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Study of the structure of Shigella flexneri o antigen I. Chemical degradation
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
155
26272
STUDY OF T H E S T R U C T U R E OF S H I G E L L A F L E X N E R I
0 ANTIGEN
I. CHEMICAL D E G R A D A T I O N E. H A N N E C A R T - P O K O R N I ,
D. D E K E G E L ,
F. D E P U Y D T
AND J. D I R K X
Institut Pasteur du Brabant, Brussels (Belgium) (Received S e p t e m b e r I l t h , 1969)
SUMMARY
The lipopolysaccharide antigen of Shigella flexneri F6S serotype 5 appears under the electron microscope as long filaments having a molecular weight of IO. lO645" lO6. This macromolecular structure is built of repeating units having a sedimentation constant, S2o,w, of lO.2 S and a molecular weight of 250000 and is linked together by hydrophobic interaction, due to the lipid part of the molecule and b y bivalent cations and carboxylic groups. These unit antigen structures are themselves polymerization products built of 1.44-S subunits linked together by weak chemical bonds of the same nature as those binding the units in the antigen molecule. The nature of the degrading agent seems to be important for obtaining the subunit fragments. Some preliminary experiments indicate that the IO.2-S unit antigen structures still retain their biological properties such as phage-receptor function and toxicity.
INTRODUCTION
During recent years, somatic antigens of enterobacteriaceae have been analysed intensively as to their chemical nature and biological properties. These studies mainly concerned two fundamental aspects of the biological nature of these antigens: which part of the molecule would be responsible for their toxic behaviour 1-5 and what fundamental building block constitutes the complicated macromolecular structures of these antigens. Many authors tried to isolate a fundamental toxic unit of somatic antigens b y carefully controlled acid or alkaline hydrolysis. The somewhat severe methods (high or low p H values, high temperature) certainly destroy covalent bindings and therefore surely alter the steric arrangements of the antigens. Two groups of workers were able to degrade their antigenic material without hydrolysis and found the fractions to differ in toxicity and molecular weight. Thus OROSZLAN AND MORA6 dissociated Serratia marcescens antigen with sodium lauryl sulphate into a nontoxic fraction having a sedimentation constant of 1.5 S. BEER AND BRAUDE7 used the same detergent on Escherichia coli antigens and isolated two toxic fractions of 9 S (30 %) and 3 S (60 %) WeS, 9 also found that different detergent substances and bovine serum albumin were able to dissociate the somatic antigen of ShigelIaflexneri into different fragments, Biochim. Biophys. Acta, 2Ol (197 o) 155-166
156
E. HANNECART-POKORNIctal.
the smallest of which had a sedimentation constant, s°2o,w of lO.2 S. Heating in the presence of citrate buffer (pH 7.3) also yielded particles of lO.2 S which were still toxic and therefore comparable to the 9-S fractions of BEER AND BRAUDE7. RUDBACH et al. 1°, using Salmonella typhosa antigen, and BEER AND BRAUDE7, using E. coli antigen, obtained 1.5-S fragments after treatment with deoxycholate and 9-S fragments by the action of organic solvents. Quite obviously the antigens and their degradation products must also be examined from the point of view of their antigenic activity and toxicity. BEUMER et al. n-~4 studied the receptor function of the somatic antigen of Shigella flexneri for different phages to which these bacteria were sensitive. This study is mainly concerned with an analysis of the macromolecular structure of the Shigella flexneri antigen. For the sake of clarity, we propose to call the smallest particle still showing a toxicity of the same order as the complete antigen a fundamental unit and to call any fragment smaller than this fundamental unit which has lost this toxic property a subunit. MATERIALS AND METHODS
Extraction of antigen The glucidolipidic antigen of Shigella flexneri serotype 5 was prepared according to BOlVlN AND MESROBEANU15-17 from bacterial cultures in BERTANI'STM LB medium at 4 °.
Purification, chemical treatments and analysis Unless otherwise stated, all denaturing and degradation reactions were carried out in media containing o.15 M NaC1 plus o.o15 M sodium citrate (pH 7.3). The reaction between the antigen and sodium deoxycholate was made following RIBI et al. 1~. Protein contents were estimated by the method of LowRY et al. 2° using a serum albumin standard. Pepsin and trypsin digestions were done as described previously s. The procedure of NOMOTO et a l Y was followed for the pronase digestion. Deoxyribonuclease and ribonuclease products were analysed by the spectrophotometric method of KUNITZ22,23. Gel filtration was used as a means of separating nucleic acid compounds from the antigen. The antigen preparation was passed onto a 2.5 cm x 50 cm column of Sephadex G-2oo which had been equilibrated with o.I M NaCI. The antigen was eluted with increasing NaCI concentration (o.i-i M) at a speed of 2o ml/h, 5-ml fractions being collected. Adsorption spectra of glucidolipidic antigen and degradation compounds were obtained in distilled degassed water. This procedure allows the spectrum to be taken down to 21o m/~ without interference from absorption by phosphate or citrate buffer. The Beckman DB recording spectrophotometer was used for the different spectrophotometrical analyses. Alkaline hydrolysis was carried out with a solution of 4 % NH2OH in alcohol (5 rain at 65 °), while the acetic acid hydrolysis was done as described previouslys. Thin-layer chromatography on silica gel and cellulose was run using aliquots after a 4-h hydrolysis at IOO° in I M HCI neutralised to pH 7. Different solvents and detectors were used. (a) Neutral sugars; elution solvent, double e]ution with a mixture
Biochim. Biophys. Mcta, 2Ol (197o) I55-166
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of ethyl aeetate-isopropanol-water (6.5:2.4:1.2, by vol.); detector solution, 3 % p-anisidine HC1 in n-butanol. (b) Nucleic acid compounds; elution solvent, butanolacetic acid- 5 % ammonia-acetone-water (3.5:1.5:1.5:2.5 : I, by vol.) ; detector, ultraviolet. (c) Amino acids; elution solvent, n-butanol-acetic acid-water (3:1:I, by vol.) ; detector solution, 0.2 % ninhydrin in n-butanol.
Physicochemical determinations Sedimentation velocity determinations of the glucidolipidic antigen were performed in a Spinco model E ultracentrifuge. The observed sedimentation coefficients reported in Svedberg units (i S = i . l O -13 cm/sec per unit field) were corrected to values corresponding to a solvent having the viscosity and density of water at 20 °. The molecular weight and the density of glucidolipidic antigen and unit lO.2 S are determined by centrifugation with schlieren optics in density gradient of CsC1 (starting density 1.4o89) for 18 h at 5074 ° rev./min using the double-sector capillarytype cell of 12 mm with an Epon centerpiece 2 ° sector. To prepare specimens for electron microscopy, the glucidolipidic antigen was suspended in distilled water at a concentration of I.O mg/ml. Carbon formvar-coated grids were floated film downwards on the surface of the suspension for a few minutes, removed and sucked dry with filter paper and shadowed with platinium-iridium at an angle of 3°o . RESULTS
Antigen purification A crude glucidolipidic antigen preparation, as seen in the electron microscope, shows (Fig. I) spherical and rod-like particles of different lengths. This polymorphism is a rather serious obstacle to any physicochemical measurement. We tried to purify the antigen which shows a great tendency to adsorb foreign substances and found the morphological heterodispersity to vary considerably with the presence of contaminating proteins. According to the extraction procedures, these
Fig. I. Laterally shadowed crude glucidolipidic antigen preparation showing rod-like particles and spherical particles of different diameters. Magnification, 40000 ×,
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158
protein values m a y be from 5 to 7 %; antigen preparations with the highest protein content also show a higher percentage of small particles in the electron microscope. We applied different protein extraction methods such as phenol extraction, enzymatic digestion and ultraeentrifugation. Although none of these procedures ever resulted in a complete elimination of proteins, we found the enzymatic digestion with trypsin and pepsin (Table I) to give the best purification. The 2 % remaining protein material seems to be firmly bound to the lipopolysaccharide part of the antigen molecule and m a y be either proteins, peptides or amino acids. Since ultracentrifugation also eliminates foreign material in excess of the 2 % which remain firmly bound to the lipopolysaccharide, we m a y assume this protein to be foreign to the antigen. As shown in Table II, different antigen fractions obtained by various chemical treatments still contain 2.5 % protein material, which was not further identified, except for the absence of aromatic amino acids. Together with the 2 % protein material, which in fact m a y be either proteins, peptides or amino acids, the glucidolipidic antigen also contains nucleic acid components (3-4 %). The ultraviolet spectrum of glucidolipidic antigen shows a slight absorption band at 260 m # (Fig. 2) which is more intense in both polysaccharides obtained after careful hydrolysis. A more severe hydrolysis (I M HC1 during 4 h at ioo°), gives even more intense absorption bands (Fig. 3). The hyperchromicity is too high to be only due to a denaturation of a polynucleic acid structure and an unmasking of nucleic acid components must take place. We tried without success to separate these hypothetical nucleic acid compounds absorbed in the glucidolipidic TABLE
I
TREATMENT
OF
GLUCIDOLIPIDIC
Methods
ANTIGEN
BY
DIFFERENT
DEPROTEIN1ZING
% of protein material before purification
% of protein material after purification
A. E n z y m a t i c r e a c t i o n I. P r o n a s e 2. P e p s i n - T r y p s i n
5.7 5.7
3.3 2.22
B. C e n t r i f u g a t i o n 4oooo r e v . / u l i n 2 × I h
5.7
3.44
TABLE II PROTEIN CONTENT
Protein material (%) G l u c i d o l i p i d i c a n t i g e n from different b a t c h e s Glucidolipidic antigen after purification
5.7, 7, 6, 5.4, 5.6, 5.8 2.3, 2. 7, 2.4, 2.1, 1. 7
P r o t e i n c o n t e n t of g l u c i d o l i p i d i c a n t i g e n f r a c t i o n s s°20, w = 17o S 2. 7 8o20, w =
10.2
G
2. 3
8°20, w =
9 I
S S
2.42 2. 3
Biochim. Biophys. dcta, 2Ol (197 o) 155-166
AGENTS
Shigellaflexneri 0 ANTIGEZ~'. I
159
antigen by passage through a Sephadex G-200 column. Nevertheless we cannot eliminate the possibility of an absorption unless a passage through an ion-exchange column is carried out. BARKER et al. 24however succeeded in separating nucleic acid compounds from polysaccharides on Sephadex G-2oo. Glucidolipidic antigen and the polysaccharides, which are smaller structures and therefore more accessible to enzymes, have been subjected to ribonuclease and deoxyribonuclease. No effect was observed in the ultraviolet spectrum on the elution pattern on Sephadex G-2oo.
!
5
3 o ~2
1 ~210
260
310
210
2603101
mJ~
Fig. 2. Absorption spectra of different glucidolipidic antigen constituents (0.2 mg/ml) in degassed distilled water. O - - O , glucidolipidic antigen; 0 - - 0 , lO.2 S; / x - - & , hydroxylamine polysaccharide; A - - A , acetic acid polysaccharide. Fig. 3. Absorption spectra of different glucidolipidic antigen constituents (0.2 mg/ml) after acid hydrolysis for 4 h. O - - C ) , glucidolipidic antigen; 0 - - 0 , lO.2 S; /X--/x, hydroxylamine polysaccharide; • - - A , acetic acid polysaccharide.
The presence of nucleic acids seems therefore rather improbable, but nucleotide compounds m a y be responsible for the 26o-m# absorption band. Thin-layer chromatography showed cytidine to be responsible for the largest part of the nucleotide compounds present. If cytidine really is bound to the antigen molecule, it seems reasonable to suppose that this nucleotide is acting in a way similar to UDP-galactose in the case of Salmonella 25, as a cofactor in the transfer of glucidic units during the biosynthesis of somatic antigens in Shigella flexneri. The fact that glucidolipidic antigen contains ketodeoxyoctonate, as established lately b y DEBUYSSCHER 2e, m a y indicate the possibility of its transfer through CMP-ketodeoxyoctonate as described b y HORECKER27. We also found the antigen to contain adsorbed f a t t y acids which are easily extracted in a Soxhlet by cold chloroform.
Study of the unit The antigen is seen in the electron microscope as long filaments (Fig. 4)- According to the nature of the nutrient medium, liquid or solidified agar or to the presence of protein material, glucidolipidic antigen has a molecular weight of IO. lO645" lO6, suggesting a presence of aggregates as usually observed with somatic antigen from enterobacteria. The chemical nature as well as the high molecular mass of t3iochim. Biophys. Acta, 2oi (197 o) i 5 5 - i 6 6
16o
E. HANNECART-POKORNI Ct al.
glucidolipidic antigen suggest furthermore the possibility of the existence of a repeating unit polymerised by weak chemical bounds. Such links may be of a different nature. (a) Ionic bonds due to the presence of fl-hydroxymyristie acid possibly linked to the macromolecular building block by the alcohol radical, leaving the carboxylic radical free (as shown by electrotitration) 2s. All phosphoric acid radicals seem to be esterified since the antigen has but a low electrophoretic mobility. (b) Hydrogen bonds due to the many OH groups of sugar radicals. (c) Hydrophobic bonds due to the longchain (C1~ C16) fatty acids. Various chemical substances were used to destroy the low-energy bonds of glucidolipidic antigen (Table II) giving fractions of 30, 21 and IO S sedimentation constants. Ionic bond- and hydrogen bond-rupturing agents did not interfere with fragmentation. Moreover their action varied with different antigen preparations. Glucidolipidic antigen is sensitive only to high pH values because of hydrolysis and saponification of the ester bonds. Detergent substances had an immediate effect on hydrophobic bonds resulting eventually in io-S fragments. Bovine serum albumin degraded glucidolipidic antigen at much lower rate and only at temperatures over 4 °° (Fig. 5). Heating of glueidolipidic antigen in citrate buffer resulted in a nonreversible degradation. The different degradation products were quite comparable to those obtained by denaturing agents (Table III). This technique of nonreversible degradation permitted an easy isolation of the different fractions. Furthermore it is quite obvious
Fig. 4- Laterally shadowed purified glucidolipidic antigen preparation showing essentially long filaments of equal diameter. Magnification, 40000 ×. Fig. 5. Laterally shadowed detergent-(cetavlon 5 mg/ml)-treated glucidolipidic antigen preparation showing lO.2 S spherical particles. Magnification, 40000 ×. Biochim. Bio~ghvs. Acta, 2Ol (197o) 155 166
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that any detergent substance present in the isolated fractions would seriously interfere with the study of immunological and phage-receptor properties of the antigen fractions. Degradation of glucidolipidic antigen may be obtained by heating (Fig. 6), the original II3-S fractions being progressively supplanted by the 3o-S fraction. At about 9°o the II3-S fraction disappears completely and a new 2I-S fraction appears together with the 3o-S fraction. At about IOO° a third transition is obtained giving rise to the IO.2-S fraction coexisting in small quantities with the residual 2I-S fraction. Any further heating at ioo ° has no effect on the io.2-2I-S equilibrium. A complete transformation into IO.2-S units may be obtained by heating at TABLE III VARIATIONS
IN
THE
PRESENCE
OF
DENATURING
Denaturing agents~
Fractions of glucidolipidic antigen
NaC1, 0. 3 M NaC1, i M Guanidine, 5 M Glucose, 5 % B o v i n e s e r u m a l b u m i n , o.I % Mechanical (Ultra-turrax) Heating Heating Poxesmol; o.i, I and 2 % B o v i n e s e r u m a l b u m i n ; (0. 5 a n d i %), 4 o°, 48 h Heating Mechanical (Ultra-turrax) Heating * I n o.15 M NaC1 plus O.Ol 5 • 5 mg/ml.
100
AGENTS
3° S
s%0.w 21 S s°20,,, ~ IO S -
-
s°20. w = 9 S
% o/these fractions
43 45 67 25 65 91 o - i oo o-ioo ioo ioo o - i oo 9 o-ioo
c i t r a t e , p H 7.3. C o n c e n t r a t i o n of g l u c i d o l i p i d i c a n t i g e n is
--
~°
/ /
60
0
20
Ovo
8~)
90 TEMPERATURE
100
Fig. 6. H e a t d e g r a d a t i o n of g l u c i d o l i p i d i c a n t i g e n (5 m g / m l ) in o.15 M NaC1 plus o . o i 5 ~V~s o d i u m c i t r a t e (p H 7.3) a l i q u o t s of t h e h e a t e d m i x t u r e were t a k e n a t d i f f e r e n t t e m p e r a t u r e s a n d perc e n t a g e of differen t f r a c t i o n s d e t e r m i n e d in t h e u l t r a c e n t r i f u g e . O - - O , 113 S; ~ k - - $ k , 3 ° S; O--O, 20 S; I - - ' , lO.2 S.
Biochim. Biophys. Acta, 2Ol (197 o) 155-166
E. HANNECART-POKORNI el al.
162
12o ° during 25 min. If heating is continued the I0.2-S fragments are degraded into 9-S units, free sugar molecules and a precipitate of fatty acids. Obviously this hightemperature treatment destroys the chemical integrity of the antigen unit. Thus glucidolipidic antigen seems to be built of repeating io.2-S units essentially linked together by hydrophobic bonds, due to the lipid part of the molecule. This IO.2-S unit may be obtained by thermal degradation of glucidolipidic antigen in a citrate buffer. No degradation is observed when glucidolipidie antigen is heated in a phosphate buffer. It thus appears that citrate degradation of glucidolipidic antigen may be a combined effect of: (a) the aliphatic chain of the citrate molecule acting on the hydrophobic links; (b) the OH group, a tertiary alcohol group; and (c) the three carboxylic groups acting ionically or by chelation. We therefore experimented with different substances having some of these characteristics alone or in combination (Table IV). It appears that neither the aliphatic chain nor the OH groups would degrade the antigen molecule. Molecules with one carboxylic group are without any effect. TABLE IV THERMIC DEGRADATION OF GLUCIDOLIPIDIC ANTIGEN WITH DIFFERENT AGENTS All s u b s t a n c e s w e r e d i s s o l v e d at a c o n c e n t r a t i o n of o.oi 5 M in o. 15 M NaC1 a n d b r o u g h t to pH 7.2 w i t h NaOH.
Substance
trormula
Sodium chloride Hexane
NaC1 CH3-(CH,~ ) 4-CHa (CH3) 3-COH H3PO 4
tert.-Butanol P h o s p h o r i c acid
Degradation, glucidolipidic antigen
2.12
Formic acid A c e t i c acid Caproic acid S u c c i n i c acid
H-COOH CHa-COOH CH3-(CH~)4-COOH HOOC-(CH2) 2-COOH
M a l e i c acid
HOOC-CH = CH-COOH
+
T a r t a r i c acid
HOOC-(CHOH)~-COOH
+
m
m
+
OH Citric acid
I
HOOC-CH2-C-CH~-COOH
+
I
COOH HOOC-(CH2)5-COOH
+
EDTA
C6H~COOt-I NH 2 CbH4-COOH (NO~) a-C6H~-OH CH~N-(CH2-COOH )
+ + +
Glycine
CH~N-(CH2COOH) 2 NH2-CH2-COOH
P i m e l i c acid B e n z o i c acid p - A m i n o b e n z o i c acid Picric acid
i
JBiochim. Biophys. Acta, 2Ol (197 o) 155-166
pK
+
7.21 12.67 3.75 4.78 4.83 4.6 5.61 1.83 6.07 2.98 4.34 3,o8 4"74 5-4 4.71 5-42 4.19 4.92 0.38 3.o3 4"44 2.34 9,69
Shigdlaflexneri 0 ANTIGEN. I
163
Molecules with two carboxylic groups and acids with aromatic radicals used in identical conditions are listed in Table IV. We m a y consider different characteristics of the molecules. (i) Polarity has no effect in itself, since the salt concentration is io times higher than the buffer concentration. Moreover, if the dipole m o m e n t of the molecule would be important, a degradation of glucidolipidic acid in the presence of trinitrophenol, which has a very low dipole moment, would not take place. (ii) I n the ionic form, the p K of different buffer molecules is not essential but the presence of two close anionic groups is a prerequisite to obtain the unit fragment. This would account for the variable activity of pimelic acid. Normally both carboxylic groups of pimelic acid are far a w a y from each other but thermic agitation m a y bring t h e m close enough to each other to act on the glucidolipidic antigen molecule. We therefore think that the chelating p r o p e r t y of the buffer substances is mainly responsible for the thermic degradation of glucidolipidic antigen. This fact suggests t h a t glucidolipidic antigen is built of smaller unit structures having a sedimentation constant of lO.2 S polymerized by hydrophobic links and bivalent cations except copper (glycine, a copper-chelating agent has no effect on the antigen). Stu:tv of the subunit RUDBACH et al. 1° succeeded in degrading the antigens of various gram-negative bacteria directly into I-S fragments b y sodium deoxycholate. Our antigen preparation resisted any further degradation than the I0.2-S unit even when the reagents such as citrate plus poxesmol were used at I 0 times higher concentrations. An acetic acid hydrolysis of the I0.2-S unit structure, however, yielded a polysaccharide having a sedimentation constant of I S, corresponding to a molecular weight of about 9000. In the presence of sodium deoxycholate our antigen is degraded into I0.2-S unit fragments (Fig. 7) which are further transformed into 1.44-S subunits (Fig. 8) when the sodium deoxycholate concentration is increased.
Fig. 7- Sedimentation pattern of glucidolipidic antigen (7.5 mg/ml) solution containing IO mg/ml sodium deoxycholate. 2 peaks at 1.44 S and lO.2 S. Fig. 8. Sedimentation pattern of glucidolipidic antigen (7-5 mg/ml) solution containing 20 mg sodium deoxycholate, i peak at 1.44 S. Both experiments (Figs. 7 and 8) were performed in a synthetic boundary cell of 12 ram with Epon centerpiece and 3 ° sector--angle 55% speed 5978o rev./min. Biochim. Biophys. Acta, 2o1 (197o) 155-166
164
E. HANNECART-POKORNI
~'l a l .
Sodium deoxycholate breaks the hydrophobic bonds by the hydrophobic part of its molecule and has the possibility of building a chelating complex using its free COO- and OH groups which, due to the great flexibility of the aliphatic chain and its free rotation around the C-I 7 atom, may very easily come close together. Although the bonds between the units and the subunits are of the same nature, the chemical conformation of the degrading agents seems to be essential. DISCUSSION
The somatic antigen of Shigellaflexneri F6S, serotype 5b, is a lipopolysaccharide with a molecular weight of IO. lO6-45 • lO8. With the electron microscope, important morphological changes are observed during its purification, the spherical structures seen in the crude extract being transformed into long filaments. The variations observed in sedimentation constant and molecular weight values suggest that the antigen molecules are formed by polymerisation of a smaller repeating unit. Various procedures have been applied to study this fundamental unit structure, such as hydrogen bond- and hydrophobic bond-breaking chemical substances (dimethylformamide, urea, guanidine, detergents) and heating. The action of these procedures was studied by light scattering and ultracentrifugation techniques. It was shown that the antigen is built of longitudinal repeating unit structures having a sedimentation constant of IO.2-S and a molecular weight of 25oooo, linked together by hydrophobic interaction due to the lipid part of the molecule as well as by bivalent cations, copper excepted and carboxylic groups, revealed by electrotitration 2s. Detergent and chelating substances alone will not depolymerize glucidolipidic antigen down to the io.2-S unit except when heating is applied (EDTA, citrate, succinate, amino acids excepted). The IO.2-S antigen unit is itself made of smaller 1.44-S fragments linked together by chemical bonds of the same nature as those binding the fundamental units. Sodium deoxycholate acting on hydrophobic links and of the chelating complexes of glucidolipidic antigen, disrupts the antigen molecule first into IO.2-S units which are then further depolymerized into the 1.44-S subunits. The configuration of the degrading agents seems to play an essential role in the depolymerization process. Both the trichloroacetic acid 15-17 and the phenol-water ~9 procedures were used to extract the antigen from Shigella flexneri; these preparations were studied simultaneously. No differences were found in the values observed for the unit and the subunit fragments. TABLE
V
COMPARISON FOR DIFFERENT THE
SODIUM
B A C T E R I A L S P E C I E S OF T H E L I P I D C O N T E N T OF T H E I R A N T I G E N S
DEOXYCHOLATE
CONCENTRATION
REQUIRED
Bacterial species
Lipid (%)
Sodium deoxycholate (%)
Salmonella enteritidis E. coli O l i i E. coli O l l 3 Bordetella
0. 4 12. 5 33.3 18
0. 5 2 2 o.5
Shigella flexneri
15
2
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2Ol (197o) 155-I66
TO O B T A I N
I-S
SUBUNITS
AND
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165
Comparing our results to those obtained by RIBI et al. TM(Table V), we observe that different concentrations of sodium deoxycholate are necessary for the degradation of antigens from different bacterial species. The same phenomenon is found when as detergent sodium lauryl sulphate is used. This substance gives two fractions, 9 S (30 %) and 3 S (60 %) with the E. coli antigen 7 and only one fraction with our antigen (lO.2 S). At first sight it would appear that the detergent concentration necessary to degrade the antigen would depend upon the amount of f a t t y acids present. According to Table V this seems not to be the case, so that quite probably the links between the subunits and units of the antigen structure, being of the same nature for both preparations, are sterically arranged in a different way. The rotatory dispersion 3° has shown the steric arrangement of the glucidolipidic antigen and the io.2-S unit to depend upon the presence in these molecules of linked f a t t y acids but the participation of the sugar side chains of the polysaccharide m a y not be excluded. The fact that our antigen preparation has a different density from those isolated from other bacterial species enforces the idea that their steric structures m a y also be different. This difference in behaviour is not only observed during their degradation process but also during the recombination of antigen structures from low-molecular-weight fractions. Starting from fractions having a sedimentation constant of about I S isolated from the following bacterial species: Serratia marcescens 6, Salmonella 19, recombined fragments are found after dialysis, having respectively a sedimentation constant of 3 S and 16 S. Using the same conditions with Shigella flexneri, we obtained after dialysis recombined fragments having a sedimentation constant of 70 S. I t seems unreasonable to consider such large fragments to be the unit structure and therefore we believe that recombination experiments are not suitable to determine the antigen unit. ACKNOWLEDGEMENTS
The authors wish to express their gratitude to Dr. J. BEUMER and Mrs. M. P. helpful suggestions and valuable criticism throughout this
BEUMER-JOCHMANSfor work.
REFERENCES I 2 3 4 5 6 7 8 9 IO II 12
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E. HANNECART-POKORNI et al.
14 J. BEUMER, M. P. BEUMER-JOCHMANS, J. DIRKX AND D. DEKEGEL, Bull. Acad. Roy. M~d. Belg. 5 (1965) 749. 15 A. BOIVlN AND L. C. R. MESROBEANU, Compt. Rend. See. Biol., 112 (1933) 76. 16 A. BolvlN AND L. C. R. MESROBEANU, Compt. Rend. Soc. Biol., 112 (1933) 611. 17 A. BOIVIN AND L. C. R. MESROBEANU, Compt. Rend. Soc. Biol., 112 (1933) lOO9. 18 G. BERTANI, J. Bacteriol., 62 (I951) 29319 E. RIBI, R. L. ANACKER, R. BROWN, W. T. HASKINS, B. MALC-REN, K. C. MILNER AND J. a . RUDBACH, J. Bacteriol., 92 (1966) 1493. 20 O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND ]~. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . 21 M. NoMoro, Y. NARAFIASHI AND a~. ~/fURAKAMI,J. Bioehem., 48 (196o) 593. 22 M. KUNITZ, J. Biol. Chem., 164 (1964) 563 . 23 M. KUNITZ, J. Gen. Physiol., 33 (195 °) 34924 S. A~. BARKER, S. M. BICK, J. S. BRIMACOMBE AND P. J. SOMERS, Carbohydrate Res., i (1966) 39325 H. NIKAIDO, Biochim. Biophys, Acta, 48 (1961) 460. 26 L. DEBUYSSCHER, in preparation. 27 B. L. HORECHER, Ann. Rev. Microbiol., 20 (1966) 253. 28 J. DIRKX, in preparation. 29 O. ~TESTPHAL, O. LUDERITZ AND V. BISTER, Z. Naturforsch., 76 (1952) I48. 3° E. HANNECART-POKORNI, D. DEKEGEL, F. DEPUYDT AND J. DIRKX, Bioehim. Biophys. Acta, 2Ol (197 ° ) 167 .
Biochim. Biophys. Acta, 2Ol (197 o) I 5 5 - I 6 6
BBA
155
26272
STUDY OF T H E S T R U C T U R E OF S H I G E L L A F L E X N E R I
0 ANTIGEN
I. CHEMICAL D E G R A D A T I O N E. H A N N E C A R T - P O K O R N I ,
D. D E K E G E L ,
F. D E P U Y D T
AND J. D I R K X
Institut Pasteur du Brabant, Brussels (Belgium) (Received S e p t e m b e r I l t h , 1969)
SUMMARY
The lipopolysaccharide antigen of Shigella flexneri F6S serotype 5 appears under the electron microscope as long filaments having a molecular weight of IO. lO645" lO6. This macromolecular structure is built of repeating units having a sedimentation constant, S2o,w, of lO.2 S and a molecular weight of 250000 and is linked together by hydrophobic interaction, due to the lipid part of the molecule and b y bivalent cations and carboxylic groups. These unit antigen structures are themselves polymerization products built of 1.44-S subunits linked together by weak chemical bonds of the same nature as those binding the units in the antigen molecule. The nature of the degrading agent seems to be important for obtaining the subunit fragments. Some preliminary experiments indicate that the IO.2-S unit antigen structures still retain their biological properties such as phage-receptor function and toxicity.
INTRODUCTION
During recent years, somatic antigens of enterobacteriaceae have been analysed intensively as to their chemical nature and biological properties. These studies mainly concerned two fundamental aspects of the biological nature of these antigens: which part of the molecule would be responsible for their toxic behaviour 1-5 and what fundamental building block constitutes the complicated macromolecular structures of these antigens. Many authors tried to isolate a fundamental toxic unit of somatic antigens b y carefully controlled acid or alkaline hydrolysis. The somewhat severe methods (high or low p H values, high temperature) certainly destroy covalent bindings and therefore surely alter the steric arrangements of the antigens. Two groups of workers were able to degrade their antigenic material without hydrolysis and found the fractions to differ in toxicity and molecular weight. Thus OROSZLAN AND MORA6 dissociated Serratia marcescens antigen with sodium lauryl sulphate into a nontoxic fraction having a sedimentation constant of 1.5 S. BEER AND BRAUDE7 used the same detergent on Escherichia coli antigens and isolated two toxic fractions of 9 S (30 %) and 3 S (60 %) WeS, 9 also found that different detergent substances and bovine serum albumin were able to dissociate the somatic antigen of ShigelIaflexneri into different fragments, Biochim. Biophys. Acta, 2Ol (197 o) 155-166
156
E. HANNECART-POKORNIctal.
the smallest of which had a sedimentation constant, s°2o,w of lO.2 S. Heating in the presence of citrate buffer (pH 7.3) also yielded particles of lO.2 S which were still toxic and therefore comparable to the 9-S fractions of BEER AND BRAUDE7. RUDBACH et al. 1°, using Salmonella typhosa antigen, and BEER AND BRAUDE7, using E. coli antigen, obtained 1.5-S fragments after treatment with deoxycholate and 9-S fragments by the action of organic solvents. Quite obviously the antigens and their degradation products must also be examined from the point of view of their antigenic activity and toxicity. BEUMER et al. n-~4 studied the receptor function of the somatic antigen of Shigella flexneri for different phages to which these bacteria were sensitive. This study is mainly concerned with an analysis of the macromolecular structure of the Shigella flexneri antigen. For the sake of clarity, we propose to call the smallest particle still showing a toxicity of the same order as the complete antigen a fundamental unit and to call any fragment smaller than this fundamental unit which has lost this toxic property a subunit. MATERIALS AND METHODS
Extraction of antigen The glucidolipidic antigen of Shigella flexneri serotype 5 was prepared according to BOlVlN AND MESROBEANU15-17 from bacterial cultures in BERTANI'STM LB medium at 4 °.
Purification, chemical treatments and analysis Unless otherwise stated, all denaturing and degradation reactions were carried out in media containing o.15 M NaC1 plus o.o15 M sodium citrate (pH 7.3). The reaction between the antigen and sodium deoxycholate was made following RIBI et al. 1~. Protein contents were estimated by the method of LowRY et al. 2° using a serum albumin standard. Pepsin and trypsin digestions were done as described previously s. The procedure of NOMOTO et a l Y was followed for the pronase digestion. Deoxyribonuclease and ribonuclease products were analysed by the spectrophotometric method of KUNITZ22,23. Gel filtration was used as a means of separating nucleic acid compounds from the antigen. The antigen preparation was passed onto a 2.5 cm x 50 cm column of Sephadex G-2oo which had been equilibrated with o.I M NaCI. The antigen was eluted with increasing NaCI concentration (o.i-i M) at a speed of 2o ml/h, 5-ml fractions being collected. Adsorption spectra of glucidolipidic antigen and degradation compounds were obtained in distilled degassed water. This procedure allows the spectrum to be taken down to 21o m/~ without interference from absorption by phosphate or citrate buffer. The Beckman DB recording spectrophotometer was used for the different spectrophotometrical analyses. Alkaline hydrolysis was carried out with a solution of 4 % NH2OH in alcohol (5 rain at 65 °), while the acetic acid hydrolysis was done as described previouslys. Thin-layer chromatography on silica gel and cellulose was run using aliquots after a 4-h hydrolysis at IOO° in I M HCI neutralised to pH 7. Different solvents and detectors were used. (a) Neutral sugars; elution solvent, double e]ution with a mixture
Biochim. Biophys. Mcta, 2Ol (197o) I55-166
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157
of ethyl aeetate-isopropanol-water (6.5:2.4:1.2, by vol.); detector solution, 3 % p-anisidine HC1 in n-butanol. (b) Nucleic acid compounds; elution solvent, butanolacetic acid- 5 % ammonia-acetone-water (3.5:1.5:1.5:2.5 : I, by vol.) ; detector, ultraviolet. (c) Amino acids; elution solvent, n-butanol-acetic acid-water (3:1:I, by vol.) ; detector solution, 0.2 % ninhydrin in n-butanol.
Physicochemical determinations Sedimentation velocity determinations of the glucidolipidic antigen were performed in a Spinco model E ultracentrifuge. The observed sedimentation coefficients reported in Svedberg units (i S = i . l O -13 cm/sec per unit field) were corrected to values corresponding to a solvent having the viscosity and density of water at 20 °. The molecular weight and the density of glucidolipidic antigen and unit lO.2 S are determined by centrifugation with schlieren optics in density gradient of CsC1 (starting density 1.4o89) for 18 h at 5074 ° rev./min using the double-sector capillarytype cell of 12 mm with an Epon centerpiece 2 ° sector. To prepare specimens for electron microscopy, the glucidolipidic antigen was suspended in distilled water at a concentration of I.O mg/ml. Carbon formvar-coated grids were floated film downwards on the surface of the suspension for a few minutes, removed and sucked dry with filter paper and shadowed with platinium-iridium at an angle of 3°o . RESULTS
Antigen purification A crude glucidolipidic antigen preparation, as seen in the electron microscope, shows (Fig. I) spherical and rod-like particles of different lengths. This polymorphism is a rather serious obstacle to any physicochemical measurement. We tried to purify the antigen which shows a great tendency to adsorb foreign substances and found the morphological heterodispersity to vary considerably with the presence of contaminating proteins. According to the extraction procedures, these
Fig. I. Laterally shadowed crude glucidolipidic antigen preparation showing rod-like particles and spherical particles of different diameters. Magnification, 40000 ×,
Biochim. Biophys. Acta, 2Ol (197o) 155-166
E. HANNECART-POKORNIet al.
158
protein values m a y be from 5 to 7 %; antigen preparations with the highest protein content also show a higher percentage of small particles in the electron microscope. We applied different protein extraction methods such as phenol extraction, enzymatic digestion and ultraeentrifugation. Although none of these procedures ever resulted in a complete elimination of proteins, we found the enzymatic digestion with trypsin and pepsin (Table I) to give the best purification. The 2 % remaining protein material seems to be firmly bound to the lipopolysaccharide part of the antigen molecule and m a y be either proteins, peptides or amino acids. Since ultracentrifugation also eliminates foreign material in excess of the 2 % which remain firmly bound to the lipopolysaccharide, we m a y assume this protein to be foreign to the antigen. As shown in Table II, different antigen fractions obtained by various chemical treatments still contain 2.5 % protein material, which was not further identified, except for the absence of aromatic amino acids. Together with the 2 % protein material, which in fact m a y be either proteins, peptides or amino acids, the glucidolipidic antigen also contains nucleic acid components (3-4 %). The ultraviolet spectrum of glucidolipidic antigen shows a slight absorption band at 260 m # (Fig. 2) which is more intense in both polysaccharides obtained after careful hydrolysis. A more severe hydrolysis (I M HC1 during 4 h at ioo°), gives even more intense absorption bands (Fig. 3). The hyperchromicity is too high to be only due to a denaturation of a polynucleic acid structure and an unmasking of nucleic acid components must take place. We tried without success to separate these hypothetical nucleic acid compounds absorbed in the glucidolipidic TABLE
I
TREATMENT
OF
GLUCIDOLIPIDIC
Methods
ANTIGEN
BY
DIFFERENT
DEPROTEIN1ZING
% of protein material before purification
% of protein material after purification
A. E n z y m a t i c r e a c t i o n I. P r o n a s e 2. P e p s i n - T r y p s i n
5.7 5.7
3.3 2.22
B. C e n t r i f u g a t i o n 4oooo r e v . / u l i n 2 × I h
5.7
3.44
TABLE II PROTEIN CONTENT
Protein material (%) G l u c i d o l i p i d i c a n t i g e n from different b a t c h e s Glucidolipidic antigen after purification
5.7, 7, 6, 5.4, 5.6, 5.8 2.3, 2. 7, 2.4, 2.1, 1. 7
P r o t e i n c o n t e n t of g l u c i d o l i p i d i c a n t i g e n f r a c t i o n s s°20, w = 17o S 2. 7 8o20, w =
10.2
G
2. 3
8°20, w =
9 I
S S
2.42 2. 3
Biochim. Biophys. dcta, 2Ol (197 o) 155-166
AGENTS
Shigellaflexneri 0 ANTIGEZ~'. I
159
antigen by passage through a Sephadex G-200 column. Nevertheless we cannot eliminate the possibility of an absorption unless a passage through an ion-exchange column is carried out. BARKER et al. 24however succeeded in separating nucleic acid compounds from polysaccharides on Sephadex G-2oo. Glucidolipidic antigen and the polysaccharides, which are smaller structures and therefore more accessible to enzymes, have been subjected to ribonuclease and deoxyribonuclease. No effect was observed in the ultraviolet spectrum on the elution pattern on Sephadex G-2oo.
!
5
3 o ~2
1 ~210
260
310
210
2603101
mJ~
Fig. 2. Absorption spectra of different glucidolipidic antigen constituents (0.2 mg/ml) in degassed distilled water. O - - O , glucidolipidic antigen; 0 - - 0 , lO.2 S; / x - - & , hydroxylamine polysaccharide; A - - A , acetic acid polysaccharide. Fig. 3. Absorption spectra of different glucidolipidic antigen constituents (0.2 mg/ml) after acid hydrolysis for 4 h. O - - C ) , glucidolipidic antigen; 0 - - 0 , lO.2 S; /X--/x, hydroxylamine polysaccharide; • - - A , acetic acid polysaccharide.
The presence of nucleic acids seems therefore rather improbable, but nucleotide compounds m a y be responsible for the 26o-m# absorption band. Thin-layer chromatography showed cytidine to be responsible for the largest part of the nucleotide compounds present. If cytidine really is bound to the antigen molecule, it seems reasonable to suppose that this nucleotide is acting in a way similar to UDP-galactose in the case of Salmonella 25, as a cofactor in the transfer of glucidic units during the biosynthesis of somatic antigens in Shigella flexneri. The fact that glucidolipidic antigen contains ketodeoxyoctonate, as established lately b y DEBUYSSCHER 2e, m a y indicate the possibility of its transfer through CMP-ketodeoxyoctonate as described b y HORECKER27. We also found the antigen to contain adsorbed f a t t y acids which are easily extracted in a Soxhlet by cold chloroform.
Study of the unit The antigen is seen in the electron microscope as long filaments (Fig. 4)- According to the nature of the nutrient medium, liquid or solidified agar or to the presence of protein material, glucidolipidic antigen has a molecular weight of IO. lO645" lO6, suggesting a presence of aggregates as usually observed with somatic antigen from enterobacteria. The chemical nature as well as the high molecular mass of t3iochim. Biophys. Acta, 2oi (197 o) i 5 5 - i 6 6
16o
E. HANNECART-POKORNI Ct al.
glucidolipidic antigen suggest furthermore the possibility of the existence of a repeating unit polymerised by weak chemical bounds. Such links may be of a different nature. (a) Ionic bonds due to the presence of fl-hydroxymyristie acid possibly linked to the macromolecular building block by the alcohol radical, leaving the carboxylic radical free (as shown by electrotitration) 2s. All phosphoric acid radicals seem to be esterified since the antigen has but a low electrophoretic mobility. (b) Hydrogen bonds due to the many OH groups of sugar radicals. (c) Hydrophobic bonds due to the longchain (C1~ C16) fatty acids. Various chemical substances were used to destroy the low-energy bonds of glucidolipidic antigen (Table II) giving fractions of 30, 21 and IO S sedimentation constants. Ionic bond- and hydrogen bond-rupturing agents did not interfere with fragmentation. Moreover their action varied with different antigen preparations. Glucidolipidic antigen is sensitive only to high pH values because of hydrolysis and saponification of the ester bonds. Detergent substances had an immediate effect on hydrophobic bonds resulting eventually in io-S fragments. Bovine serum albumin degraded glucidolipidic antigen at much lower rate and only at temperatures over 4 °° (Fig. 5). Heating of glueidolipidic antigen in citrate buffer resulted in a nonreversible degradation. The different degradation products were quite comparable to those obtained by denaturing agents (Table III). This technique of nonreversible degradation permitted an easy isolation of the different fractions. Furthermore it is quite obvious
Fig. 4- Laterally shadowed purified glucidolipidic antigen preparation showing essentially long filaments of equal diameter. Magnification, 40000 ×. Fig. 5. Laterally shadowed detergent-(cetavlon 5 mg/ml)-treated glucidolipidic antigen preparation showing lO.2 S spherical particles. Magnification, 40000 ×. Biochim. Bio~ghvs. Acta, 2Ol (197o) 155 166
Shigellaflexneri 0 ANTIGEN. I
161
that any detergent substance present in the isolated fractions would seriously interfere with the study of immunological and phage-receptor properties of the antigen fractions. Degradation of glucidolipidic antigen may be obtained by heating (Fig. 6), the original II3-S fractions being progressively supplanted by the 3o-S fraction. At about 9°o the II3-S fraction disappears completely and a new 2I-S fraction appears together with the 3o-S fraction. At about IOO° a third transition is obtained giving rise to the IO.2-S fraction coexisting in small quantities with the residual 2I-S fraction. Any further heating at ioo ° has no effect on the io.2-2I-S equilibrium. A complete transformation into IO.2-S units may be obtained by heating at TABLE III VARIATIONS
IN
THE
PRESENCE
OF
DENATURING
Denaturing agents~
Fractions of glucidolipidic antigen
NaC1, 0. 3 M NaC1, i M Guanidine, 5 M Glucose, 5 % B o v i n e s e r u m a l b u m i n , o.I % Mechanical (Ultra-turrax) Heating Heating Poxesmol; o.i, I and 2 % B o v i n e s e r u m a l b u m i n ; (0. 5 a n d i %), 4 o°, 48 h Heating Mechanical (Ultra-turrax) Heating * I n o.15 M NaC1 plus O.Ol 5 • 5 mg/ml.
100
AGENTS
3° S
s%0.w 21 S s°20,,, ~ IO S -
-
s°20. w = 9 S
% o/these fractions
43 45 67 25 65 91 o - i oo o-ioo ioo ioo o - i oo 9 o-ioo
c i t r a t e , p H 7.3. C o n c e n t r a t i o n of g l u c i d o l i p i d i c a n t i g e n is
--
~°
/ /
60
0
20
Ovo
8~)
90 TEMPERATURE
100
Fig. 6. H e a t d e g r a d a t i o n of g l u c i d o l i p i d i c a n t i g e n (5 m g / m l ) in o.15 M NaC1 plus o . o i 5 ~V~s o d i u m c i t r a t e (p H 7.3) a l i q u o t s of t h e h e a t e d m i x t u r e were t a k e n a t d i f f e r e n t t e m p e r a t u r e s a n d perc e n t a g e of differen t f r a c t i o n s d e t e r m i n e d in t h e u l t r a c e n t r i f u g e . O - - O , 113 S; ~ k - - $ k , 3 ° S; O--O, 20 S; I - - ' , lO.2 S.
Biochim. Biophys. Acta, 2Ol (197 o) 155-166
E. HANNECART-POKORNI el al.
162
12o ° during 25 min. If heating is continued the I0.2-S fragments are degraded into 9-S units, free sugar molecules and a precipitate of fatty acids. Obviously this hightemperature treatment destroys the chemical integrity of the antigen unit. Thus glucidolipidic antigen seems to be built of repeating io.2-S units essentially linked together by hydrophobic bonds, due to the lipid part of the molecule. This IO.2-S unit may be obtained by thermal degradation of glucidolipidic antigen in a citrate buffer. No degradation is observed when glucidolipidie antigen is heated in a phosphate buffer. It thus appears that citrate degradation of glucidolipidic antigen may be a combined effect of: (a) the aliphatic chain of the citrate molecule acting on the hydrophobic links; (b) the OH group, a tertiary alcohol group; and (c) the three carboxylic groups acting ionically or by chelation. We therefore experimented with different substances having some of these characteristics alone or in combination (Table IV). It appears that neither the aliphatic chain nor the OH groups would degrade the antigen molecule. Molecules with one carboxylic group are without any effect. TABLE IV THERMIC DEGRADATION OF GLUCIDOLIPIDIC ANTIGEN WITH DIFFERENT AGENTS All s u b s t a n c e s w e r e d i s s o l v e d at a c o n c e n t r a t i o n of o.oi 5 M in o. 15 M NaC1 a n d b r o u g h t to pH 7.2 w i t h NaOH.
Substance
trormula
Sodium chloride Hexane
NaC1 CH3-(CH,~ ) 4-CHa (CH3) 3-COH H3PO 4
tert.-Butanol P h o s p h o r i c acid
Degradation, glucidolipidic antigen
2.12
Formic acid A c e t i c acid Caproic acid S u c c i n i c acid
H-COOH CHa-COOH CH3-(CH~)4-COOH HOOC-(CH2) 2-COOH
M a l e i c acid
HOOC-CH = CH-COOH
+
T a r t a r i c acid
HOOC-(CHOH)~-COOH
+
m
m
+
OH Citric acid
I
HOOC-CH2-C-CH~-COOH
+
I
COOH HOOC-(CH2)5-COOH
+
EDTA
C6H~COOt-I NH 2 CbH4-COOH (NO~) a-C6H~-OH CH~N-(CH2-COOH )
+ + +
Glycine
CH~N-(CH2COOH) 2 NH2-CH2-COOH
P i m e l i c acid B e n z o i c acid p - A m i n o b e n z o i c acid Picric acid
i
JBiochim. Biophys. Acta, 2Ol (197 o) 155-166
pK
+
7.21 12.67 3.75 4.78 4.83 4.6 5.61 1.83 6.07 2.98 4.34 3,o8 4"74 5-4 4.71 5-42 4.19 4.92 0.38 3.o3 4"44 2.34 9,69
Shigdlaflexneri 0 ANTIGEN. I
163
Molecules with two carboxylic groups and acids with aromatic radicals used in identical conditions are listed in Table IV. We m a y consider different characteristics of the molecules. (i) Polarity has no effect in itself, since the salt concentration is io times higher than the buffer concentration. Moreover, if the dipole m o m e n t of the molecule would be important, a degradation of glucidolipidic acid in the presence of trinitrophenol, which has a very low dipole moment, would not take place. (ii) I n the ionic form, the p K of different buffer molecules is not essential but the presence of two close anionic groups is a prerequisite to obtain the unit fragment. This would account for the variable activity of pimelic acid. Normally both carboxylic groups of pimelic acid are far a w a y from each other but thermic agitation m a y bring t h e m close enough to each other to act on the glucidolipidic antigen molecule. We therefore think that the chelating p r o p e r t y of the buffer substances is mainly responsible for the thermic degradation of glucidolipidic antigen. This fact suggests t h a t glucidolipidic antigen is built of smaller unit structures having a sedimentation constant of lO.2 S polymerized by hydrophobic links and bivalent cations except copper (glycine, a copper-chelating agent has no effect on the antigen). Stu:tv of the subunit RUDBACH et al. 1° succeeded in degrading the antigens of various gram-negative bacteria directly into I-S fragments b y sodium deoxycholate. Our antigen preparation resisted any further degradation than the I0.2-S unit even when the reagents such as citrate plus poxesmol were used at I 0 times higher concentrations. An acetic acid hydrolysis of the I0.2-S unit structure, however, yielded a polysaccharide having a sedimentation constant of I S, corresponding to a molecular weight of about 9000. In the presence of sodium deoxycholate our antigen is degraded into I0.2-S unit fragments (Fig. 7) which are further transformed into 1.44-S subunits (Fig. 8) when the sodium deoxycholate concentration is increased.
Fig. 7- Sedimentation pattern of glucidolipidic antigen (7.5 mg/ml) solution containing IO mg/ml sodium deoxycholate. 2 peaks at 1.44 S and lO.2 S. Fig. 8. Sedimentation pattern of glucidolipidic antigen (7-5 mg/ml) solution containing 20 mg sodium deoxycholate, i peak at 1.44 S. Both experiments (Figs. 7 and 8) were performed in a synthetic boundary cell of 12 ram with Epon centerpiece and 3 ° sector--angle 55% speed 5978o rev./min. Biochim. Biophys. Acta, 2o1 (197o) 155-166
164
E. HANNECART-POKORNI
~'l a l .
Sodium deoxycholate breaks the hydrophobic bonds by the hydrophobic part of its molecule and has the possibility of building a chelating complex using its free COO- and OH groups which, due to the great flexibility of the aliphatic chain and its free rotation around the C-I 7 atom, may very easily come close together. Although the bonds between the units and the subunits are of the same nature, the chemical conformation of the degrading agents seems to be essential. DISCUSSION
The somatic antigen of Shigellaflexneri F6S, serotype 5b, is a lipopolysaccharide with a molecular weight of IO. lO6-45 • lO8. With the electron microscope, important morphological changes are observed during its purification, the spherical structures seen in the crude extract being transformed into long filaments. The variations observed in sedimentation constant and molecular weight values suggest that the antigen molecules are formed by polymerisation of a smaller repeating unit. Various procedures have been applied to study this fundamental unit structure, such as hydrogen bond- and hydrophobic bond-breaking chemical substances (dimethylformamide, urea, guanidine, detergents) and heating. The action of these procedures was studied by light scattering and ultracentrifugation techniques. It was shown that the antigen is built of longitudinal repeating unit structures having a sedimentation constant of IO.2-S and a molecular weight of 25oooo, linked together by hydrophobic interaction due to the lipid part of the molecule as well as by bivalent cations, copper excepted and carboxylic groups, revealed by electrotitration 2s. Detergent and chelating substances alone will not depolymerize glucidolipidic antigen down to the io.2-S unit except when heating is applied (EDTA, citrate, succinate, amino acids excepted). The IO.2-S antigen unit is itself made of smaller 1.44-S fragments linked together by chemical bonds of the same nature as those binding the fundamental units. Sodium deoxycholate acting on hydrophobic links and of the chelating complexes of glucidolipidic antigen, disrupts the antigen molecule first into IO.2-S units which are then further depolymerized into the 1.44-S subunits. The configuration of the degrading agents seems to play an essential role in the depolymerization process. Both the trichloroacetic acid 15-17 and the phenol-water ~9 procedures were used to extract the antigen from Shigella flexneri; these preparations were studied simultaneously. No differences were found in the values observed for the unit and the subunit fragments. TABLE
V
COMPARISON FOR DIFFERENT THE
SODIUM
B A C T E R I A L S P E C I E S OF T H E L I P I D C O N T E N T OF T H E I R A N T I G E N S
DEOXYCHOLATE
CONCENTRATION
REQUIRED
Bacterial species
Lipid (%)
Sodium deoxycholate (%)
Salmonella enteritidis E. coli O l i i E. coli O l l 3 Bordetella
0. 4 12. 5 33.3 18
0. 5 2 2 o.5
Shigella flexneri
15
2
Biochim. Biophys. Acta,
2Ol (197o) 155-I66
TO O B T A I N
I-S
SUBUNITS
AND
Shigellaflexneri 0 ANTIGEN. I
165
Comparing our results to those obtained by RIBI et al. TM(Table V), we observe that different concentrations of sodium deoxycholate are necessary for the degradation of antigens from different bacterial species. The same phenomenon is found when as detergent sodium lauryl sulphate is used. This substance gives two fractions, 9 S (30 %) and 3 S (60 %) with the E. coli antigen 7 and only one fraction with our antigen (lO.2 S). At first sight it would appear that the detergent concentration necessary to degrade the antigen would depend upon the amount of f a t t y acids present. According to Table V this seems not to be the case, so that quite probably the links between the subunits and units of the antigen structure, being of the same nature for both preparations, are sterically arranged in a different way. The rotatory dispersion 3° has shown the steric arrangement of the glucidolipidic antigen and the io.2-S unit to depend upon the presence in these molecules of linked f a t t y acids but the participation of the sugar side chains of the polysaccharide m a y not be excluded. The fact that our antigen preparation has a different density from those isolated from other bacterial species enforces the idea that their steric structures m a y also be different. This difference in behaviour is not only observed during their degradation process but also during the recombination of antigen structures from low-molecular-weight fractions. Starting from fractions having a sedimentation constant of about I S isolated from the following bacterial species: Serratia marcescens 6, Salmonella 19, recombined fragments are found after dialysis, having respectively a sedimentation constant of 3 S and 16 S. Using the same conditions with Shigella flexneri, we obtained after dialysis recombined fragments having a sedimentation constant of 70 S. I t seems unreasonable to consider such large fragments to be the unit structure and therefore we believe that recombination experiments are not suitable to determine the antigen unit. ACKNOWLEDGEMENTS
The authors wish to express their gratitude to Dr. J. BEUMER and Mrs. M. P. helpful suggestions and valuable criticism throughout this
BEUMER-JOCHMANSfor work.
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