THE JOURNAL OF BIOLOGKAL CHEMISTRY Vol. 251, No. 10, Issue of May 25, pp. 2967-2975, 1976

Printed in U.S.A.

The Polysaccharides from Heterocyst and Spore Envelopes of a Blue-Green Alga METHYLATION ANALYSIS AND STRUCTURE OF THE BACKBONES*

(Received for publication, September 29, 1975, and in revised form, December 29, 1975)

LILIANA CARDEMIL$ AND C. PETER WOLK§ From the MSUIERDA Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824

Lindberg’s combined gas chromatography-mass spectrometry techniques for the analysis of partially methylated alditol acetate sugar derivatives were used to study the structures of polysaccharides from the envelopes of heterocysts and’spores of Anabaena cylindrica. Polysaccharides from both envelopes are highly branched. Glucose, mannose, galactose, and xylose are at terminal positions, whereas glucose and mannose are at internal positions in these polymers. The molar percentages of the 11 partially methylated alditol acetate derivatives observed were approximately the same for both envelopes, suggesting that the envelope polysaccharides may be identical or almost identical.

Smith degradation (periodate oxidation followed by reduction with sodium borohydride and mild acid hydrolysis) of the polysaccharides from the two kinds of envelopes removes the side branches without measurable fragmentation of the backbones. Gas chromatographic analysis of partially methylated alditol acetate derivatives of the sugars showed that the backbones of both envelope polysaccharides consist of glucose (Glc) and mannose (Man) linked by 1 --f 3 glycosidic bonds. Disaccharides, trisac- charides, and tetrasaccharides obtained from the backbone polysaccharides by partial acid hydrolysis were fractionated by column chromatography and separated by high voltage paper electrophoresis. Analysis of these oligosaccharides established that the backbone polysaccharides from both heterocysts and spores consist of repetitions of the structural unit Glc ---t Glc + Glc + Man, and that all linkages in the backbones are in the p configuration.

Vegetative cells of certain filamentous blue-green algae can differentiate into two morphologically and physiologically distinguishable types of cells: heterocysts, which are important in algal nitrogen fixation; and spores, which are perennating structures capable of germination (1). Deposition of a thick envelope exterior to the wall of a vegetative cell is the most conspicuous, and can be the first, morphological sign of differentiation. The envelope surrounding a heterocyst is perforated by a large pore at each point of attachment to a vegetative cell, whereas the envelope surrounding a spore is entire.

Geitler suggested, on the basis of morphological observa- tions, that the processes underlying differentiation of spores and heterocysts are evolutionarily (2) and ontogenically (3) related. Differently stated, these algae, Anabuena cylindricu Lemm. for example, are suitable for study of the relationships

*This investigation was supported by the United States Energy Research and Development Administration under Contract E(ll-l-1338.

$Present address, Facultad de Ciencias, Departamento de Biologia, Universidad de Chile; Santiago, Chile.

8 To whom inquiries concerning this paper should be addressed.

between alternative differentiations in a single organism. We have approached such a study by inquiring whether there are chemical similarities between the molecules forming the enve-

lopes of heterocysts and spores. Both envelopes contain amino compounds, lipids, and carbo-

hydrate. The spore envelope contains 6 times more amino compounds, as per cent of dry weight, than does the heterocyst envelope. Glucose, mannose, galactose, and xylose constitute, and are present to approximately the same percentages in, the carbohydrate portions of the two envelopes (4). The two envelopes might, therefore, have the same polysaccharide molecule as their main component. Alternatively, the same sugar residues might form structurally different polysaccha- rides in the two envelopes. The polysaccharides from the two envelopes can be compared further by identifying the linkages between the sugar residues. By combining mass spectrometric and gas chromatographic data (5), we have identified the glycosidic linkages in the polysaccharides from the envelopes of heterocysts and spores of A. cylindricu. We have analyzed these polysaccharides also by examining the products of their chemical degradation. We thereby determined the sequence of a portion of their sugar residues.

2967

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2968 Heterocyst and Spore Polysaccharides

MATERIALS AND METHODS

Preparation of Envelopes

Anabaena cylindrica was grown in photosynthetic fermenters as described previously (6). Filaments used for heterocyst envelope analysis were collected twice weekly, with subsequent additions of fresh medium, while spores were collected after 3 weeks of culture without intervening addition of fresh medium. Filaments were concen- trated by centrifugation at 48,200 x g (RC-2B centrifuge, Ivan Sorvall, Inc., Norwalk, Corm.). Vegetative cells were broken by cavitation (6). Published techniques were used for the isolation of heterocysts (7) and spores (8). Envelopes were isolated as described by Dunn and Wolk (4). The isolated envelopes were extracted with a mixture of chloroform/ methanol (2/l, v/v), lyophilized, and maintained in a vacuum desicca- tor at 60” until used.

Sugar Composition Analysis

Sugar composition was determined by gas chromatography of the alditol acetate derivatives as follows: 2 mg of envelopes were hydro- lyzed with 1 ml of 2 N trifluoroacetic acid in sealed vials at 121’ for 60 min. The sugars were converted to alditol acetates (9). The final acetic anhydride solution containing the mixture of alditol acetates was injected directly into the .2.2.4 column that is described below. Chromatography was performed with a Perkin-Elmer 900 gas chro- matograph (Perkin-Elmer Corp., Norwalk, Corm.) temperature pro- grammed at 1.5”/min from 130~180”, with a helium flow rate of 60 mllmin.

Methyl&ion Analysis

Three milligrams of dried heterocyst envelopes and 5 mg of dried spore envelopes were methylated by Hakomori’s method (10) as described by Sandford and Conrad (11).

The methylated envelopes were extracted with a mixture of chloro- lorm/methanol (l/l, v/v). The residual material was dialyzed against water, dried, remethylated, and extracted with chloroform/methanol. The chloroform/methanol extracts were concentrated, separately (see “Results”) or in combination, to a volume of 2 ml and separated from water-soluble reagents of low molecular weight either by dialysis against several changes of distilled water for 48 hours or by passage through a Sephadex LH20 column (1.3 cm inside diameter x 70 cm; Pharmacia Fine Chemicals, Inc., Piscataway, N.J.). In the latter case, fractions of 1.5 ml were collected. Those fractions which contained sugars, as shown by a positive reaction to the anthrone reagent (12), were pooled and dried in air at 60”.

The dried methylated polysaccharide was hydrolyzed with 1 ml of 2 N trifluoroacetic acid for 75 to 80 min at 121” in sealed tubes, and the partially methylated aldoses were converted to the partially methyl- ated alditol acetate derivatives (9). For mass spectrometry the partially methylated aldoses were reduced with NaBD, (Merck, Sharp and Dohme of Canada, Ltd., Montreal) instead of NaBH,. The presence of deuterium at the anomeric carbon provides a distinguisha- ble difference between the fragmentation patterns derived from C,- and Cd-linked hexoses (5).

The qualitative identification of each partially methylated alditol acetate component shown by gas chromatography was made from the following information: (a) The position of methoxy groups in the methylated alditol acetate as obtained by mass spectral analysis (5); (b) the sugar composition of the unmethylated envelopes (9); and (c) the relative retention times of the components on three chromato- graphic columns (see below). The three columns were calibrated, using known standards, against retention times relative to terminal glucose (5, 13) for the same columns and the same temperature programs. Standard partially methylated alditol acetate derivatives were pre- pared from sycamore cell walls (13), xyloglucan (14), maltose (Pfan- stiehl Laboratories, Inc., Waukegan, Ill.), arabinogalactan (Pfaltz and Bauer, Inc., Flushing, N.Y.), and yeast mannan (Sigma Chemical Co., St. Louis, MO.). The positions of the peaks derived from the envelope polysaccharides were then compared, by linear interpolation between the positions of the standards, with published retention times of partially methylated alditol acetate derivatives (5, 13).

Quantitative analysis was performed by integration (Autolab Com- puting Integrator, System .IV, Spectra-Physics, Santa Clara, Calif.) of the gas chromatographic peaks. So as to express the data as mole per cent of the recovered carbohydrate, each peak area was divided by the molecular weight of the appropriate partially methylated alditol acetate derivative and the sum of these ratios was normalized to 100.

Gas Chromatography

Gas chromatography, when not combined with mass spectrometry, was performed with the Perkin-Elmer 900 gas chromatograph with helium as carrier. Acetic anhydride solutions containing a mixture of the partially methylated alditol acetate derivatives were injected directly into the chromatographic columns.

Three different glass columns (2 mm inside diameter x 1.22 m) were used. They contained, on a stationary phase of Gas-chrom Q (80 to 100 mesh): (a) Three per cent ECNSS-M, a nitrite silicone-polyester co-polymer (Applied Science Laboratories, Inc., Ann Arbor, Mich.). Chromatography on this column was performed isothermally at 155”, with a helium flow rate of 60 ml/mm. (b) Two-tenths per cent poly(ethylene glycol adipate), 0.2% poly(ethylene glycol succinate), and 0.4% silicone GE-XEGO. This column, denoted .2.2.4, was temper- ature-programmed at l”/min from llO-180”, with a flow rate of 60 ml/min. (c) Three per cent silicone polymer OV-225 containing methyl, phenyl, and cyanopropyl groups (Applied Science Laborato- ries, Inc.). Chromatography was performed isothermally at 170”, with a flow rate of 30 ml/min (for identification of the branched sugars), or by temperature programming at 2”/min from 170-200”, with a flow rate of 60 ml/min. The latter program, which resolved branched sugars and also provided sharp peaks with approximately equal base widths, was the only program which provided closely reproducible relative peak areas. Accurate quantification of the branched sugars is impor- tant, because the total amount of branches should balance the terminal sugars.

Mass Spectrometry

Combined gas chromatography-mass spectrometry was performed in an LKB-9000 instrument (LKB Instruments, Inc., Rockville, Md.), interfaced with a PDP8/I computer. Glass, tubular columns of 2 mm inside diameter and 1.22 m length containing either 3W OV-225 on Gas-chrom Q (100 to 120 mesh) or 3% SP-2401, a trifluoropropyl silicone (Supelco, Inc., Bellefonte, Pa.) on Supelcoport (100 to 120 mesh) were used. The .2.2.4 column previously described was also used in order to identify 3.mannose’ as a 3-hexose. Oven temperature was rapidly increased from 110 to 170” immediately after the 3-mannose peak was recorded. When gas chromatography was used as the inlet to the mass spectrometer, the carrier gas was helium at a flow rate of 25 to 30 ml/min. The temperature program for the OV-225 and .2.2.4 columns was as described above. The SP-2401 column was tempera- ture programmed at S”/min from 160-230°C.

The ion source was at 240”, and the scanning voltage was 70 eV. Background was subtracted, and the data prepared in the form of bar graphs or fragmentograms, by means of a computerized data system (15).

Infrared Spectroscopy

Infrared spectra were recorded on a Perkin-Elmer 621 spectropho- tometer using a 0.025.mm KBr demountable cell. The sample for infrared spectroscopy was a 10% solution of permethylated heterocyst or spore envelopes in Ccl,.

Smith Degradation (16)

Periodate Oxidation-Sixty milligrams of lipid-free heterocyst or spore envelopes were oxidized at 5”, in darkness, using 160 mg of sodium metaperiodate dissolved in 50 ml of sodium acetate buffer (0.1 M, pH 5). The rate of the reaction was determined by measuring the absorption at 223 nm, the absorption maximum for periodate (17). A solution containing 160 mg of sodium metaperiodate in 50 ml of sodium acetate buffer was used as a blank. For these measurements, 10.~1 aliquots of the reaction mixture (freed of particulate matter by centrifugation) were taken at regular time intervals and diluted to 2.5 ml with distilled water. The reaction was run for 9 or 6 days (see “Results”). Periodate remaining in the reaction mixture was destroyed

‘We denote each partially methylated alditol acetate derivative by indicating the linkages (in addition to C-l) by which the corresponding glycosyl residue of the original sample was connected to other sugars. Thus, we write 3-mannose instead of 1,3,5-tri-O-acetyl-2,4,6-tri-O- methyl mannitol. Partially methylated alditol acetate derivatives which were obtained from sugar derivatives that occupied nonreducing terminal positions in the polysaccharides, that is, which were glycosidi- tally linked to other sugar residues only at C-1, are denoted by T- (e.g. 1,5-di-0-acetyl-2,3,4,6-tetra-O-methyl glucitol is denoted T-glucose).

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Heterocyst and Spore Polysaccharides 2969

by the addition of a few drops of a 0.1 M solution of sodium arsenite (NaAsO,), after which no reaction could be obtained with KI starch. The reaction mixture was then dialyzed against several changes of distilled water, and the volume reduced to 60 ml with a rotary evaporator.

Sodium Borohydride Reduction-Ninety milligrams of NaBH, were stirred with 60 ml of the above reaction mixture for 10 hours at room temperature (approximately 23”), after which 1 N HCl was added dropwise to destroy excess NaBH,.

Mild Acid Hydrolysis-The pH of the solution was adjusted to 0.5 with HCl, and the solution stirred for 6 to 8 hours at room temperature. After hydrolysis, the solution was centrifuged and a small pellet was separated from a clear yellow supernatant liquid. The pellet was washed with 0.5 ml of 0.05 N acetic acid two or three times by centrifugation. The combined supernatant liquids, but not the pellet, reacted positively to the anthrone reagent (12). The pellet was then discarded. The combined supernatant fluids were concentrated to 5 ml in a rotary evaporator, and passed through anion and cation exchange columns in order to remove Cl-, Na+, and other ions present. The anion exchange resin was AG3-X4, 100 to 200 mesh, chloride form (Bio-Rad Laboratories, Richmond, Calif.), converted to hydroxyl form with 0.5 N NaOH solution. The cation exchange resin was Dowex 5OW-X8, 100 to 200 mesh, hydrogen form (Bio-Rad Laboratories). These columns were packed in glass tubes 1.2 cm inside diameter and 15 cm long. Samples were eluted with distilled water, and fractions of 2 ml were collected. Fractions reacting positively with anthrone were pooled, and concentrated to a volume of 5 ml.

Fractionation of Products of Smith Degradation

A I- or 2.ml aliquot of the above solution was passed through a Bio-Gel P2 column, 400 mesh (Bio-Rad Laboratories; 1.3 cm inside diameter x 100 cm long), maintained at 50”, in order to separate mono- or oligosaccharides present. The sample was eluted with distilled water (7 ml/hour). Fractions of 1.5 ml were collected and tested for the presence of sugars with the anthrone reagent. The fractions that reacted positively with the reagent were pooled. A solution containing 2 mg each of glucose, maltose, raffinose, and stachyose was used as standard in order to define the fractions corresponding to particular molecular weights. The void volume of the column was determined with arabinogalactan (Pfaltz and Bauer, Inc., Flushing, N.Y.). Because all of the sugar content of the reaction mixture came in the void volume of the column, the steps using ion exchange resins and gel filtration were subsequently replaced with dialysis against distilled water.

The fractions collected from the Bio-Gel P2 column, following pooling, or, alternatively, dialyzed portions, were dried in a rotary evaporator. The dried material was acidified with 0.5 ml of 0.05 N

acetic acid, and then washed with absolute methanol and evaporated to complete dryness at 60” several times in order to remove the borate ions present as a result of the NaBH, reduction. The dried polysaccha- ride was resuspended in 2 ml of distilled water and lyophilized. Sugar composition analysis and methylation analysis of this polysaccharide material were performed as described above.

Partial Acid Hydrolysis of Backbone Polysaccharide

Fifteen milligrams of backbone were hydrolyzed with 1 ml of either 0.5 N trifluoroacetic acid or 0.5 N H,SO, at 100” for 30 min in sealed vials. The hydrolysis was stopped by neutralization of the solution with 5 N NaOH. The neutralized solution was passed through the Bio-Gel P2 column as described above, except that l-ml fractions were collected. The tubes corresponding to oligosaccharide fractions of the same molecular weight were pooled, dried at 60”, and stored in uacuo at 60”.

Paper Electrophoresis

Different oligosaccharides of the same molecular weight were separated on Whatman No. 3MM paper by means of an E-C 453 pressure plate paper electrophoresis apparatus (Milton Roy Company, St. Petersburg, Fla.), 20 x 45 cm, at 20 V/cm. The electrophoresis apparatus was connected to a Lauda K-2/R water-circulating pump (Lauda Instruments Division, Brinkmann Instruments Inc., Westbury, N.Y.) maintained at 5”. The buffer was 0.2 M sodium borate, pH 10. For qualitative analyses, the paper was cut in strips 6.3 cm wide and 60 cm long. For quantitative analysis and recovery of oligosaccharides, the paper was cut in sheets 20 cm wide and 60 cm long. The paper was equilibrated with buffer solution for 1 hour before application of

samples. Twenty-five microliters of each sample were applied on the strips (6.3 x 60 cm) and 250 ~1 on the sheets (20 x 60 cm). The duration of electrophoresis was 6 hours for the disaccharides, 10 hours for the trisaccharides, and 14 hours for the tetrasaccharides.

Oligosaccharides were detected on the paper strips by means of silver nitrate-alcoholic sodium hydroxide (18). Oligosaccharides local- ized by this means (reaction at the margin of the sheets (20 x 60 cm)) were eluted with distilled water for 24 hours. The solution containing an oligosaccharide was concentrated to 1 to 2 ml and passed through the anion and cation exchange columns described previously. The solution was then dried, acidified with 0.5 ml of 0.05 N acetic acid, and washed several times with methanol at 60” for removal of borate ions.

Analysis of Oligosaccharides

Four different analyses were performed in order to identify and to sequence the oligosaccharides: (a) sugar composition analysis; (b) methylation analysis; and (c) reducing end analysis, performed by reduction of the oligosaccharide with 1 to 2 mg of NaB3H, (185 mCi/mmol, New England Nuclear, Boston, Mass.) in 1 ml of 1 N

NH,OH solution. After 1 hour of reduction the excess of NaB3H, was destroyed with glacial acetic acid, and the oligosaccharide dried, and washed and dried several times with absolute methanol at 60”. The tritiated oligosaccharide was then hydrolyzed with 1 ml of 2 N trifluoroacetic acid for 1 hour at 121’ in a sealed vial. The products of hydrolysis were reduced with NaBH, in 1 N NH,OH. The resulting sugar alcohols were separated by paper electrophoresis in 0.05 M sodium borate solution, pH 9.2, at 20 V/cm (18) for 6 to 8 hours. Twenty-five microliters of the sample solution (concentration, approxi- mately 1 mg/ml) were applied to strips (6.3 x 60 cm) of paper (Whatman No. GMM) previously equilibrated for 1 hour with the buffer solution. The sugar alcohols were detected with silver nitrate- alcoholic sodium hydroxide reagent modified by the addition of 4% pentaerythritol (Eastman Kodak Co., Rochester, N.Y.) to the final 0.5 N NaOH alcoholic spray (18). The strip of paper was developed at the margins and the remainder of the paper was scanned for radioactivity in a model 7200 Radiochromatogram Scanner (Packard Inst. Co., Downers Grove, Ill.). (d) Analysis of the anomeric configuration of 1 + 3 linkages, by enzymatic hydrolysis of the three disaccharides, and the trisaccharide Glc + Glc + Glc, obtained by partial acid hy- drolysis of the backbones from the polysaccharides of heterocyst and spore envelopes. The enzymes and reaction conditions used were:

1. cu-Glucosidase partially purified from Saccharomyces cereoisiae (19; obtained from Sigma Chemical Co., St. Louis, MO.). This enzyme was used in a 0.1 M sodium phosphate solution, pH 6.8, at 37”. The enzyme was shown to be active on maltose (Pfanstiehl Laboratories, Inc., Waukegan, Ill.) and maltotriose (Aldrich Chemical Co., Inc., Milwaukee, Wise.), and inactive on cellobiose (Eastman Organic Chemicals, Rochester, N.Y.).

2. /3-Glucosidase purified from almonds (20; obtained from Sigma Chemical Co.), used in 0.1 M sodium acetate, pH 5, at 37’. The enzyme was shown to be active on cellobiose and inactive on maltose and maltotriose.

3. ol-Mannosidase purified from jack bean meal (21). This enzyme was used in a 0.05 M solution of sodium acetate, pH 4.5, at 37”. The enzyme was shown to be active on p-nitrophenyl-a-n-mannopyrano- side and mannosyl-a-(1 + 2).mannose and inactive on p-nitrophenyl- @-o-mannopyranoside. The enzyme and the nitrophenyl man- nopyranosides were a generous gift of Dr. Yu-Teh Li (Tulane Univer- sity, New Orleans, La.). The mannosylmannose was generously provided by Dr. Clinton E. Ballou (University of California, Berkeley, Calif.).

4. fl-Mannosidase purified from Polyporus sulfureus (22), also from Dr. Li. This enzyme was used in a 0.05 M solution of glycine. HCl, pH 3, at 37”. The enzyme was shown to be active on p-nitrophenyl-P-n-man- nopyranoside and inactive on p-nitrophenyl-n-n-mannopyranoside and mannosyl-a-(1 --* 2)-mannose.

5, 6. p-Mannosidase-containing fl-mannanases S339Q from Penicil- lium ochrochloron and S339K from Penicillium uerruculosum (23), obtained as a generous gift from Dr. E. T. Reese (United States Army Natick Laboratories, Natick, Mass.). These enzymes were used in 0.05 M sodium acetate solution, pH 4.5, at 50”. The enzymes were shown to be active on p-nitrophenyl-fl-n-mannopyranoside and inactive on p-nitrophenyl-cu-o-mannopyranoside.

1. Lx1 + 3))Endoglucanase S176N partially purified from Rhizopus arrhizus QM-1032 (24), also from Dr. Reese. The enzyme was used at 50”, in 0.01 M sodium acetate solution, pH 5.

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2970 Heterocyst and Spore Polysaccharides

One-half millicrram of enzvme (exceut for the mannosidases. which were provided by Dr. Li as solutions) -and 1 mg of Glc + Glc,’ Glc + Man. Man - Glc. or Glc - Glc - Glc, obtained bv oartial acid hydrolysis of backbone, were dissolved in 1 ml of the -appropriate buffer. The sample was shaken for 24 hours in a water bath at the temperature indicated above. The course of the enzyme reaction was assayed as follows: After 0, 3, 6, and 24 hours of incubation, lo-p1 aliquots of the reaction mixture were diluted with 0.99 ml of distilled water and tested by means of the Nelson test (25) for reducing end equivalents released.

Laminaribiose (glucosyl-&(I - 3).glucose) and nigerose (glucosyl- n-(1 + 3).glucose) were generously provided by Dr. Reese, and maltopentaose by Dr. A. Kivilaan (Michigan State University). Structures were modeled with CPK space-filling atomic models (Schwarz/Mann, Orangeburg, N.Y.).

RESULTS

Heterocyst and spore envelopes have the same sugar compo- nents and in the same amounts (Table I). Our results are very close to those reported by Dunn and Wolk (4) except that the amount of galactose found here is higher (6.3 i- 1.6% as compared with 3 + 1% reported previously).

The methylated polysaccharide was separated into chloro- form/methanol-soluble and chloroform/methanol-insoluble portions. The soluble and insoluble portions accounted for two-thirds and one-third, respectively, of the total polysaccha- ride content of the envelopes as estimated by the anthrone test. All of the sugar content of the insoluble portion became soluble in chloroform/methanol when remethylated, and in terms of methylation analysis was identical with the portion soluble in chloroform/methanol after the first methylation. Infrared spec- troscopy of the methylated polysaccharide showed no peak at 3 pm. Comparison with the spectrum of a 10% v/v solution of methanol in chloroform showed that a measurable peak would have been observed in the region 2800 to 3400 A if 2% of the hydroxyl groups which were originally free in the polysaccha- ride had remained unmethylated.

The ECNSS-M and OV-225 columns resolve 9 of the 11 partially methylated alditol acetate components of the enve- lopes (Fig. 1, a and c). The .2.2.4 column partially resolves two peaks at relative retention times of 1.33 and 1.35 (Peaks 4a and 4b, Fib. lb). The SP-2401 column partially resolves two other peaks in the region of terminal (T-) hexoses (Peaks 2a and 2b, Fig. Id).

Peaks 1 to 9 appear in the same order on the four columns (Fig. 1, a to d). Mass spectrometric analysis performed after chromatography with the OV-225 and SP-2401 columns identi- fied these peaks as: T-pentose (Peak I), T-hexose (Peak 2a), T-hexose (Peak 2b, SP-2401 column), T-hexose (Peak 3), 3-hexose (Peaks 4a and 4b), 4-hexose (Peak 5), 2,3-hexose

TABLE I

Analysis of sugar composition of envelopes of heterocysts and spores

Each mole per cent is a mean value * standard deviation, derived from three different preparations of envelopes. The corresponding mole percentages derived from methylation analysis (Table II) are presented in parentheses, for the sake of comparison.

Sugar

Xylose Galactose Mannose Glucose

Mole per cent of envelope polysaccharide

Heterocyst Spore

5.6 i 1.2 (3.6 i 0.6) 5.0 i 1.8 (4.5 i 0.7) 6.2 i 1.4 (8.1 + 1.0) 6.4 + 1.5 (8.2 zt 1.2)

22.4 i 2.0 (19.0 * 1.5) 23.8 i 2.5 (20.5 zt 2.0) 65.7 i 2.5 (69.6 i 2.3) 64.7 i 3.0 (66.8 zt 2.2)

(Peak 8), and 2,3,4-hexose (Peak 9). The molecular fragments of each of these partially methylated alditol acetate sugars are as reported by Lindberg (5). In the fragmentograms, 3-hexose and 4-hexose were easily distinguished because the methyl sugars had been reduced with NaBD,: the 3-hexose (Peak 4) gives fragments of m/e=161 and 234 whereas the 4-hexose (Peak 5) gives fragments of m/e= 162 and 233.

Gas chromatographic analysis identified the T-sugars. Peak 1 (T-pentose) is T-xylose, as shown by chromatographic identity with T-xylose present in xyloglucan and sycamore cell walls. Peak 2a (T-hexose) is T-glucose, as shown by chromato- graphic identity with T-glucose from maltose. Peak 2b (T-hex- ose) from column SP-2401 is T-mannose. It has the same retention time as T-mannose present in yeast mannan, and is chromatographically resolvable from T-glucose and T-galac- tose present in standard polysaccharides. Peak 3 (T-hexose) is T-galactose, as shown by chromatographic identity with T- galactose present in arabinogalactan and xyloglucan. Peak 5 (4-hexose) is 4.glucose, as shown by chromatographic identity with 4-glucose present in maltose, sycamore cell walls, and xyloglucan.

Each of the other partially methylated alditol acetate derivatives was identified by determining its retention times relative to T-glucose as described under “Materials and Methods,” using columns ECNSS-M, .2.2.4, and OV-225 (iso- thermal program, Fig. 1~). The relative retention times for all of the partially methylated alditol acetate components of the heterocyst envelope are identical, within experimental error, to those reported by Talmadge et al. (13) and Lindberg (5) for the same partially methylated alditol acetate derivatives and the same columns. The X-mannose peak (Peak 4b, Fig. lb) is resolved only by the .2.2.4 column. The mass spectrum of this peak is characteristic of a 3-hexose. Its relative retention time, 1.35, differs by only 0.02 unit from the relative retention time of S-glucose.

The mole per cent compositions of the partially methylated alditol acetate components of the heterocyst and spore enve- lope polysaccharides, determined by quantification of peak areas using the OV-225 column as described under “Materials and Methods,” are presented in Table II. Results essentially identical with those of Table II were obtained when the methylated, chloroform/methanol-soluble polysaccharide was subjected to the methylation procedure one and two additional times. The compositions of the polysaccharides from the envelopes of the two cell types are equal, within experimental error.

The amount of each sugar present in the methylated polysaccharide (Table II) accounts for the total amount of that sugar present in the envelopes as given by sugar composition analysis (Table I). For every mole of the doubly branched sugar, 2,3,4-glucose, there must be 2 mol of T-sugars. Hence, its mole per cent was added twice in the balance sheet, Table II, in which branches and terminals are compared.

Periodate oxidation of the polysaccharides is complete at 6 days, after which time no further decrease in absorption at 223 nm is observed. The entire sugar content of the reaction mixture passes through the Bio-Gel P2 column with the void volume. Smith degradation of 60 mg of dried heterocyst envelopes containing 55 mg of polysaccharide (based on sugar composition analysis of 1 mg of envelope material) yields 30 mg (approximately 55%) of a white lyophilized polysaccharide. The recovery from spore envelopes is lower: only 20 mg

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FIG. 1. Gas chromatograms of the partially methylated alditol acetate derivatives obtained from the polysaccharide of the heterocyst envelope. a, The ECNSS-M column was used for determinations of relative retention times; b, the .2.2.4 column was used for resolution of 5glucose and 3-mannose; c, the OV-225 column was used isother- mally (illustrated) for determinations of relative retention times, and was temperature-programmed for quantification of peak areas; and d, the SP-2401 column was used for separation of T-glucose and T-mannose. The programs used for the different columns are described under “Materials and Methods.” Peaks 1 to 9 are identified in Table II. Peak 10, 0acetyl inositol, the internal standard, comes at 180”, and is therefore not seen in a and c, which were performed isothermally at 155° and 170”, respectively. The mass spectrum of the unidentified

TIME ,m,nutar~

(d)

TABLE II

Mole per cent composition of hetwxyst and spore envelope polysaccharides

Each value presented is the mean + standard deviation, derived from methylation analysis of three different envelope preparations. Peak numbers refer to the peaks in the gas chromatograms of Fig. 1.

3 T-Galactose

4a Z-Glucose 4b SMannose 5 ,4-Glucose

6 2,3-Mannose 7 3,4-Glucose

8 3,6-Glucose

3.6 zt 0.6 22.0 * 1.0 39.9%

6.2 + 1.0 Terminals 8.1 i 1.0

10.6 i 0.6 7.0 i 0.6 30.5%

12.9 i 0.6 Linears

5.8 + 1.0 29.9% 5.3 * 0.8 + 12.8

6.0 + 1.2 42.7% 12.8 * 1.3 Branches

4.5 * 0.7 23.8 i 1.0 43.3% 6.8 + 1.5 Terminals 8.2 i 1.2

10.7 i 0.7 7.5 i 0.8

29.3%

11.1 * 0.8 Linears

6.2 * 1.0 21.4% 4.9 * 0.9 + 9.7

6.6 * 1.0 37.1% 9.7 A 1.0 Branches

(approximately 49%) of lyophilized material is obtained from

60 mg of dried spore envelopes, containing 41 mg of polysac- charide. Glucose and mannose, in molar ratio approximately 3/l (73.7 * 1.7Rl26.3 * 1.7% and 73.0 f 1.4%/27.0 i 1.4% in material derived from the envelopes of heterocysts and spores,

respectively) were found in these polysaccharides. Methylation analysis showed the presence of only two sugars: 1,3-linked glucose (3.Glc) and 1,3-linked mannose (3.Man). Because terminal sugars were not detected, branches accounted for less

than 2% of the sugars present. We therefore refer to these polysaccharides as “backbones” of their respective envelope polysaccharides.

The products of partial acid hydrolysis of the backbones, fractionated by means of a Bio-Gel P2 column, corresponded in their elution volumes to mono-, di-, tri-, tetra-, and pentasac- charides (Fig. 2). Thirty per cent of the total sugar content was found in the void volume of the column. When rehydrolyzed

under the same conditions, the residual 30% was broken down to mono-, di-, tri-, and tetrasaccharides (24%) and to higher oligosaccharides, the, great majority of which passed through the Bio-Gel P2 column after the void volume.

Di-, tri-, and tetrasaccharides having the same electropho- retie mobilities (Fig. 3) were obtained in very close to the same ratios from the backbones of spore and heterocyst envelope polysaccharides. Paper electrophoresis separated the disaccha- ride fractions from the envelopes of both types of cells into three disaccharides (Fig. 3), Di-I, -11, and -111, having, respec-

tively, the following electrophoretic mobilities: 0.243, 0.312, and 0.382 pm s1 (V/cm)-‘. The trisaccharide fractions were both separated into four trisaccharides (Fig. 3), Tri-I, -11, -111, and -IV, having electrophoretic mobilities of 0.125, 0.160, 0.201, and 0.243 Frn s-’ (V/cm))r. The two tetrasaccharide fractions were also separated into four components, Te-I, -11, -111, and -IV, having respective electrophoretic mobilities of

0.079, 0.109, 0.139, and 0.188 pm s’ (V/cm)-I. The disaccharides were identified by sugar composition

peak between Peaks 1 and 2 in the chromatogram taken with the .2.2.4 column lacks the fragment with m/e = 43 which is characteristic of partially methylated alditol acetate derivatives.

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2972 Heterocyst and Spore Polysaccharides

analysis (Table III) and by reducing end analysis as: Glc + Man (D&I), Man -+ Glc (Di-II), and Glc + Glc (Di-III). The trisaccharides were subjected to sugar composition analysis (Table III), methylation analysis (Table IV) and, where necessary, reducing end analysis (Fig. 4). Based on these analyses, the trisaccharides were identified as: Glc - Glc -+ Man (Tri-I), Glc -+ Man -+ Glc (Tri-II), Man ---f Glc -+ Glc (Tri-III), and Glc -+ Glc + Glc (Tri-IV). The tetrasaccharides were analyzed only for their sugar composition. All contain glucose and mannose, in molar ratio 3/l.

The disaccharides Glc + Man (Di-I; Fig. 5a) and Glc - Glc (Di-III; Fig. 5c) were hydrolyzed by P-glucosidase but not by a-glucosidase. Similarly, the trisaccharide Glc + Glc - Glc (Tri-IV) was completely hydrolyzed by P-glucosidase, but was not measurably hydrolyzed by a-glucosidase (Fig. 5d). Man + Glc (D&II) was hydrolyzed by P-mannosidase, but not by ol-mannosidase (Fig. 5b). P-Mannosidase-containing P-manna- nases from both Penicillium ochro-chloron and Penicillium uerruculosum also readily cleave the linkage of this disaccha- ride. In confirmation of these results, it was found (a) that Di-III has the same electrophoretic mobility as laminaribiose, and is separable from nigerose by electrophoresis; and (b) that

‘I Void Volume

IO 20 30 40 50 60 70 60 90

Fraction Number FIG. 2. Fractionation, by chromatography on a column of Bio-Gel

P2, of the products of the partial acid hydrolysis of the backbone of the pplysaccharide from heterocyst envelopes. Fractions of 1.0 ml were collected and assayed for sugar with the anthrone reagent. The arrows show the peak fractions in which, in a prior run, glucose, maltose, maltotriose, stachyose, maltopentaose, and arabinogalactan (void volume) were eluted.

DISACCHARIDES

approximately 93% of the backbone from heterocyst envelope polysaccharide is hydrolyzed to mono-, di-, and trisaccharides in 1.5 hours by the /3(1 - 3)endoglucanase from Rhizopus arrhizus.

DISCUSSION

The results of methylation analysis of the polysaccharides from the envelopes of heterocysts and spores lead us to the following conclusions.

1. Xylose, galactose, mannose, and glucose occupy terminal positions in the polymers, whereas the internal portion of the polysaccharides is composed of mannose and glucose.

2. Forty-five to forty-eight per cent of the sugars, including all of the branched sugars and a majority of the linear sugars, in the polysaccharides have C-3 involved in a glycosidic bond.

3. The polysaccharides are highly branched. About 30% of the sugar residues are branched (Table II), and there is an unusually large amount (10 to 13%) of a doubly branched sugar, 2,3,4-Glc. Doubly branched sugars have been found in small amounts in other polysaccharides (13). Five types of evidence, taken together, demonstrate that the polysaccha- rides are completely methylated, so that the high frequency of appearance of branched and doubly branched sugars is not an artifact of undermethylation. First, the chromatograms (Fig. 1, b and d) show no peaks corresponding to unmethylated alditol acetate derivatives. If the polysaccharides had been only partially methylated, small peaks of 0-acetylmannitol, -galac- titol, and -glucitol would have been expected, with retention times of from 1 to 3 min less than 0-acetylinositol. Second, the infrared spectrum of the methylated polysaccharide shows no absorption, attributable to free hydroxyl groups, at 3 Grn. Third, the results of quantitative analysis are closely reproduc- ible (Table II). Fourth, remethylation did not increase the level of methylation of the chloroform/methanol-soluble polysaccha- ride; in particular, the amount of doubly branched sugars did not change significantly. Fifth, the amount of terminal sugars balances the amount of branched sugars (Table II).

4. Both polysaccharides have the same components and in approximately the same proportions.

We have analyzed these polysaccharides further by examin- ing the products of their chemical degradation. We began with Smith degradation (16) because this procedure, which involves periodate oxidation, would not degrade sugars linked by C-3 bonds which comprise about 47% of the sugar residues in the envelope polysaccharides. Sugar residues with vicinal hydroxyl

TRISACCHARIDES TETRASACCHARIDES

HETEROCYST SPORE HETEROCYST SPORE HETEROCYST SPORE FIG. 3. Paper electrophoretograms of

oligosaccharides obtained by partial acid hydrolysis of the backbones from heterocyst and spore envelope polysac- charides. In order of increasing electro- nhoretic mobilitv are found disaccha- rides Di-I, -11, and -111; trisaccharides Tri-I, -11, -111. and -IV: and tetrasaccha- rides Te-I, -11, -111, and -IV. The arrow marks the origin.

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Heterocyst and Spore Polysaccharides 2973

TABLE III

Summary of analyses performed on the oligosaccharides derived by partial acid hydrolysis of heterocyst-polysaccharide backbone

Ratios between the amounts of the different oligosaccharides of a given chain length were calculated, following elution of the oligosaccharides from electrophoretograms, from gas chromatographic determinations of the amounts of their component sugars. Very similar ratios were obtained by quantifying the amounts of the eluted oligosaccharides by use of the anthrone reagent, with oligosaccharides derived from the backbone polysaccharides of both heterocysts and spores.

Analyses

Methods of analysis

Disaccharides

a. Sugar composition b. Reducing end (NaBaH,)

Backbone oligosaccharides

Trisaccharides

a. Sugar composition b. Reducing end c. Methyl&ion analysis

Tetrasaccharides

Sugar composition

Sequence Di-I = Glc *Man Di-II = Man + Glc Di-III = Glc - Glc

Ratio

1.0 1.1 1.6

Tri-I = Glc + Glc + Man Tri-II = Glc + Man + Glc Tri-III = Man + Glc + Glc Tri-IV = Glc + Glc --t Glc

Ratio

1.0 1.0 1.0 1.0

Te-I Te-II Te-III Te-IV

All have Glc/ Man = 3/l

Ratio

1.1 1.0 1.0 1.0

TABLE IV

Approximate relative peak areas in gas chromatograms (‘l.2.2.4” column) of partially methylated alditol acetate derivatives from each of trisaccharides isolated, by paper electrophoresis, from backbone of

heterocyst-envelope polysaccharide

The identification of the nonreducing terminal sugars was based in part on the results of sugar composition analysis.

Trisaccharide T-Glc T-Man 3.Glc 3.Man

I 1 0 1 1 II 1 0 1 1 III 0 1 2 0 IV 1 0 2 0

groups, such as terminal sugars, or internal sugars linked at positions 1 and 4, or at positions 1 and 2, would, however, be oxidized. The dialdehyde sugar derivatives which were ob- tained by periodate oxidation (26-28) were then converted to polyalcohols by means of reduction with sodium borohydride. The linkages between the degraded and the nonoxidized sugars were then cleaved selectively by mild acid hydrolysis (29).

Because the only sugar-containing products of Smith degra- dation of heterocyst and spore envelope polysaccharides are found in the void volume of the Bio-Gel P2 column, those products have a molecular weight in excess of 1800, i.e. contain at least 11 sugar residues. When those products were subjected to methylation analysis, terminal sugars were undetectable, i.e. present at a frequency of less than 2% of the total sugar content. The Nelson test showed the presence of 0.2% reducing end sugar residues. Due to the limited solubility of those products, this test may have underestimated their content of reducing sugars. We conclude that the polysaccharide products of Smith degradation of the envelope polysaccharides contain between 50 and 500 sugar residues. Because branched sugars were also not detectable, the polysaccharide products consti- tute the linear, internal portions, or backbones, of the highly branched envelope polysaccharides from which they were derived.

Methylation analysis showed the presence of only 3-linked glucose and 3-linked mannose in these backbone polysaccha- rides. The observed ratio .of glucose to mannose was 2.8/l for the backbone polysaccharide derived from heterocyst enve- lopes and 2.7/l for the corresponding backbone from spores. These ratios are in fairly good agreement with the ratios (2.7/l

+

t-1

TRISACCHARIDE II

+ -

I I

25 15 5 Distance (cm)

FIG. 4. Reducing end analyses of trisaccharides Tri-I and -11. Bands of glucitol and mannitol are shown at the base of each electrophoreto- gram. NaBaH, has reduced a mannosyl residue in Tri-I and a glucosyl residue in Tri-II. Tri-III and Tri-IV were not subjected to reducing end analysis because Tri-III lacks 3-Man (Table IV) and Tri-IV lacks mannose, so that both trisaccharides must have glucose at the reducing end.

and 2.3/l, respectively) between total 3-linked glucose and total S-linked mannose from the intact polysaccharides from heterocyst and spore envelopes, as given by methylation analysis (Table II), and are consistent with a 3/l ratio of glucose to mannose in the backbone polysaccharides.

Because 4-Glc is broken down by the Smith degradation

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2974 Heterocyst and Spore Polysaccharides

procedure, and because oligosaccharides are not recovered,

4-Glc cannot be a frequent constituent of the backbone of the original polysaccharide. The absence of branched sugars fol- lowing Smith degradation implies that in the intact polysac- charides, the branches consist of T-sugar linked to the back- bone either directly or through the 4-Glc residues. Although we do not know whether sequences of seriate 4-Glc residues are present in the intact envelope polysaccharides, the stoichiome- try of4-Glc to total X-linked mannose (Table II) suggests that a single 4-Glc residue may be a constituent in a basic subunit

(see below) of the intact polysaccharides, and would be present in a side branch.

As shown by methylation analysis of the products of Smith degradation, all T-sugar and 4-Glc residues had been de- graded. On the other hand, there appears to have been negligible degradation of other portions of the original envelope polysaccharides. Thus, about 53% of the original polysaccha- ride from heterocyst envelopes is comprised of T-sugars plus 4-Glc (Table II), whereas about 45% of the polysaccharide originally present is lost during the Smith degradation proce- dure. The lower recovery of backbone from spores, as per cent of envelope material, is consistent with the higher content of

amino compounds in their envelopes (4). The presence of both glucose and mannose in oligosaccha-

rides derived from partial acid hydrolysis implies that the

backbones do not consist of separate glucan and mannan chains, and the fact that no di-, tri-, or tetrasaccharide contains 2 mannosyl residues implies that the backbones do not consist of a random distribution of glucosyl and mannosyl moieties. The presence of a single mannosyl residue within each tetrasaccharide implies that the backbones consist of a repeating unit Glc - Glc --t Glc + Man, an interpretation which is consistent with the structures and relative frequencies

TIME Cllrsl TIME (iTsI

FIG. 5. Enzymatic hydrolysis of disaccharides Di-I, -II, and -III, and trisaccharide Tri-IV isolated following partial acid hydrolysis of the backbones of the polysaccharides from the envelopes of heterocysts (circles) and spores (triangles). The appearance of reducing end groups, as per cent of total sugar residues present, is shown as a function of time of hydrolysis of a, Glc - Man @-I); c, Glc - Glc (Di-III), and d, Glc ~+ Glc - Glc (Tri-IV) by @-glucosidase from almonds (0, A) and by niglucosidase from yeast (0, A); and as a function of time of hydrolysis of b, Man - Glc (Di-II) by fl-mannosi- dase from Polyporus sulfureu~ (0, A) and by a-mannosidase from jack beans (0, A).

of di- and trisaccharides which were observed (Table III). Because Glc + Man, Glc + Glc, and Glc + Glc + Glc are completely hydrolyzed by /I-glucosidase and not measurably by a-glucosidase, and because Man - Glc is hydrolyzed by @-mannosidase but not by oc-mannosidase, we conclude that all of the sugars in the backbones are P-linked.

We have found no differences between the envelope polysac- charides from heterocysts and spores. As we have shown here, the backbone of both consists of p(1 + 3).linked glucose and mannose with a repeating sequence of Glc + Glc - Glc -Man. Moreover, all of their other glycosidic linkages are present in the same frequency, within experimental error. Although it remains to be determined whether or not the branches are linked with the same anomeric configurations to the same

sugar residues in the backbones of the two polysaccharides, the evidence available to date seems sufficient to suggest, as a working hypothesis, that the envelope polysaccharides from the two types of differentiated cells are essentially identical. We propose that, if the polysaccharides from the two sources are essentially identical, their structures resemble, to a first approximation, the structure presented in Fig. 6. The structure as presented, and its fully methylated derivative, have been

shown to be sterically possible, using space-filling atomic models. Only a portion of the mannosyl residues in the back- bone is substituted, and certain of the terminal sugars are present in amounts not stoichiometric with the backbone sugars. Therefore, even if the sequence of substitutions of glu- cosyl residues in the backbone were correct, the “basic” structural unit illustrated would not be, in the strict sense, a “repeating” subunit. It is also possible that the envelopes con- tain a mixture of slightly differing polysaccharides, each of which has a strictly repeating subunit.

Whether or not these envelope polysaccharides are produced, in small amounts, by vegetative cells is unknown. The vegeta-

c T-S 0

FIG. 6. Possible structure of the polysaccharides from the envelopes of heterocysts and spores of Anabaena cylindrica. Linkages to terminal sugars (T-S) indicated by dashed lines can he present in only certain of the “basic” units illustrated. On the basis of approximate stoichiom- etry (Table II), it may be suggested that the irregularly appearing substituent on the mannosyl residue in the backbone is T-Man; that the 2,3,4-Glc in the polysaccharide has, as regular substituents, T-Glc and (linked to the C-2 or C-4) Glc-(1 - 4).Glc; and that the irregularly appearing substituents on the other two backbone glucosyl residues are, to a large extent, galactose at position 6 and xylose at position 4. Because the sequence of substitutions of backbone glucosyl residues is unknown, an arbitrarily chosen sequence is illustrated. Unlinked carbon atoms 6 are omitted from the drawing for the sake of simplification.

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Heterocyst and Spore Pol.ysaccharides 2975

tive cells of our cultures, in contrast to those grown in more concentrated inorganic growth medium (4), do not produce evident sheath material. Sheath polysaccharide, when formed, is much richer in xylose (21%) and poorer in glucose (SO%), than are the envelope polysaccharides of the differentiated cells, and also contains a trace of fucose (4). In addition, the wall of a vegetative cell contains only between 1.6 and 3.2% as much glucose as does the envelope of a heterocyst (4, 30). The heterocyst envelope is deposited in approximately one-fifth of a vegetative cell doubling time (30). Thus, if a vegetative cell forms a polysaccharide comparable to that in the envelope of heterocysts, it does so at a rate less than 1% as rapid as the rate of synthesis of the polysaccharide in a heterocyst. It follows that if the polysaccharides in the envelopes of heterocysts and spores are identical, it is probable that the same set of polysaccharide-synthesizing enzymes is produced or activated during the alternative differentiation processes. Such a finding would be consistent with the idea (2, 3) of evolutionary and ontogenic relationships between the two processes.

AchnoLuZedgments-Dr. Wolfgang D. Bauer, who was to have been co-author of the portion of this paper dealing with methylation analysis of the intact polysaccharides, generously

withdrew his name in response to the editor’s stipulation that the original pair of manuscripts be combined. We are very grateful to Dr. Bauer for introducing us to the methods of methylation analysis, and for his comments on the manuscript. Substantial help and advice were received from the research group headed by Professor C. C. Sweeley of the M.S.U. Department of Biochemistry. In particular, the help of Mr. Jack Harten, who ran all of the mass spectra, is warmly acknowledged. We thank Doctors Yu-Teh Li, E. T. Reese, C. E. Ballou, and A. Kivilaan for enzymes, glycosides, and oligosaccharides.

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L Cardemil and C P WolkMethylation analysis and structure of the backbones.

The polysaccharides from heterocyst and spore envelopes of a blue-green alga.

1976, 251:2967-2975.J. Biol. Chem. 

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