ethyleneglycol indicates that these two hexoses were connected by a (1 + 2)-linkage, in agreement with the observation that they consumed 3 moles of periodate when MMP was oxidized.
The polysaccharide fragment was characterized by repeating the Smith degradation on MMP isolated from cells grown in the presence of L-[methyl-%]methionine.
The periodate-resistant fragment contained only 3-O-methylmannose, and its specific activity was 1.192 times greater than that of the intact polysaccharide.
This increase in specific activity resulted from the loss of the nonradioactive mannose residues, and the change corresponded to the loss of 1.92 mannose residues from the total of 12 hexose units.
Therefore, MMP appears to be composed of 10 residues of 3-0-methylmannose and 2 residues of mannose, in agreement with the size determined by sedimentation equilibrium, and the ratio determined by other procedures.
Complete methylation of Smith-degraded [rnet?~yl-~~C]MMP and hydrolysis of this product yielded a mixture of methylated mannose derivatives which was separated by paper chromatography as before (Fig. 7). Again the major radioactive component was 2,3,6-tri-0-methylmannose (92.45 %), while smaller amounts of 2,3-di-0-methylmannose (5.25 To) and 2,3,4,6tetra-O-methylmannose (2.30%) were observed. If MMP were a linear molecule, the removal of the 2 mannose residues at the The ratio of the total radioactivity in 2,3,6-tri-Omethylmannose to that in 2,3,4,6-tetra-0-methylmannose was 40, which is far from the ratio of 9 expected if the tetra-o-methylmannose came from a terminal 3-0-methylmannose unit. It is possible, however, that the specific activities of the individual 3-0-methylmannose residues in MMP differed greatly along the chain. Therefore, the ratio of 2,3,6-tri-0-methylmannose to 2,3,4,6-tetra-0-methylmannose was determined by gas chromatography of their methyl glycosides (Column 2) which gave a value of 23. Again, the value was too high to allow for the presence of one 3-0-methylmannose residue at the nonreducing end of each polysaccharide chain, although it is difficult to explain the presence of any tetramethylether in this experiment. Immunochemical IdentiJication of a~(1 --t %)-linked ilfannobiose Unit at "Nonreducing" End of MMP-The preceding experiments strongly suggested that the 2 mannose residues in the polysaccharide occurred together at a "nonreducing" end and were connected by an ar-(1 + 2).linkage.
From the proton magnetic resonance spectrum and high optical rotation of the polysaccharide, all linkages appeared to have the o( configuration. To confirm these findings, MMP and Smith-degraded MMP were tested as inhibitors of the precipitin reaction between Kloeckera brevis phosphomonoester mannan and the antiserum obt,ained against whole cells of this yeast. It has been shown (29) that the antibodies formed against K. brevis cells are directed against the mannan coat of these cells, and that the majority of the antibodies are specific for a mannosylphosphate group in diester linkage attached to a side chain of the mannan.
However, antibodies are also formed against the neutral side chains of the mannan, which consist of cr-(1 + 2)linked mannose residues (30). These side chains can be isolated by acetolysis, which breaks the oc-(1 ---f 6)-linkage of the backbone and yields C\I-( 1 + 2)linked disaccharides (Mz) and trisaccharides @!I,). When Mz and Ma were tested as inhibitors of the precipitin reaction between K. brevis serum and phosphodiester mannan, only slight inhibition (20%) was observed.
However, with phosphomonoester mannan (in which the mannose residues linked to phosphate were removed) for the antigen in the precipitin reaction, only antibodies directed against the neutral side chains are measured, and the observed inhibition by MB and M3 was increased. MMP and Smith-degraded MMP were compared with XI2 and MS as inhibitors of the precipitin reaction between phosphomonoester mannan and the anti-K. brevis serum. As seen in Fig. 8, MMP was as inhibitory as MS, and each was more effective than MC Smith-degraded MMP, in which the mannose side chain had been removed, was inactive.
The specificity of the precipitin reaction is seen from the fact that M4 from Hansenula angusta, which contains all (~(1 + 2)linkages, is a much more effective inhibitor than is M4 from Xaccharomyces cerevisiae, which contains two LY-(1 --f 2) -linkages with an a-(1 + 3)-linkage at the nonreducing end. These results clearly confirm the presence of at least two ol-(1 --f 2)-linked mannose residues at a terminal position of MMP, and provide strong evidence that they have the D configuration.
structure of,VJJP-The facts that Smith degradation of MMP does not expose a 3-0-methylmannose residue at the nonreducing end of the polysaccharide, that MMP does not have a reducing end, and that no aglycon has yet been found attached at the reducing end to make the substance nonreducing, raised thegossibility that MMP was a macrocyclic polysaccharide. Consistent Issue of November 25, 1971 G. R. residues. The mannose residues are linked or-(1 -+ 2) and the 3-0-methylmannose residues are linked ~(1 -+ 4). In Form ZZZ, the triangle represents a postulated, but unidentified, nonreducing aglycon. with these facts would be a structure ( Fig. 9) in which the ten 3.0-methylmannose residues were linked ~(1 ---f 4) in a cyclic chain, to which was attached the ~(1 + 2)linked mannobiose side chain at position 6 of one of the 3-0-methylmannose residues (Form I). To account for the small amount of 2,3,4,6-tetra-Omethylmannose and 2,3-di-0-methylmannose observed on complete methylation of Smith-degraded MMP, another form could be present (11) in which one 3-O-methylmannose residue was outside the macrocyclic chain.
It is also possible that an acyclic form (III) is present in which an unidentified aglycon is attached to the reducing end. However, the presence of a major proportion of Form III is not consistent with the virtual absence of 2,3, 4,6-tetra-0-methylmannose that was observed on methylation of Smith-degraded MMP, and the presence of 2,3-di-o-methylmannose on methylation of intact LIMP.
Indeed, a branch point in an acyclic form (III) would require the presence of still larger amounts (2 moles per mole of MMP) of 2,3,4, B-tetra-o-methylmannose after methylation.
A precedent for the proposed macrocyclic form of MMP are the Shardinger dextrins (31) trins are composed of 6 or more n-glucose units in ~(1 -+ 4)linkage, with the largest ring thus far isolated being the decamer. Regardless of the accuracy in t'hc postulation of a macrocyclic ring for MMP, it is noteworthy t'hat this is the only polysaccharide we are aware of in which mannose is found in a long chain in ~(1 + 4)-linkage.
Such a chain, as in oc-(1 -+ 4)-glucans, leads nat'urally to a coiled conformation and is a prerequisite for the formation of the cyclodextrins.
Several interesting features of the ring structure proposed for MMP are seen in the space-filling models in Fig. 10. The formation of a macrocyclic ring from ~(1 + 4)-linked 3-O-methylmannose residues positions all of the 3-O-methyl groups on one side of the ring (Fig. 10A) and all of the hydroxymethyl groups on the other side of the ring (Fig. 10B). This leads to a very hydrophobic surface on one side of the ring (Fig. 1OA) and a very hydrophilic surface on the other side (Fig. IOB). Another interesting feature of the model is the prominence of the a-(1 --) 2)linked mannobiose side chain, which forms the only region of structural complexity in the otherwise uniform chain. These two features assume some importance in view of the reported stimulatory effect of methylmannose polysaccharides on the fatt'y acid syn-Vol. 246, nTo. 22 thetase of M. phlei (8). If, as seems reasonable, the polysaccharide interacts with the enzyme complex in a specific way to alter its activity (as reflected in a reduced K, for acetyl-CoA) (S), the mannobiose side chain could provide a sight for recognition by the sjinthetase complex in much the same way as it did for the mannan-specific antibody.
Once specifically bound to the synthetase complex, one side or the other of the macrocyclic ring could associate with a complementary domain of the protein surface, thus changing the catalytic activity of the total complex. While highly conjectural, this hypothesis is open to a direct test by binding and structure modification studies.

g-O-~~ethyl-o-lMannose in Other
Bacteria-The occurrence 3-0-methyl-n-mannose was first reported in a polysaccharide fraction isolated from Streptomyces griseus (9). The polysaccharide, which was not characterized, was found to incorporate radioactivity from L-[methyl-14C]methionine, to be somewhat included on Sephadex G-25, and to lack acidic or basic groups; all of which are properties of MMP isolated from M. phlei.

Methylmannose
has also been reported in the 0-antigenic lipopolysaccharides of Klebsiella and Escherichia coli (32), and in hydrolysates of the mycelium, arthrospore cells, and cell walls of the fungus Coccidioides immitis (33). Thus, the distribution of Y-0-methylmannose is much wider than that of 6-O-methyl-Dglucose, which has been found only in the lipopolysaccharide of Mycobacterium species (6).