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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 172, 510-523 (1976)
lmmunochemical Studies on Blood Groups
Purification, Chemical and lmmunochemical Properties of Blood Group-Active
Glycoproteins from Horse Gastric Mucosae I,*
WALTER NEWMAN AND ELVIN A. KABAT
Division of Chemical Siology and the Departments of Microbiology, Human Genetics and Deuelopment, and Neurology, College Physicians and Surgeons, Columbia University, and the Neurological Institute,
Presbyterian Hospital, New York, New York 10032
Received July 3, 1975
Digestion of the gastric mucosae of 10 horses with pepsin or Pronase was followed by phenol/ethanol fractionation. Chemical and immunochemical examination of the frac- tions showed the mucosae to possess various combinations of A, B and H activities. Most were B-active, three had weak A activity, one had strong H activity and the remainder were weakly H-active; one mucosa possessed neither A, B nor H activity. Digestion with pepsin or Pronase of different portions of the same mucosa yielded products equivalent in serological and most chemical properties. Materials digested by Pronase tended to have less peptide nitrogen than those treated with pepsin. Fractions with the strongest serological activities contained significantly higher amounts of carbohydrate and lesser amounts of peptide nitrogen than those with weak A, B or H activity or with no activity. All mucosae, independent of their A, B or H activity, reacted with concanavalin A. The fractions precipitable by 10% ethanol from 90% phenol reacted most strongly.
The intervening 25 years since blood group substances from horse gastric mu- cosae were last studied (1) have seen the development and refinement of powerful techniques for isolation, purification and identification of small amounts of oligosac- charides. Especially important are the NaOH-NaBH,-catalyzed elimination and reduction from blood group-active mate- rials of the carbohydrate portion linked to serine and threonine (2, 31, the develop- ment of new methods for calorimetric (4, 5) and gas-liquid chromatographic (5-7) and mass spectrometric (8, 9) analyses of con- stituent sugars, the development of chro- matographic methods (5) and, most re-
1 Supported by grants from the National Science Foundation, No. BMS-72-02119A02 and 32543 X-l, and a Program Project Grant from the National Institutes of Health, No. 5P0 GM 18153-05.
2 From Part I of a dissertation submitted by W. Newman in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University, New York. This article is No. LIX of a series; the previous article appeared in Biochemistry (1973) 12, 5355- 5360.
cently, of high pressure liquid chromatog- raphy (10) for resolution of complex mix- tures of monosaccharides (11,12) and oligo- saccharides (13).
Horse gastric mucosae possess A and B activities and were shown to be a potent source of blood group B materials when there was interest in absorbing the alloag- glutinins from group 0 blood prior to trans- fusion (14, 15). Early studies demonstrated the overall chemical and immunochemical similarity of B substances from horse mu- cosae (1, 16) to those from human sources (17, 18). Horse blood groups based upon hemagglutination reactions with horse erythrocytes (19) bear no known relation to the A and B substances from human tis- sues and secretions or to those from horse gastric mucosae. Horse saliva contains A and B substances, but the activities in sa- liva do not correlate with those from the mucosa of the same horse (15, 16).
This study deals with the isolation, puri- fication and chemical and immunochemi- cal characterization of blood group sub- stances from horse gastric mucosae. H ac- tivity, which had not previously been rec-
510 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
HORSE BLOOD GROUP-ACTIVE GLYCOPROTEINS 511
ognized in horse materials, was found by using lectins from Ulex europeus and Lo- tus tetragonolobus. Despite these addi- tional assays, one mucosa showed no A, B or H activity. While preparing blood group substances from horse mucosae (1) a tenta- tive identification of fucose in the dialy- sates was made. It seemed that the condi- tions of peptic digestion (pH 1.8-2.0, 37”C, 72 h) might have been sufficient to hydro- lyze some fucose (20). Accordingly, prod- ucts of peptic digestion of the same as well as different mucosae were compared with those isolated by digestion with Pronase, a mixture of proteases from Streptomyces griseus (211, at neutral pH. No dialyzable fucose and no significant differences in yield or blood group activity were found. Blood group B- and H-active glycoproteins of high serological activity were isolated by both procedures. After purification, Pronase-digested materials contained less peptide nitrogen than did those from pep- sin digestion. Many of the fractions con- tained considerably more fucose than had been found previously (1). Some mucosae showed weak A activity and all had vary- ing amounts of Con A3 activity.
MATERIALS AND METHODS Gastric mucosae from 10 individual horses were
obtained from Roth Packing Co., Philadelphia, Pa., and were stored on Dry Ice prior to use. They were numbered 7-16 for continuity with an earlier study in this laboratory on six horse mucosae (1). The amounts of each used are in Table Ia.
Pronase was from Calbiochem, Grade B, Lot 73716, 43,000 PUK units/g; pepsin from Worthing- ton, 2x crystallrzed. The buffer for Pronase diges- tions was 0.2 N Tris-HCl, pH 7.8, 0.008 M CaCl,. For pepsin digestion 0.02 M citrate-HCl, pH 2.0, was used. Mucosae were homogenized in a Waring Blen- dor at 4°C with the buffer in which they were to be digested. Mucosae 8 and 11, however, were homoge- nized in distilled water at 4°C. Digestions were car- ried out in a total volume of 1-2 liters in the pres- ence of toluene at 37°C for 72 h with adjustment to pH 2.0 twice daily for pepsin and to pH 7.8 for Pronase digestion. Mucosae 7, 9 and 10 were di- gested by Pronase and 12-16 by pepsin. Mucosae 8 and 11 were divided in half, the appropriate buffers added and one-half digested by pepsin, the other by
3 Abbreviations used: N, nitrogen; Fuc, 6-deoxy- galactose; GalNAc, 2-acetamido-2-deoxygalactopyra- nose; GlcNAc, 2-acetamido-2-deoxyglucopyranose; Gal, galactopyranose; ConA, concanavalin A.
Pronase. The amount of enzyme used was based on the weight of mucosa; on day 1, 1 mg of Pronase per 5-7 g (wet weight) of gastric mucosa was added. On day 2, an additional 20 or 40 mg of Pronase were added to each flask. For digestion with pepsin, 1 mg per 5-7 g (wet weight) of mucosa was added; an additional 15 mg were added on day 2.
After treatment with enzyme, digests were di- alyzed against 0.1 N NaCl at 4°C for 8 h (22) and dialysates tested for methylpentose (23). The digests were filtered through glass wool and 2-3 g of sodium acetate plus 2-3 volumes of 95%’ ethanol were added to the clear filtrates. The flocculent white precipi- tate was centrifuged, washed with 95% ethanol, dried over P,O, in a desiccator, dissolved in distilled water, centrifuged to remove insoluble material and lyophilized. It was then extracted with 90% phenol as schematized in Fig. 1 (17, 24). The phenol-insolu- ble fraction from the Pronase-digested portion of mucosa 8 was further digested with pepsin. On day 1, 5 mg of pepsin was added to 125 mg of phenol- insoluble fraction in 10 ml of citrate-HCI buffer. An additional 5 mg of pepsin was added on day 2. After a 72-h digestion at 37°C (with toluene), the material was reisolated by ethanol precipitation in the pres- ence of sodium acetate. Refractionation was carried out as shown in Fig. 1.
All fractions from phenol/ethanol were assayed for A, B and H activities by hemagglutination inhibi- tion and for Con A-precipitating ability. The most active B fractions from hemagglutination inhibition were tested for their capacity to precipitate anti-B. Hemagglutination inhibition assays were performed with a Takatsy microtitrator (Cooke Engineering Co., Alexandria, Va.). B activity was assayed using a 1:50 dilution of anti-B serum 310, (25) which had been produced by immunization of an A individual with purified horse B substance, horse 4, 25% (1); A activity with a 1:50 dilution of anti-A serum Chris D, (26); H activity with a 109 saline extract of Ulex europeus seeds and 2% suspension of human B, A or 0 erythrocytes, respectively. The antibody or lectin used contained 4-8 hemagglutinating units. Le and H activities of the combined B-active and non-B- active materials (see Results) were kindly assayed by Dr. R. E. Rosenfield of Mount Sinai Hospital, New York, using an Autoanalyzer (27, 281.
Concanavalin A was isolated from jack bean meal according to Ref. (291 by adsorbing the lectin on Sephadex G-50 and eluting with methyl a-n-gluco- side. Quantitative precipitin curves for the various fractions with Con A were determined relative to dextran B 1355-S-4 (30) as standard. Tubes con- tained varying amounts of antigen and 8.1 pg of Con A N in 60 ~1 of 1 M NaCl; the total volume was adjusted to 260 ~1 with 0.018 M phosphate buffer, pH 7.2, in 0.9% saline. The N content of the washed precipitates was determined by the ninhydrin method after digestion with sulfuric acid (2). The lectin from Lotus tetrogonolobas (33) had been puri-
512 NEWMAN AND KABAT
Enzylw? digested ma;erial Dialysis at 4 against 0.1 M NaCl, the” distdled Water
Jf
C%ntrifllge Add sodium acetate and 2 volumes 95 percent ethanol
Dried ethanol precipitate Extract with 90 percent phenol
phenol’i”soluble supernatant *, ~i$W&itb 1057,etha”ol 1 t
1;; ;jzizi;
supernatant : precipitate with 20% ethanol in 90% phenol
pkclpitate supernatant
0 s”pel’nata”t
phenol insoluble precipitate with 2
I‘ 10% ethanol in
pkcipitate 90% phenol
p+ata”t
supernatant precipitate 10% 2x precipitate with with 10% ethanol
3 20% ethanol i” in 90% ph no1 90% phenol
pkxipitate I
supematant precipitate super atant 20% from 2nd 10% 10% from 1st F
4 20% precipitate with , 5 f$+t+in
precipitate supematant 20% 2x I
6 precipitate with 25% ethanol i” 90% phenol
pkcipitate 25%
7
supenlatant
FIG. 1. Outline of the procedure for fractionation with 90% phenol and ethanol (17) of pepsin- or Pronase-digested blood group glycoproteins. The seven fractions are numbered in order of increasing ethanol required for precipitation from 90% phenol.
tied by adsorption on polyleucyl hog A+H substance and elution with LFUC. Each tube contained 6.2 pg of lectin N in a total volume of 225 ~1. JS phenol insolu- ble (21, an HLeh-active material from human ovar- ian cyst fluid was used as standard. Quantitative precipitin reactions with anti-B serum 310, and with Lotus lectin were performed on a microscale (23). Tubes containing antigen and antiserum were incu- bated at 37°C for 1 h and kept at 4°C for 5-7 days and centrifuged in the cold, and the precipitates were washed twice with 0.4 ml of chilled saline. Nitrogen in the washed precipitates was determined as above (2).
Calorimetric methods for determination ofhexosa- mines, N-acetylhexosamines, galactosamine, meth- ylpentose (fucose) and hexose (galactose) have been described previously (2, 4, 23). Sialic acid was deter- mined by the thiobarbituric acid method (34).
Reference blood group substances used were Beach phenol insoluble (35), a B-active material from human ovarian cyst fluid; hog 25 (36) and hog 33B (37) from H-active hog gastric mucosae; MSM, an A-active material from human ovarian cyst (2) and N-l 20% from 2nd 10% (381, an Lea-active mate- rial from human ovarian cyst fluid.
RESULTS
Table Ia shows the yields, compositions and serological activities of various frac- tions from phenol/ethanol purification (Fig. 1). Mucosae generally yielded consid- erable amounts of three to five of the possi- ble seven fractions shown; the remaining fractions were disregarded if less than 10 mg or if of negligible carbohydrate con- tent. Yields of purified blood group sub- stances were approximately the same for each mucosa; l-2 mg/g wet weight (No. 7- 11) or 2-6 mg/g dry weight (No. 12-16) whether pepsin or Pronase was used for digestion.
Table II shows the A, B, H, Lea and Leh activities of the phenol/ethanol fractions in Table Ia. Two horse blood group materials were obtained for further study by combin- ing fractions from Table Ia. One, B-active, consists of the most active B fractions, de- noted in Table Ia by an asterisk (*); the
TABLE Ia
ANALYTICAL COMPOSITION OF HORSE BLQOD GROUP FRACTIONS
Fraction Yield (ma)”
Percent composition (weight)
Total Methyl- Hexose N pentose (galac-
(fucose) tose) -
Horse 7, Pronase di- gested, starting weight (wet) 431 g (strong B, weak A, H)
Phenol insoluble* 10% 2x* 10% from 1st 20%*
Horse 8, pepsin digested, starting weight (wet) 202 g (strong B, weak A, H)
10% 2x’ 20% from 2nd LO%* 20% 2x 25%
Horse 8, Pronase di- gested, starting weight (wet) 202 g (strong B, weak H)
Phenol insoluble* 10% 2x* 20% from 2nd 10%
Horse 8, Pronase-digested phenol-insoluble frac- tion, pepsin digested, 132 mg (strong B, weak H)
Phenol insoluble* Horse 9, Pronase di-
gested, starting weight (wet) 170 g (strong B, weak H)
Phenol insoluble* 10% 2x* 20% from 2nd 10% 10% from 1st :!O% 20% 2x*
Horse 10, Pronase di- gested, starting weight (wet) 326 g (in- active)
Phenol insolublet 10% Phenol insolublet 10% 2xt 20% from 2nd lO%t 10% from 1st 20% t 20% 2xt
Horse 11, pepsin digested, starting weight (wet) 130 g (strong H)
10% 2xt 20% from 2nd 10% t 20% zxt 25?Jt
126 (113) 4.5 9.4 25.9 19.5 22.0 7.3 3.0 193 (161) 5.7 7.4 24.5 25.7 22.3 11.7 3.7
84 (68) 6.1 9.2 28.4 21.2 16.7 10.1 4.5
38 (34) 5.9 9.4 22.6 18.0 14.7 8.1 4.5 153 (122) 5.8 10.4 26.0 19.3 17.3 9.3 4.3
19.5 6.7 5.6 19.4 23.9 20.0 10.9 4.8 13.0 7.5 5.4 19.3 16.5 16.5 5.3 6.2
132 (46) 5.1 11.3 26.2 19.4 22.8 7.7 3.6 47 (27) 5.4 6.6 23.9 18.4 16.2 7.3 4.0 17 6.5 5.6 20.2 20.9 18.4 7.9 4.9
65 (46) 4.4 10.6 27.6 22.5 26.8 8.3 2.7
130 (96) 5.4 9.6 25.8 17.1 17.8 9.5 4.1 91 (70) 5.7 6.7 23.3 22.8 18.9 10.6 3.9 18 6.0 9.4 25.2 17.9 12.3 10.4 4.6 38 6.4 11.0 27.4 16.0 10.8 10.5 5.2 98 (60) 5.9 10.1 27.7 18.3 12.5 11.7 4.5
87 (68) 5.1 2.0 12.5 16.7 22.0 3.5 3.8 10 (4) 4.6 1.6 12.6 16.1 22.1 2.1 2.9
120 (103) 7.7 3.3 14.1 19.1 15.5 3.6 6.2 14 (5) 6.9 3.6 13.7 15.3 12.3 4.1 5.7
154 (138) 8.8 4.2 14.0 14.8 11.2 4.0 7.6 68 (50) 8.7 4.2 15.1 16.1 11.9 3.3 7.4
28 (17) 5.8 117 (98) 5.9 84 (75) 6.9 44 (34) 7.3
8.3 21.2 7.2 17.3 7.5 20.4 6.9 22.4
-
Hexos- N-acetyl- Galac- Pep&de amine” hexos- tosamine
amine
25.7 20.8 8.8 3.8 25.4 22.7 10.1 3.9 29.3 24.7 12.2 4.6 19.0 11.2 14.3 5.8
513
TABLE Ia-Continued
Fraction Yield (mg)”
Percent composition (weight)
Total Methvl- Hexose Hexes- N-acetvl- Galac- Peptide N pent&e (galac- amine” hexos- tosamine
amine
Horse 11, Pronase di- gested, starting weight (wet) 130 g (strong H)
Phenol insolublet 104 2xt 20% from 2nd 10% t 10% from 1st 20% t 25Yct
Horse 12, pepsin digested, starting weight (dry) 33 g (moderately strong HI
Phenol insolublet 10% 2xt 20% from 2nd 10% t 20% 2xt
Horse 13, pepsin digested, starting weight (dry) 31 g (strong B, weak H)
Phenol insoluble* 10% 2x* 2O’X from 2nd 10% * 20% 2x
Horse 14, pepsin digested, starting weight (dry) 46 g (strong B, Hl
10% 2x* Horse 15, pepsin digested,
starting weight (dry) 31 gm (moderate B, weak A and H)
Phenol insoluble 104 2x 20%. 2x 25%
Horse 16, pepsin digested, starting weight (dry) 42 g (weak Hf
Phenol insoluble? 10% phenol insolublet 10% 2x1 20% from 2nd lO%t 204 2xt
95 (76) 5.3 3.3 15.5 24.6 23.4 8.8 3.4
19 (9) 5.7 7.2 20.6 21.2 19.6 8.6 4.1 53 (38) 6.1 6.8 23.6 23.3 19.1 8.7 4.3 24 (16) 6.3 4.4 19.9 27.4 24.1 7.7 4.8 39 (30) 6.8 4.8 21.3 30.4 27.7 7.3 4.4
18 (10) 9.2 2.7 14.4 17.7 15.0 3.1 7.8 24 (12) 5.8 3.2 19.3 19.1 17.8 5.4 4.3 75 (58) 6.5 4.8 17.7 24.1 22.8 8.0 4.6 27 (18) 7.8 4.2 15.8 27.4 22.8 9.1 5.7
44 (30) 8.9 5.6 17.2 18.3 17.0 5.1 7.5 63 (39) 7.2 5.0 18.5 20.0 17.3 6.9 5.6 57 (43) 6.3 5.2 17.0 20.4 14.5 10.5 4.7 36 8.6 3.0 14.4 15.7 11.2 7.2 7.4
99 (80) 6.4 5.6 21.6 27.6 23.2 11.5 4.3
29 8.6 2.8 15.0 13.2 12.0 4.6 7.6 29 6.9 3.4 18.0 14.3 12.1 3.8 6.0 14 8.8 3.3 15.9 12.4 8.5 4.4 7.8 15 8.7 3.7 18.0 14.0 7.4 7.3 7.6
18 (7) 17 (71
108 (841 18 (6) 50 (36)
9.1 2.0 12.7 10.4 10.0 2.4 8.3 6.1 0.7 14.7 8.9 8.2 1.6 5.4 6.5 3.8 18.0 22.5 20.3 5.4 4.7 5.0 1.1 10.7 6.6 5.5 1.6 4.5 7.7 2.3 15.3 12.6 8.8 5.6 6.7 5.9 2.8 13.4 12.3 6.4 3.1 4.9 25% t 15 (6)
ifucose) iose)
n Omitted are those fractions of less than 10 mg yield or with negligible amounts of carbohydrate. Values in parentheses under the yield indicate amounts pooled into either the B-active or the non-B-active materials for further study in the following two papers. In addition 281 mg from horse 2 (1) was added to the B-active sample and the following amounts were added to the non-B-active sample: 172 mg of horse, 3, 151 mg of horse 5, and 600 mg of horse 6 (1).
n In the hexosamine assay N-acetylglucosamine and N-acetylgalactosamine give equal color intensities whereas in the N-acetylhexosamine assay N-acetylgalactosamine gives 31% of the color intensity of N- acetylglucosamine.
r Calculated as difference between percentage of total N and percentage of hexosamine N. * Indicates most active B fractions by hemagglutination inhibition, Table II. See also Fig. 2 for precipita-
tion of these fractions by anti-B serum 310,. These fractions were later combined (391. t Indicates non-B active fractions combined. See Fig. 2 and Table II for immunochemical properties.
514
HORSE BLOOD GROUP-ACTIVE GLYCOPROTEINS 515
TABLE Ib
SUMMARY OF RANGE IN ANALYTICAL COMPOSITION OF FRACTIONS FROM TABLE Ia FOR ALL FRACTIONS, MOST
ACTIVE B FRACTIONS” AND FRACTIONS OBTAINED IN LARGEST YIELD FROM EACH MUCOSA
Fractions Percent composition (weight)
Total Methyl- Hexose Hexos- N-acetyl- Galactos- Peptide N pentose (galac- amine hexos- amine N
(fucose) tose) amine
All fractions Low 4.4 0.7 10.7 5.5 6.6 1.6 2.7 High 8.3 11.3 28.4 27.7 30.4 14.3 8.3
Most active B fractions Low 4.4 5.0 17.0 12.5 17.1 5.1 2.7 High 8.9 10.6 28.4 26.8 27.6 11.7 7.5
Largest fractions Low 5.1 3.3 14.0 11.2 14.8 4.0 3.4 High 8.8 11.3 26.2 23.4 27.6 11.7 7.6
m Indicated in Table Ia by an asterisk (*).
other, non-B-active, includes materials in Table Ia with a dagger (t). Their proper- ties are described more fully in the follow- ing article (39). As shown at the bottom of Table II, no Lewis activities were detected in these materials, while H activity with the Ulex lectin was comparable to the hu- man standard, JS phenol insoluble.
Hemagglutination inhibition data, Ta- ble II, on the individual fractions from Ta- ble Ia indicate that various combinations of A, B and H activities were present in each mucosa as follows: strong B and H in mucosa 14; strong B, weak H in 9 and 13; strong B, weak A and H in 7 and 8; strong H in 11; weak H in 16; moderately strong H in 12; moderate B, A and H in 15, and mucosa 10 was inactive. Within each mu- cosa, individual fractions showed some var- iation in activity. Six of ten mucosae had B activity; five of these, mucosae 7, 8, 9, 13 and 14, yielded materials comparable in potency to the human B standard. A and H activities, except for H-active mucosa 11, were always less than their standards. Mu- cosae with A. or B activity also showed weak H activity, except for strongly B- active mucosa 14, which was also strongly H-active. The weak A activity of mucosa 8 was found only in pepsin-digested frac- tions. Pronase-digested mucosa 7 showed weak A activity.
Certain fractions from pepsin and Pro- nase digestions had high serological activi- ties (Tables II and III). The main fractions
of Pronase-digested mucosa 8 (phenol insol- uble and 10% 2 x 1 were less active in inhibi- tion of B hemagglutination than the pep- sin fractions (10% 2x and 20% from 2nd 10%) while, in inhibition of H hemaggluti- nation, the most active fractions from the Pronase-digested portion of mucosa 11 were as potent as those from pepsin treat- ment. In phenol/ethanol fractionation, B- active mucosae 7, 8, 9 and 13 (Tables Ia and II) showed a distribution pattern that differed somewhat depending on the en- zyme used in digestion. Pepsin treatment tended to give the largest yields and the most active materials in the 10% 2x and 20% from 2nd 10%; with Pronase these were generally the phenol insoluble and 10% 2x. Fractions from the non-B-active pepsin-digested mucosae (No. 11, 12, 15 and 16) and an inactive Pronase-digested mucosa (No. 10) tended to be more uni- formly distributed.
Table Ia demonstrates the wide range of calorimetric values for the 50 fractions ana- lyzed. The range in values for fractions from the same mucosa was always less than for fractions from different mucosae. Table Ib summarizes data from Table Ia on the ranges in analytical composition for all fractions, the most active B fractions and those from each mucosa obtained in high- est yield regardless of their serological ac- tivity. The ranges of values for hexosa- mine, N-acetylhexosamine and galactosa- mine were substantially narrower both for
TAB
LE
II
HE
MA
GG
LUTI
NA
TIO
N
INH
IBIT
ION
A
SS
AY
S O
F H
OR
SE
BLQ
OD
GR
OU
P F
RA
CTI
ON
S F
OR
A,
B,
H,
Le”
AN
D L
eh
AC
TIV
ITIE
S
Min
imum
co
ncen
tratio
n @
g/m
l) gi
ving
co
mpl
ete
inhi
bitio
n of
hem
aggl
utin
atio
n”
Sta
ndar
d B
A
H
Bea
ch
phen
ol
inso
lubl
e M
SM
Hog
25
H
og
25
Hog
33
B
__-~
_ 6.
3 3.
1 6.
3 1.
5 6.
8 1.
6 0.
8 0.
8 6.
3 6.
3 12
.0
Hor
se
7 (P
rona
se)
Phe
nol
inso
lubl
e 10
% 2
x 10
% f
rom
1s
t 20
%
Hor
se
8 (p
epsi
n)
10%
2x
20%
fro
m
2nd
10%
20
% 2
x 25
%
Hor
se
8 (P
rona
se)
Phe
nol
inso
lubl
e 10
% 2
x 20
% f
rom
2n
d 10
%
Hor
se
8 (p
epsi
n-P
rona
se)
Phe
nol
inso
lubl
e H
orse
9
(Pro
nase
) P
heno
l in
solu
ble
10%
2x
20%
fro
m
2nd
10%
10
% f
rom
1s
t 20
%
20%
2x
Hor
se
10 (
Pro
nase
) P
heno
l in
solu
ble
10%
phe
nol
inso
l. 10
% 2
x 20
% f
rom
2n
d 10
%
10%
fro
m
1st
20%
20
% 2
x H
orse
11
(pe
psin
) 10
% 2
x 20
% f
rom
2n
d 10
%
20%
2x
25%
15
4.5
20
7.5
4.5
5
30
>960
24
0 12
0 4.
5 28
5 36
14
3 5
159
159
317
2.4
1.2
2.1
151
303
133
2.5
2.5
2.1
108
158
131
6.8
13.7
10
21
9 10
9 31
8 41
41
18
>1
310
655
580
33
33
79
73
33
33
40
73
19
18
31
>123
0 >1
230
8.2
33
37
523
>104
5 >1
170
54
54
62
>173
0
62
62
62
73
62
34
17
~108
0 >1
280
68
8.4
2.7
16.7
16
.7
>107
0 26
8 5.
5 1.
4 >1
400
88
18.1
19
.1
>116
0 14
5 74
18
.6
>119
0 29
7 43
43
>1
370
172
88
73
149
171
>139
0 >1
390
>139
0 >1
390
>139
0 >1
390
>126
0 >1
260
>126
0 >1
260
630
>114
0 >1
140
>114
0 57
0 57
0 >1
040
>104
0 >1
040
>104
0 >1
040
>118
0 >1
180
>118
0 >1
180
>118
0 >1
140
>114
0 >1
140
> 11
40
>114
0
>115
0 >1
150
>115
0 9
>103
0 >1
030
>103
0 16
>1
250
>125
0 >1
250
20
>129
0 21
290
>129
0 81
9 9 16
20
20
.- ._
-
,. ,._
-
._ -
-
> 10
00
>104
0 >1
350
>109
0 >
1000
16
16
10.3
41
43
43
16
8
6.1
12.3
--&se
11
(P
rona
se)
-
Phe
nol
inso
lubl
e >l
OO
O
>lO
OO
16
16
10
% 2
x >1
040
>104
0 16
8
20%
fro
m
2nd
10%
>1
350
>135
0 5
11
10%
fro
m
1st
20%
>1
090
>109
0 3
17
25%
>
1000
11
000
16
31
Hor
se
12 (
peps
in)
Phe
nol
inso
lubl
e :
1080
>
1080
,1
08o
34
34
10%
2x
> 14
30
>143
0 >1
430
22
45
20%
fro
m
2nd
10%
>1
450
> 14
50
>145
0 23
23
20
% 2
x >1
160
>116
0 >1
160
18
36
Hor
se
13 (
peps
in)
Phe
nol
inso
lubl
e 8
8 >1
020
64
64
10%
2x
41
10
>132
0 83
83
20
% f
rom
2n
d 10
%
43
11
>137
0 17
1 34
3 20
% 2
x 16
7 84
>1
340
>134
0 H
orse
14
(pep
sin)
10
% 2
x 6.
1 6.
1 >1
570
12
25
Hor
se
15 (
peps
in)
Phe
nol
inso
lubl
e 30
5 38
38
10
% 2
x 73
73
73
20
% 2
x 26
5 13
3 25
%
>115
0 57
5 57
5 H
orse
16
(pe
psin
) P
heno
l in
solu
ble
>121
0 15
2 15
2 10
% P
heno
l in
solu
ble
>126
0 63
0 63
0 10
% 2
x >1
360
85
85
20%
fro
m
2nd
10%
>1
290
>129
0 >1
280
20%
2x
>124
0 >1
240
> 12
40
25%
13
40
>340
>3
40
-~__
---
Le”
Le”
H
Con
cent
ratio
n (~
glm
l)
Le”,
Leh
and
H
activ
ities
H
orse
no
n-B
-act
ive
bloo
d gr
oup
subs
tane
(p
ool)
- 1.
9 0.
70
111
Hor
se
B-ac
tive
bloo
d gr
oup
subs
tanc
e (p
ool)
- 19
.0
0.95
13
0 S
tand
ard
hum
an
HLe
” su
bsta
nce,
JS
phe
nol
inso
lubl
e 2.
2h
0.09
3 1.
3 11
0 S
tand
ard
hum
an
Lea
subs
tanc
e,
N-l
Lea
20%
fro
m
2nd
10%
0.
062
6.2
- 95
o Se
vera
l ex
perim
ents
w
ere
perfo
rmed
; ac
tiviti
es
are
expr
esse
d re
lativ
e to
the
en
dpoi
nt
for
the
stan
dard
bl
ood
grou
p su
bsta
nce
assa
yed
at
the
sam
e tim
e,
sinc
e tw
ofol
d se
rial
dilu
tion
valu
es
may
di
ffer
by
a fa
ctor
of
2.
b N
umbe
rs
repr
esen
t m
illilit
ers
of b
lood
gr
oup
subs
tanc
e ne
eded
fo
r 50
% in
hibi
tion
of h
emag
glut
inat
ion
by
1 m
l of
ant
ibod
y.
Ant
i-Lea
di
lute
d 1:
300
for
use;
ant
i-Leb
di
lute
d 1:
lOO
for
use;
U
ler
extra
ct
dilu
ted
1:30
for
use.
D
ash
(-)
indi
cate
s no
act
ivity
se
en.
Goa
t an
ti-Le
a an
d -L
eb w
ere
kind
ly
sup-
pl
ied
by
Dr.
Don
ald
Mar
cus
(52)
.
38
19
73
146
265
133
144
72
>121
0 >1
210
>126
0 ~1
260
1136
0 >1
360
>129
0 >
1290
>
1240
>1
240
>940
>3
40
518 NEWMAN AND KABAT
TABLE III
BLOOD GROUP B ACTIVITY AND METHYLPENTOSE CONTENT OF FRACTIONS OF Two INDIVIDUAL HORSE GASTRIC MUCOSAE OBTAINED BY
PEPSIN AND PRONASE DIGESTIONS
Pepsin digested Pronase digested
Yield Methyl- Methyl- Hemag- Yield Methyl- Methyl- Hemag- (mg) pentose pentose glutin- (mg) pentose pentose glutin-
(o/o) (mg) ation in- (%) (mg) ation in- hibition” hibition”
Horse 8 (B-active) Phenol insoluble 10% Phenol insol. 10% 2x 20% from 2nd 10% 10% from 1st 20% 20% 2x 25% Total
Standard B
Horse 11 (H-active) Phenol insoluble 10% Phenol insoluble 10% 2x 20% from 2nd 10% 10% from 1st 20% 20% 2x 25% Total
Standard H
3.7 5.7
38 153
6 19.5 12
238
3 3
28 117
2 84 44
281
11.7 0.6 6.5 5.8 2.7 4.0 5.1
3.4 2.3 8.3 7.2 4.4 7.5 6.9
-
0.43 0.03 2.50 8.90 0.16 0.78 0.61
13.41
0.10 0.07 2.32 8.42 0.09 6.30 3.04
20.34
>1230 132 >1140 46
2.4 47 2.5 17
303 4 6.8 9
41 7 262
6.3b
n.d.’ n.d. 9
16 n.d. 20 81
6.3d
95 12 19 53 24 2
39 244
10.8 0.8 9.1
11.3 2.7 4.0 5.8
3.3 2.7 7.2 6.8 5.8 7.4 4.8
14.25 19 0.37 139 4.30 8.2 1.92 54 0.11 >1360 0.40 211 0.41 670
21.76 6.3*
3.14 16 0.32 32 1.37 16 3.60 5 1.39 9 0.15 nd. 1.87 16
11.84 6.3d
n Minimum concentration @g/ml) giving complete inhibition of hemagglutination. b Standard B substance, Beach phenol insoluble. c Not done. d Standard H substance, hog 25.
the most active B materials and for those fractions obtained in highest yield. B-ac- tive fractions generally had higher fucose and gala&se as compared with non-B-ac- tive materials; the strong H-active mucosa 11 had somewhat lower fucose and the weak A, B, H or inactive mucosae had the lowest fucose. A number of fractions was analyzed for sialic acid, none was found.
Mucosae 7, 8, 9, 13 and 14 with strong B and/or H activities had substantially higher carbohydrate contents than did the others and showed somewhat lower pep- tide nitrogen. Strong serological activity correlated with the higher proportion of carbohydrate and lower peptide content in- dependently of method of digestion even though the Pronase-digested materials tended to have lower peptide nitrogen
than the pepsin-digested fractions. The weak A activity of mucosae 7,8 and 15 was not associated with higher amounts of ga- lactosamine than found in non-A-active materials.
Table III and Figs. 2 and 3 compare frac- tions prepared by pepsin and Pronase digestion from portions of the same mu- cosa. All fractions, including those in low yield for both 8 and 11 are included in Table III. Mucosa 8 is strongly B-active and weakly A- and H-active; mucosa 11 is strongly H-active. For each, the yields of purified blood group substances from pep- sin or Pronase digestions were about the same. Methylpentose, the residue most likely to be hydrolyzed under the condi- tions of peptic digestion is essentially unaf- fected, and no methylpentose was detected
HORSE BLOOD GROUP-ACTIVE GLYCOPROTEINS
200~1 HUMAN ANTI-B SERUM,SUBJECT 3103 Total Volumr~3OOpl
519
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 0 2 4 6 8 IO 0 2 4 6 8 ,o 12
MlCRclGRAMS nNT,GEN noLm
FIG. 2. Precipitation by human anti-B (subject 310,) of the most active B fractions and standard human blood group B substance, Beach phenol insoluble. Arrows and numbers refer to micrograms of antigen required to precipitate 50% of the maximum amount of N precipitated by the Beach standard. (01, Phenol insoluble; (01, 10% 2x, (V), 20% from 2nd 10%; (r), 10% from 1st 20%; (01, 20% 2x. In (e) are precipitation data with the B-active and non-B-active combined materials.
B LOTUS ECTIN 6.2pg N Total Volume- 225)d
3 HORSE II PEPSIN CYGESTED HORSE II PROMSE ‘VGESTED
f FIG. 3. Quantitative precipitin curves of the lec-
tin from Lotus tetrugonolobus with fractions from pepsin- or Pronase-digested portions of strongly H- active mucose 11. (01, Standard H substance, JS phenol insoluble (2); (O), phenol insoluble; CO), 10% 2x; (V), 20% from 2nd 10%; (r), 10% from 1st 20%; CO), 20% 2x and (+l, 25%.
in dialysates of pepsin or Pronase digests. Highly B-active substances were obtained from mucosa 8 by either enzyme, though the pepsin-treated materials were slightly more active in inhibition of hemagglutina- tion. H-active blood group substances of equal potency were obtained from both por- tions of mucosa 11.
The most active B fractions in inhibition of hemagglutination, designated by an as- terisk (*) in Table Ia, were tested with the same anti-B serum for their precipitating abilities. The results are shown in Fig. 2. Figure 2a is a precipitin curve with the standard human B substance, Beach
phenol insoluble. The quantity of antigen precipitating 50% of the nitrogen precipi- tated by the Beach standard is indicated for each curve by an arrow and a number. Fractions from both digestions are as ac- tive as the standard. The somewhat weaker inhibition of B hemagglutination by Pronase-digested fractions from mucosa 8 as compared with pepsin-digested por- tions is associated with precipitation of somewhat less total nitrogen at the maxi- mum, although. no difference was noted in the quantities needed to precipitate 50% of the total specific precipitate nitrogen. The phenol-insoluble fraction from Pro- nase digestion of mucosa 3 (Fig. 24 was unchanged in precipitating ability by sub- sequent peptic digestion (Fig. 2d). The non-B-active material (Fig. 2e) precipi- tated a small amount of antibody nitrogen but much less than any of the B-active substances.
Figure 3 shows results of the precipita- tion of Lotus lectin by pepsin- and Pro- nase-digested fractions of H-active mucosa 11; both were equal in precipitating ability to the standard human H substance, JS phenol insoluble. In antigen excess the Pro- nase-digested portions tended to go into the inhibition zone more readily.
Table IV demonstrates the effect of pep- sin digestion on the phenol-insoluble frac- tion of the Pronase-digested portion of mu-
520 NEWMAN AND KABAT
cosa 8; the solubility of the material in 90% phenol was not altered; all active material remained phenol insoluble. Some protein was removed as indicated by the lower total and peptide nitrogen. No significant changes in the hemagglutination inhibit- ing (Table II) or precipitating power (Fig. 2d) were found, nor were there significant changes in sugar composition.
Figure 4 shows precipitation curves with Con A and all fractions of Table Ia relative to a standard dextran. There is substantial variation in the degree of reactivity of the individual horse fractions with Con A. This was unrelated to method of digestion (Figs. 4b, c, f and g); some are almost as good as the standard dextran (Figs. 4f, g, h and j) while others are considerably less active. In most instances the 10% 2x frac- tions are the most potent (Figs. 4a, d, f, j and 1). There is no association between Con A reactivity and blood group specific- ity. Some fractions, most frequently the phenol insoluble (Figs. 4a, c and d) and 25% (Figs. 4f, k and 1) showed negligible or
low Con A activity. No consistent associa- tion was observed between Con A reactiv- ity and either hexosamine or N-acetylhex- osamine content.
DISCUSSION
The findings confirm earlier studies (1, 16) showing that some horse gastric mu- cosae are a good source of blood group B- active substances roughly similar in their chemical and immunochemical properties to human saliva (1) and ovarian cyst blood group B substances (17, 40, 41). Other mu- cosae yielded strongly H-active, weakly A- or H-active and inactive products. No Lewis activities were found. All precipi- tated with Con A and thus are similar to blood group substances from hog gastric mucosae (32, 42). H activity in horse mu- cosae was not detected in an earlier study (11, but the reagent used was goat anti- Shiga serum absorbed with A,B cells and not the Ulen europeus orLotus tetragonolo- bus lectins. Hence the three of six mucosae designated as inactive might have been H-
TABLE IV
EFFECT OF PEPSIN DIGESTION ON PHENOL-INSOLUBLE FRACTION OF PRONASE-DIGESTED B-ACTIVE HORSE 8
Yield (mg) after pepsin digestion and refractionation (starting material 125 mgl
Phenol in- 10% 104 2x 20% from 2nd 10% from 20% 2x Total Recoverv soluble Phenol in-
soluble 10% 1st 20% (VI) ”
65.2 2.8 7.8 0.3 1.9 2.1 80.1 64
Percent composition of phenol-insoluble fractions
Total Peptide Methyl- Hexose Hexos- N-ace- Galactos- N N pentose (galactose) amine ty1- amine
hexos- amine
Pronase digested 5.1 3.6 11.3 26.2 19.4 22.8 7.7 Pepsin + Pronase digested 4.4 2.7 10.6 27.6 22.5 26.8 8.3
Minimum concentration (pg/ml) of each phenol insoluble fraction required for complete inhibition of hemagglutination
Activity B A H
Pronase digested Pepsin + Pronase digested Standard
19 >I230 68 17 >1280 68
6.3" 1.6" 6.3'
II Standard B substance, Beach phenol insoluble. D Standard A substance, MSM. f Standard H substance, hog 25.
HORSE BLOOD GROUP-ACTIVE GLYCOPROTEINS 521
CONCANAVALIN A 8.INg N Total Volume = 260~11
nOmE 7 HORSE 8 8 HORSE HORSE 9 PRONASE DIGESTED PEPSIN DIGESTED PRONASE OIGESlEO PRONASE DIGESTED
PEPSIN DIGESTED
7 PEPSIN DIGESTED
. ---
PEPSIN DlGES
FIG. 4. Quantitative precipitin curves of concanavalin A with blood group fractions from horse mucosae (Tables Ia and II). Dextran B 1355-S-4 is included as a standard in (a) (0) (30). CO), Phenol insoluble; (x), 10% phenol insoluble; (01, 10% 2x; CO), 20% from 2nd 10%; (r), 10% from 1st 20%, CO), 20% 2x and (+), 25%.
active. Only one of ten mucosae in the present study showed no A, B or H activ- ity.
The range of analytical values for the fractions as outlined in Table Ib is quite large, in agreement with earlier findings (1). The most highly B-active fractions tended to show higher carbohydrate con- tents, especially methylpentose and galac- tose. In the previous investigation (1) only one horse B fraction of 30 mg had a methyl- pentose content of 9.3%, the next highest value being 6.9%. Table Ia shows that at least six H-active fractions from three mu- cosae isolated in amounts of 84-153 mg had methylpentose contents of 9.2-11.3%. The next highest in methylpentose content was H-active mucosa 11; inactive mucosa 10 had the lowest values. This decrease suggests the existence of a genetic system involving precursor I, H- and B-active horse mucosae analogous to that in hu- mans (43-46). Moreover, no evidence of fucose being split off was found either by examination of dialysates from digestions or by differences in methylpentose con- tents of the pepsin- and Pronase-treated purified substances from individual mu- cosae. Pepsin digestion appears satisfac-
tory, but Pronase digestion has been used effectively with porcine submaxillary mu- tin (471, ovarian cyst and other glycopro- teins (48, 49) and is as satisfactory as pep- sin for horse gastric mucosae.
Yields of purified blood group substance are comparable to those in the previous study (1); no differences related to method of digestion were observed. Horse mate- rials differ from human ovarian cyst blood group substances of the same specificity in containing more peptide nitrogen and less methylpentose. The range in carbohydrate composition of different cysts (18) is nar- rower than that for horse mucosae.
It should be emphasized that a substan- tial contribution to the wide range in com- position of different mucosae is made by the different A, B, H and Con A specific- ities, each of which is associated with dif- ferent sugars.
The most active B materials are compa- rable to the standard human ovarian cyst substance in quantitative precipitation. Highly H-active blood group substance from mucosa 11 was as potent as human H substance in quantitative precipitation with the Lotus lectin and equivalent to hog H substance in inhibition of hemagglu-
522 NEWMAN AND KABAT
tination with the Ulex lectin. The most active B materials were isolated most of- ten in the phenol-insoluble and 10% 2x fractions from Pronase digests and from the 10% 2x and 20% from 2nd 10% frac- tions of pepsin digests. This difference based upon method of digestion was not seen in substances with A or H activities or with no activity. The most active Pro- nase-digested B materials tended to show somewhat lower peptide nitrogen.
Mucosae with strong B and/or H activ- ites contain significantly higher amounts of carbohydrate and lower amounts of pep- tide nitrogen than those with weak A or H activities. The high H reactivity with the Lotus lectin in all but one fraction from mucosa 11 would indicate the presence of type 2 chains (44) in horse mucosae with the following structure: ~Fucal - 2nGalPl -+ 4oGlcNAc.... (33).
Results with Con A extend earlier obser- vations on its reactivity with hog stomach A and H materials (32, 421, human gastric aspirates, saliva (50) and group 0 erythro- cyte membrane glycoprotein (51) and its failure to react with human ovarian cyst blood group substances (50). Most horse fractions reacted with Con A, probably in- dicating, as for other blood group sub- stances (32, 421, the presence of terminal nonreducing a-linked DG~cNAc; heteroge- neity of fractions with respect to this pa- rameter is present since the 10% 2x frac- tions were generally the most active, with considerable variation among the others and some showed negligible activity (Fig. 4).
Of interest is the finding that pepsin digestion of the phenol-insoluble fraction from Pronase-digested mucosa 8 did not alter the solubility in 90% phenol; after refractionation all active material re- mained phenol insoluble. Pepsin digestion of crude blood group substance will usually solubilize phenol-insoluble material (17). Purified blood group substance that is al- ready insoluble in 90% phenol presumably contains a minimum of peptide material; the additional peptide removed by pepsin left its insolubility in phenol unaltered. Consistent with an earlier study on horse mucosae Cl), little phenol-insoluble mate-
rial was isolated from peptic digests of horse gastric mucosae.
ACKNOWLEDGMENT
The expert technical assistance of Mr. Jerry Liao is gratefully acknowledged.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10. 11.
12.
13.
14.
15.
16.
17.
18.
19.
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