Oct. 5 , 1953
STRUCTURAL UNITSINVOLVED IN BLOODGROUPA
AND
R SPECIFICITY
[CO\TRIBETIOS F R O M THE DEPARTMESTSO F NEUROLOGY AND MICROBIOLOGY, COLLEGE O F P H Y S I C I A N S COLUMBIA UXIVERSITY, A S D THE x E U R O L O C I C A L INSTITUTE, P R E S B P T E R I A S HOSPITAL]
ASD
51.50 SVRGEONS,
Immunochemical Studies on Blood Groups. XVII. Structural Units Involved in Blood Group A and B Specificity' BY ELVINA. KABAT AND SIDNEY LESKOWITZ RECEIVEDMARCH8, 1955 An oligosaccharide fraction obtained by paper chromatography of the dialyzable material released by mild acid hydrolysis of hog blood group A substance shows the ability specifically t o inhibit the precipitation of anti-A by A substance. N o inhibition was obtained with a similar material from hog 0 ( H ) substance. Of the monosaccharide constituents of the blood group substances only S-acetylgalactosamine showed slight inhibition. The disaccharide galactosido-l+4-P-S-acetplglucosamine also inhibited weaklL-. Inhibition of precipitation of anti-B by human saliva or horse stomach B substances was produced in order of decreasing effectiveness on a molar basis by (1) raffinose, melibiose and stachyose; (2) a-methylgalactoside; ( 3 ) galactose; (4)8-methylgalactoside. A variety of other sugars did not inhibit. The inhibition data indicate that B specificity is determined by an oligosaccharide with a terminal non-reducing galactose linked to the next sugar in 1+ 6-a-galactosidic linkage; this second sugar is probably N-acetylglucosarnine. Comparison of the molar concentrations required for inhibition in the blood group systems as compared with those for dextran-anti-dextran suggests that the oligosaccharide side chains determining blood group specificity are probably n o larger than tri- t o penta- or hexasaccharides.
Recent studies from this Laboratory2have shown that the dialyzable materials liberated by mild acid hydrolysis (9H 1.5-2.0 a t 100" for 2 hours) of the blood group A, 0 and B substances from hog and human sources have glucosamine-galactosamine ratios which correlate with their blood group specificity. In the present communication it is shown that the oligosaccharide fractions of such dialysates possess groupings which determine blood group specificity in that the oligosaccharide fraction from hog A substance inhibits the precipitation of anti-A by hog or human A substance, while a similar material from hog 0 substance shows no such effect on anti-9. Comparisons are made of the relative potency of various monosaccharides and oligosaccharides of known structure which inhibit the A-anti-A and the B-anti-B precipitin reactions. Inferences are then drawn as to the structure of a portion of the oligosaccharide units which determine A and B specificity.
Experimental Dialyzable Oligosaccharides from Mild Acid Hydrolysates of Blood Group Substances.-A sample of hog mucin substance (Fr2) containing a mixture of hog A and 0 ( H ) substances as well as an A (Hog 39) and an 0 ( H ) (Hog 33) sample each from individual hog stomach linings were studied. A neighed sample of the substance was dissolved in a measured volume of dilute HC1 (pH 1.6) to give a solution containing 10 or 15 mg. of substance per ml. and heated for 2 hours in a boiling water-bath in a tube sealed with a self sealing rubber cap (the pressure was released by puncturing the cap Kith a hypodermic needle). The solution was then transferred to a cellophane sausage casing and dialyzed against at least 5 changes of distilled water for 5 days. The combined dialysates vere brought to pH 6 with dilute S a O H in the case of the hog mucin substance (Fr2) and then concentrated under nitrogen and reduced pressure a t 40' and lyophilized. Since this procedure gave considerable quantities of salt, the dialysates of the other t1r.o substances IT ere lyophilized without neutralization. The non-dialysable residues ( P l ) were also recovered by lyophilization. Measured amounts of dialysate were spotted on sheets of Whatman #1 filter paper nhich were developed in a descending chromatogram with ethyl acetate-propanol(1) (a) T h i s investigation was carried o u t under t h e William J. Matheson Commission a n d t h e Oflice of S a v a l Research (Contract Nonr 266 (1311, K a v y Department, Washington, D . C. Reproduction in whole or in p a r t is permitted for a n y purpose of t h e United States Government. (b) Presented a t t h e American Assn. of Immunologists, San Francisco, S p r i l 11-15, 1935. Cf.Fedevntion Proc., 14,467 (1955). (2) S. Leskowitz a n d E . A. K a b a t . TEISJ O U R N A L , 76, 5060 (1954).
water (1:7:2)3 as the solvent. After about 18 hours, the papers were removed, dried and redeveloped4 with the same solvent mixture. After the second drying guide strips were cut off, bathed in a 2 % hexane solution of aniline, dried, then bathed in a 2 % solution of trichloroacetic acid in hexane, dried, and heated in a n oven a t 100' for 10 minutes to locate the reducing sugars as brown spots. By reference to the guide spots the main papers were cut to obtain four bands in the case of hog mucin substance (Fr2) and three bands for hogs 39 and 33. These bands corresponded to materials of RF 0.64, 0.32, 0.23, and a streak to the origin for Fr2. For Hog 33 and 39 the bands had RF 0.55, 0.3, and a streak to the origin. These represented fucose and N-acetylhexosamine, galactose and glucosamine, and oligosaccharides, respectively.5 The papers corresponding to each band were extracted with 20 ml. of water. Aliquot portions were examined directly and after hydrolysis with 2 N HC1 for 2 hours a t 100". Analytical M2thods.-Reducing sugar n-as determined by the Hagedorn- Jensen method,Bahexosamine by the ElsonMorgan method6b)after evaporating samples hydrolyzed with HCl to dryness in a vacuum desiccator,; methylpentose by the Dische and Shettles methods and N-acetylhexosamine as recently described by Aminoff, Morgan and Watkins.g Glucosamine-galactosamine ratios were measured by the method recently described from this Laboratory.lo Immunochemical Studies.-The micro modification of the quantitative precipitin method of Heidelberger and MacPhersonll was used. The capacity of various fractions obtained from the dialyzates of the blood group hydrolysates and of various monosaccharides and oligosaccharides of known structure to inhibit precipitation was measured by adding known quantities of the substance to be tested, or of saline, to a measured volume of antiserum, incubating a t 3 i " for 30 minutes, and then adding an appropriate amount of blood group substance. After mixing, the tubes were again incubated at 37' for one hour and placed in the refrigerator for one Twek with mixing twice daily. The tubes were centrifuged, the precipitates washed twice in the cold with saline and analyzed for N with the Folin-Ciocalteu tyrosine reagent.11,12 The percentage inhibition by any 13) S . Baar and J . P. Bull, S a l i i v e , 172,4 1 1 (1953) (4) A. Jeanes, C . S . Wise and R . J . Dimler, A n a l . C h i n . , 23, 41.5 (1951). ( 3 ) E. A. K a b a t , H . Baer, A . E . Bezer and V. K n a u b . J . E x p . W e d . , 88, 43 (1948). (6) ( a ) H. C. Hagedorn a n d B. N. Jensen, B i o c h r m . Z . . 136, 46 (1923); ( h ) L. A . Elson a n d W.T. J . Morgan, Biochrm. J . . 27, 1824 (1933). (7) J. P. Johnston, A. G . Ogston a n d J . E. Stanier, The A n a l y s t , 7 6 , 8 8 (1951). (8) Z. Dische a n d L. B. Shettles, J . Bioi. Chcm., 179, 59.5 (1948). (9) D . Aminoff, W . T . J. Morgan a n d W. M . Watkins, Biochcm. J . , 51, 379 (1952). (10) S. Leskowitz a n d E. A. K a b a t , THISJ O U R N A L , 76, 4887 (1954). (11) M. Heidelberger a n d C. F. C. MacPherson, S c i e i i r e , 97, 403 (lY43); 98, 63 (1943). (12) E. A. K a b a t a n d A l . M . M a y e r , "Experimental Immunochemistry," Chas. C Thomas, Springfield, Illinois, 1948.
TABLE I PROPERTIES O F
DIALYSATE 1 ~ R A C . I . I O S SOH I'AISED
O S M I L D ACID
I~YIJKOLYSIS
+
Amouiit substance used, g. Conditions of hydrolysis Concn. of s o h . hydrolyzed, Son-dialyzable P I fractioii I)ial>,zablematerial," g. Band designation
I .08
BLOOD CrKOLJP
ptl I
io x
tj,
pti I
I U O " , 2 hi..
0 'ti
ti,
0 392 1U:j". 2 hr
1.5 7
lr, 4 0 . I!i2 0.2'
iJ Xl!)
sCBSl'.l\C!E5
Hog 811 B2 A
!I : N j
pbl 1 ti, IOO", 2 h r . I I I ~ 'inl. .
O F I-rOC,
Hug 33 B 2 0 (H)
Hog mucin Pr2 A 0 (HI
I). I1
172 22
C o n i p ~ ~ ~ i toii ~l,ands ~ i i ohtainrd o n paper chrurnatugraphy of dialysate Icthyl acetate-propanol-water -4 B C D A n C A
RF Methylpentose, mg. Reducing sugar/ Unhydrolyzed, mg. as glucose \Hydrolyzed,' mg. Hexosamine hydrolyzed, mg. Glucosamine/galactosamine Equivalent S-acet!lheuosamiiIr," rng. Iiihibition studies Sample added, mg. reducing sugar 011 hydrolysis % inhibition
0 . 2 3 0.0' 06 2.1 12.9 18.3 22 42 11.2 19 3.9 3 1
0.55 16.2 32 3fi
0.:30 2.1 24 21 10.ii
O.0h 5 8
28
50
0.55 1G.4 33 85 92
21 "2 Xl 8.4 ,i5 10.4 1K 8,2 ?le 2.3 1.5 ml. KX,; XI+ g. Hog 3O(A) --Tot:rI vol. X O nil.
(1: 7 . 2 rj
C
0.3
3.1 30 31 18 T i l
4 4"
2 2 0.1 0 . 8 1.7 1.8 1 2 0 0 17 59 2 -1 " By difference. * A streak trailing from the origin toward the next faster movi:ig band was takeii. After hydrolysis a t 100' for 2 hours in 2 NHCI. d Maximum values of equivalerit S-acctylhexosarniiie wcre obtaitied after 4 iiiiiiiites' heatiiig with SLI&OZ.~ e Analyses by M r . Joel Goodman. concentration of inhibitor was computed from the difference in the amount of specific precipitate formed in the presence of the inhibitor and that formed in saline, a quantitative a d a p t a t i c ~ n ' ~of . ' ~the qualitative hapten inhibition procedure developed by Land~teinerI5.l~ to the quantitative measurement of the degree of inhibition. Antisera to hog A substance and to human A saliva were used. The antisera to hog A substance, K S I and KN217 were prepared by injection of individuals of group B with hog -4 substance.I8 The antiserum to human saliva A substance, PM4, was prepared by injection of purified human saliva A substance into an individual of blood group B.Ig The antiserum to B substance, Als., described in reference 20, was from a woman of group 0 who had 3 marked post-partum rise in anti-B following delivery of a B infant with erythroblastosis and kernicterus; it was kindly provided by Drs. R . E. Rosenfield and A. B. Gutman of -Mount Sinai Hospital. For the oligosaccharide inhibition studies, 1.5 ml. of KS, (30 p g. A b N ) , 1.0 ml. of KSu (23 p g. AbN) and PMa (22 p g. AbN) and 0.5 ml. of a 2 3 dilution of Als. (30 p g. AbhT) were used; the total volumes were 3.0 ml. for K N , , 2.5 ml. for K?VZand PALand 2.0 ml. for -41s. In addition to the fractions obtained by mild acid hydrolysis, dialysis and paper chromatographic separation of the hog A and 0 substances, various mono- and oligosaccharides were used in the inhibition studies. Among these ~ and p-( [ O ] ~ ~-0.5') D methylgalactowere e-([ a I z 0+179") pyranosides, melibiose ( [ a *OD ] f132.3 O (final)),21 and neolactose ( [ a ] * ' D +35.5')2* kindly supplied by Dr. Kelson K . Richtmyer, National Institutes of Health, stachyose by I k . Dexter French Iowa State College, galactosido-l ---t --f
.~~ ( 1 3 ) 1.. Pauling, I ) . Pressman. 1) 13. C'ampt,rll and C . lkedss, T H I S JUI'RXAI., 64, :
