July 5 , 1955
~R.l~lUNOCIIEhlISTRY AXD TIIE S r R U C T U R E O F
LUNGG A L A C T A N
3511
[CONTRIBUTION FROM THE DEPARTMENTS OF MEDICINEAND OPHTHALMOLOGY, COLLEGEOF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY, AND THE DEPARTMENT O F CHEMISTRY, THEOHIO STATE UNIVERSITY]
Immunochemistry and the Structure of Lung Galactan' EY
~ ~ I C H A EHEIDELBERGER,~ L ZACHARIAS
DISC HE,^ \V. EROCKN E E L Y 3
AND
M. L.
wOLFROM3
RECEIVED JUNE 24, 1954 Since the immunologically specific polysaccharide of Type X I V pneumococcus (S X I V ) contains galactose, and antibodies t o Type X I V pneumococcus are directed against S XIV, t h e cross reactivity of lung galactan in Type XTV antiserum was tested. Nearly 30y0 of the antibody was precipitated, indicating a close relationship between the galactose linkages in the two carbohydrates. T h e galactan also precipitated antiserum t o Type I1 pneumococcus. Since the Type I1 polysaccharide contains glucuronic acid and no galactose it seemed probable t h a t the uronic acid known t o be a minor constituent of lung galactan is actually glucuronic acid. This was verified by paper chromatography. Colorimetric assays of the uronic acid : galactose ratios in the lung galactan and in its cross reaction precipitates showed t h a t the initial ratio of cu. 1:50 wag not maintained. Thus the lung galactan was not a homogeneous polysaccharide or had been altered in its isolation. The galactogluco-xylan of tamarind seed also gave a cross reaction with Type X I V antipneumococcal horse serum but this was not as strong as t h a t exhibited b y the lung galactan. These results demonstrate the value of quantitative immunological methods as a n aid t o the determination of chemical structure.
Quantitative immunochemical methods have been of great service in the clarification of structural chemical relationships, but their application t o natural products has been restricted by the small number of reference substances of known constitution and fine structure capable of reacting with antibodies to form precipitates. The type-specific polysaccharides of pneumococcus constitute a large group of such materials of great potential utility, but thus far the constituent sugars and their modes of lintage are known in only a few instances. An indirect approach to the chemistry of these substances involves the testing of more easily accessible polysaccharides of known constitution against various antipneumococcus sera, since any cross reactivity found would indicate a measure of structural relationship. The recent isolation of a galactan from the residues of beef lung^^)^ after the separation of heparin and the finding that the galactan6 gave a precipitate with Type XIV antipneumococcus serum prompted the joint investigation described in the present paper. The unexpected reactivity of the galactan in Type I1 antipneumococcus serum provided additional instances of the power of immunochemical methods. Because the specific polysaccharide of Type XIV pneumococcus contains galactose' it appeared appropriate to test the reactivity of the lung galactan with Type XIV antiserum in the hope that the structural relationships of the galactan and S XIV might be sufficiently close to permit cross reactivity, i.e., precipitation of a t least a portion of the antibody. Qualitative tests showed that precipitation (1) A preliminary account of this work appeared in Fedcrafion P r o c . , 13, 226, 496 (1954). (2) Columbia University. (3) T h e Ohio State University. Acknowledgment is made t o t h e assistance rendered by Dr. R. A. Gibbons, of this Laboratory, in revising t h e manuscript for publication. (1) A I . L. Wolfrom, D. I. Weisblat, J. V. Karabinos a n d 0. Keller, Arch. Biochem., 14, 1 (1917). ( 5 ) M. L. Wolfrorn, G. Sutherland and M. Schlamowitz, THIS JOURNAL, 74, 4883 (1952). (6) Designated pneumogalactan in reference 5. In this reference the statement is made t h a t the d a t a uniquely established t h e sequence of o-galactose units as -1-+6~1-G-. This is not correct a n d t h e 3-1 d a t a allow three other possibilities: -1+G-l-G--, -1-+3f1-+3--, -1-3~1-+6--. 3+1-6+1 G-l--6-l 0-1 (7) W. F. Goebel, P. B. Beeson and C. L. Hoagland, J . Bioi. Chem., 129, 455 (1939).
actually occurred. However, there was also marked precipitation of antiserum to Type I1 pneumococcus. It is known that the specific polysaccharide of this type (S 11) contains a repeating unit composed of rhamnose, glucose and glucuronic acid.B-10 The reaction in Type I1 serum could therefore be accounted for only if the unknown uronic acid reported to be a minor constituent of the lung galactan5 turned out to be glucuronic acid. Identification of the uronic acid was thereupon undertaken and confirmation of the serological prediction ensued. Quantitative data on the precipitin reactions in Type XIV and Type I1 antipneumococcal sera are given in Table I. TABLE I PRECIPITATION OF 1.0 ML. ANTIPNEUMOCOCCAL TYPEX I V AND TYPEI 1 HORSE SERABY LUNGGALACTAN IN THE COLD
0.1
.15 .3
103
92b
42 179
157
257,c 232d
207
.45
1
95e 150
1.5
2 3
256 242
54' 59
io
175
4 6
759 181 a After absorption with pneumococcal C-substance this serum contained 1061 pg. of antibody nitrogen precipitable b y S 11; of this 20 pg. was precipitated by glycogen and the supernatants were used for the analyses with galactan. All values are calculated t o 1 ml. of C-absorbed serum. * D a t a in this subcolumn were obtained with 1.0 ml. of a 1 + 2 dilution of serum with one-half quantities of galactan and were doubled. Supernatants from the precipitations with 2 and 3 mg. gave only 2 pg. of ?: with tamarind seed polysaccharide. c T h e supernatant gave 664 pg. of antibody N with S XIV. 664 257 = 921 pg.; direct analysis of 1.0 ml. of serum with S X I V gave 920 pg. Actually run with 2.5 ml. of antiserum and 2.5 mg. of galactan; calculated t o 1 ml. e The supernatants, with enough lung galactan t o bring them u p t o t h e 3 mg. level, each gave 61 pg. of precipitate N. f C-Absorbed serum gave 52 pg. of N with 1 mg. of galactan. 0 One-half quantities actually used.
+
(8) B. R. Record and M. Stacey, J . Chem. SOL.,1561 (1948). (9) S. B. Beiser, E. A . K a b a t and J . M. Schor, J. I m m u n o f . , 69, 297 (1952). (10) M. Stacey, Endeavour, 12, 38 (1953).
The question then arose as to whether the glucuronic acid was an integral part of the lung galactan, or whether it was contained in an impurity, the more SO as the electrophoretic diagram of the galactan had indicated an inhomogeneity.j It has been possible to provide an irnmunochemical solution of this problem, also, based on the following reasoning. Precipitation of the galactan in Type XIV antiscrurn ~ - o u l dbe mediated through a portion or all of the D-galactose in its repeating units, while precipitation in the Type I1 serum would be through recurring glucuronic acid residues. If the D-galactose and glucuronic acid were components of the same polysaccharide, the ratios of these sugars in the precipitates formed in the two antisera should bt. the same. On the other hand, if the D-galaCtOSC and glucuronic acid occurred as portions of different polysaccharides, or in different proportions i n diffcwrit polysaccharides, these ratios should differ i n the two c:ises. The latter alternative was fou1id to obtain. These two successful applications of i~mnunochelnical methods do not by any means exhaust the p'issibiljtjes, From the results of the action of periodate and from hydrolysis of the methylated gal:tctan it was conclu~lcd~~ that roughly one-third of the D-grtlaCtOSe was singly bound in glycosidic liillage to ont' o l the other D-galactose units, onethird was doubly bouild, and one-third triply. As for 1he Type X I i 7 pneuiiiococcal polysaccharide, all that is known of its structure is that there are stated to be three galactose residues to one of Nacetylglucosamine.i These data are insufficient to permit any decision as to what structures the two polysaccharides have in common. Recently, however, the structure of tamarind seed polysaccharide was described." The principal feature of interest in the present connection is that all of the D-galactose in a methylated portion WE recovered as the 2,5,4,G-tetrainethyl ether, indicating that there is only one kind of D-galactose linkage in this polysaccharide, namely, that of single units attached to the D-Xylose or D-ghXoSe residues which make up the principal chain. A sample of the polysaccharide was obtained through the kindness of Dr. F. E. Hr;Iui!s, The Institute of Paper Chemistry, Applet ( J n , \Vise., and it was found to precipitate Type X 11' antipncu~nococcusseruiii. Quantitative datn I J I I the reactiuii are given in Table TI.
Experimental
Materials and Methods.-The lung galactan6 used was descrilml in reference .5. Tamarind sced polysaccliari(lc (jellose) was prepared as in reference 11. Antisera were generously furnished by the Bureau of Laboratories, Xeiv York City Department of Health, and by t h e Division of Laboratories aiid Research of the Department of Healtli, State of New York. Estimations of antibody nitrogen were carried out in duplicate a t 0-2" according t o Heidelberger and Kendall,l*with the final determination in the casc of the smaller quantities by the Markham m e t l ~ o d . ' ~ Identification of Glucuronic Acid in Lung Galactan by Paper Chromatography.-An amount of 150 mg. of lung galactan6 of highest purity5 was suspended in 20 ml. of anhydrous methanol containing 1& ,: hydrogen chloride and allowed t o react a t room temperature (25-30") for 1 week. 'J'he centrifuged, esterified material (120 mg.) was dissolvcd i i i 3 nil. of water and the solution was added under stirriiig to a solution of 200 mg. of sodiuin borohydride in 5 i i i l . OF \later. After 1 0 inin., the soltitioil was inadc O,.? Xiii liydrochloric :wit1 i i i a t o t d volume of 23 ml. uiid this L\ lieated in a boiliiig water-bath ('18")for 20 hr. T h e cooler1 solution \vas then neutralized with sodium hydroxide aiid concentrated uuder reduced pressure t o 5 m l . An aliquot of this material was subjected t o ascending paper chromatoyraphy, following the technique of Partridge.'* The upper layer of 1-butano1:acetic acid:water (1:I : ? parts by vol.! mixture was emplo>-ed as developer. Authentic samples of D-galactose, D-plUCOSe and D-glucurono-?-lactone were chromatographed simultaneously on the same paper sheet. The indicator spray reagent was a 3 5 ; solution of aiiilinc acid phthalate in I-butanol and the color was formed 1)y heating the sheet in an oven a t 105' for 10-13 min. The hydrolyzate showed three distinct spots correspouding iii position t o those given by the authentic specimcn.; of Dglucose, D-galactose and D-giucurono-r-lactone. The 11galactose spot was just above t h a t of D-glucose mliiIcs t h a t of the D-gluCUrOnO-r-lactOile was \vel1 above t h a t of u-g:ll:ictose. The D-glucurono-?-lactone control showed a secontl spot corresponding t o the free acid.'l I n tlie hytlrol?.zatc.. this spot was obscured by t h a t of D-galactose. -4nother portion of the lung galactan (100 m g . ) was 11ydrolyzed directly with 6 ml. of 0.5 AVhydrochloric acid at 98" for 20 hr. T h e neutralized and concentrated hydrolvzate nas then chroinatographed as above. Two spots resulted: a dark one corresponding in position t o that of 11galactose and a fainter one corresponding t o thxt of 11glucurono-y-lactone. Galactose and Glucuronic Acid Content of Specific Fr cipitates of Lung Galactan with Tvue XIV and Type I1 Antipneumococcal Horse Sera.-Reait-ion mixtures multiply. One or more portions were analyzed and the others were combined for t h e sugar a was found that the antibody could be quantitat bilized and the galactan quantitatively extracted from t ' e washed specific precipitates with 55; trichloroacetic :ici 1 for 1,j inin. at room temperature. T h e residual atiti'~oc'y showed a carbohydrate content normal for serum gloLuliti\. (;alactose was determined in the extracts by the cyitciiic-H2S01 reaction of galactose called SCyRI .I5 Tri:ilicatc a n a l ~ ~ swere e s made on 0.4-ml. aliquots of the trichloroacetir acid extracts (29.6 ml. total vol. for the Type XIY prccipiTABLE 11 tates from six 2.5-ml. portions of Type X I \ ' antiserum anrl 11.I ml. total volume for the Type I1 precipitates from ieveti PRECIPII'ATION O F 1.0 h I L . T Y P E X I V ANTIPNEUMOCOCCUS 5 m I . portions of Type I1 antiserum). Internal standards IIORSE S E R U M 635 B Y TAMARIND S E E D POLYSACCIIARIDE AT containing an additional 40 pg. of galactan (also in duplicate) were employed as reference standards, all reading< 0-2 O being made a t 605 m p against a blank containing all reagenti Atilt. N Amt. h' but no galactan. A control containing galactall hut no pol>saccharide precipitated, polysaccharide precipitated, added, mg, !Jg. added, mg, fig. cysteine was also included, but the extinction due t o thv action of sulfuric acid alone on galactan was small. Thr 0.1 86 2 5Sa total hexose content was also checked by the PCyIZI reac.2 92 4 31" tion15 with the result that no significant amount of liexmc .4 94,02" 0 2" other _ _ than galactose was found. 1 77," 81" (12) 11. Heidelberger and 1'. 13. Kendall, 3. Esptl. , \ I d , 6 1 , i i ' l One-half quantities actually used. The supernatants (1935). (13) R. M a r k h a m , Riochrm. J . , 36, 790 ( 1 9 4 2 ) ; see a1-o. I ( A frrmi t h e analyses rvith 0.4 mg. gave 148 pg. of additional Kabat and 1\1. hl. RIayer, "llxperimentnl I ~ I I I I ~ J I ~ ~ ;:3
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2617
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1 1 1 ) S A I . Partridge, HiorhPrt? J . , 42, 238 (1948). ( 1 5 ) 7,. I i i h r h y I , 13. S I i c ~ t l t ~. i-r.i q l \ l : ~ r ~ l t . O i , , I O . .4wit
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