Immunochemical studies on blood groups

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

172, 510-523 (1976)

lmmunochemical Purification,

Studies

on Blood Groups

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 fractions 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 mucosae were last studied (1) have seen the development and refinement of powerful techniques for isolation, purification and identification of small amounts of oligosaccharides. Especially important are the NaOH-NaBH,-catalyzed elimination and reduction from blood group-active materials of the carbohydrate portion linked to serine and threonine (2, 31, the development of new methods for calorimetric (4, 5) and gas-liquid chromatographic (5-7) and mass spectrometric (8, 9) analyses of constituent sugars, the development of chromatographic methods (5) and, most re1 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, 53555360.

cently, of high pressure liquid chromatography (10) for resolution of complex mixtures of monosaccharides (11,12) and oligosaccharides (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 alloagglutinins from group 0 blood prior to transfusion (14, 15). Early studies demonstrated the overall chemical and immunochemical similarity of B substances from horse mucosae (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 tissues and secretions or to those from horse gastric mucosae. Horse saliva contains A and B substances, but the activities in saliva do not correlate with those from the mucosa of the same horse (15, 16). This study deals with the isolation, purification and chemical and immunochemical characterization of blood group substances from horse gastric mucosae. H activity, which had not previously been rec510

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

HORSE

BLOOD

GROUP-ACTIVE

ognized in horse materials, was found by using lectins from Ulex europeus and Lotus tetragonolobus. Despite these additional assays, one mucosa showed no A, B or H activity. While preparing blood group substances from horse mucosae (1) a tentative identification of fucose in the dialysates was made. It seemed that the conditions of peptic digestion (pH 1.8-2.0, 37”C, 72 h) might have been sufficient to hydrolyze some fucose (20). Accordingly, products 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 pepsin digestion. Many of the fractions contained considerably more fucose than had been found previously (1). Some mucosae showed weak A activity and all had varying 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 Worthington, 2x crystallrzed. The buffer for Pronase digestions 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 Blendor at 4°C with the buffer in which they were to be digested. Mucosae 8 and 11, however, were homogenized in distilled water at 4°C. Digestions were carried out in a total volume of 1-2 liters in the presence 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 digested 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-deoxygalactose; GalNAc, 2-acetamido-2-deoxygalactopyranose; GlcNAc, 2-acetamido-2-deoxyglucopyranose; Gal, galactopyranose; ConA, concanavalin A.

GLYCOPROTEINS

511

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 dialyzed 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 precipitate 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-insoluble 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 phenolinsoluble 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 presence 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 inhibition 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-Bactive 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-glucoside. Quantitative precipitin curves for the various fractions with Con A were determined relative to dextran B 1355-S-4 (30) as standard. Tubes contained 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 C%ntrifllge Add sodium acetate and 2 volumes 95 percent ethanol Jf Dried ethanol precipitate Extract with 90 percent phenol phenol’i”soluble 1 t

*,

supernatant ~i$W&itb supernatant

1;; ;jzizi; 0 phenol insoluble 2 pkcipitate 10% 2x 3 pkxipitate 20% from 2nd 10% 4

1057,etha”ol : precipitate

pkclpitate

s”pel’nata”t precipitate 10% ethanol I‘ 90% phenol supernatant precipitate 20% ethanol 90% phenol supematant

with 20% ethanol in 90% phenol

with in

supernatant

p+ata”t precipitate with 10% ethanol in 90% ph no1

with i” I precipitate 10% from 1st

20% ,

5

precipitate 20% 2x 6

pkcipitate

super atant F precipitate with f$+t+in

supematant I precipitate with 25% ethanol i” 90% phenol supenlatant

25%

7

FIG. 1. Outline of the procedure for fractionation with 90% phenol and ethanol (17) of pepsinor 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 insoluble (21, an HLeh-active material from human ovarian 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 incubated 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 ofhexosamines, N-acetylhexosamines, galactosamine, methylpentose (fucose) and hexose (galactose) have been described previously (2, 4, 23). Sialic acid was determined 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 material from human ovarian cyst fluid.

RESULTS

Table Ia shows the yields, compositions and serological activities of various fractions from phenol/ethanol purification (Fig. 1). Mucosae generally yielded considerable amounts of three to five of the possible seven fractions shown; the remaining fractions were disregarded if less than 10 mg or if of negligible carbohydrate content. Yields of purified blood group substances were approximately the same for each mucosa; l-2 mg/g wet weight (No. 711) 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 combining fractions from Table Ia. One, B-active, consists of the most active B fractions, denoted in Table Ia by an asterisk (*); the

TABLE

Ia

ANALYTICAL COMPOSITION OF HORSE BLQOD GROUP FRACTIONS Fraction

Yield (ma)”

Percent composition Total N

Horse 7, Pronase digested, 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 distarting gested, weight (wet) 202 g (strong B, weak H) Phenol insoluble* 10% 2x* 20% from 2nd 10% Horse 8, Pronase-digested phenol-insoluble fraction, pepsin digested, 132 mg (strong B, weak H) Phenol insoluble* Horse 9, Pronase distarting gested, 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 digested, starting weight (wet) 326 g (inactive) 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

Methylpentose (fucose)

Hexose (galactose)

(weight)

Hexosamine”

N-acetylhexosamine

-

Galactosamine

Pep&de

126 (113) 193 (161) 84 (68)

4.5 5.7 6.1

9.4 7.4 9.2

25.9 24.5 28.4

19.5 25.7 21.2

22.0 22.3 16.7

7.3 11.7 10.1

3.0 3.7 4.5

38 (34) 153 (122) 19.5 13.0

5.9 5.8 6.7 7.5

9.4 10.4 5.6 5.4

22.6 26.0 19.4 19.3

18.0 19.3 23.9 16.5

14.7 17.3 20.0 16.5

8.1 9.3 10.9 5.3

4.5 4.3 4.8 6.2

132 (46) 47 (27) 17

5.1 5.4 6.5

11.3 6.6 5.6

26.2 23.9 20.2

19.4 18.4 20.9

22.8 16.2 18.4

7.7 7.3 7.9

3.6 4.0 4.9

65 (46)

4.4

10.6

27.6

22.5

26.8

8.3

2.7

130 (96) 91 (70) 18 38 98 (60)

5.4 5.7 6.0 6.4 5.9

9.6 6.7 9.4 11.0 10.1

25.8 23.3 25.2 27.4 27.7

17.1 22.8 17.9 16.0 18.3

17.8 18.9 12.3 10.8 12.5

9.5 10.6 10.4 10.5 11.7

4.1 3.9 4.6 5.2 4.5

87 (68) 10 (4) 120 (103) 14 (5) 154 (138) 68 (50)

5.1 4.6 7.7 6.9 8.8 8.7

2.0 1.6 3.3 3.6 4.2 4.2

12.5 12.6 14.1 13.7 14.0 15.1

16.7 16.1 19.1 15.3 14.8 16.1

22.0 22.1 15.5 12.3 11.2 11.9

3.5 2.1 3.6 4.1 4.0 3.3

3.8 2.9 6.2 5.7 7.6 7.4

28 117 84 44

5.8 5.9 6.9 7.3

8.3 7.2 7.5 6.9

21.2 17.3 20.4 22.4

25.7 25.4 29.3 19.0

20.8 22.7 24.7 11.2

8.8 10.1 12.2 14.3

3.8 3.9 4.6 5.8

(17) (98) (75) (34)

513

TABLE

Fraction

diHorse 11, Pronase starting gested, 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) (moderately 33 g 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 25% t

Yield (mg)”

Ia-Continued Percent composition

Total N

Methvlpent&e ifucose)

95 19 53 24 39

(76) (9) (38) (16) (30)

5.3 5.7 6.1 6.3 6.8

3.3 7.2 6.8 4.4 4.8

18 24 75 27

(10) (12) (58) (18)

9.2 5.8 6.5 7.8

2.7 3.2 4.8 4.2

44 (30) 63 (39) 57 (43) 36

8.9 7.2 6.3 8.6

99 (80)

29 29 14 15

18 (7) 17 (71 108 (841 18 (6) 50 (36) 15 (6)

Hexose (galaciose)

15.5 20.6 23.6 19.9 21.3

Hexesamine”

(weight) N-acetvlhexosamine

24.6 21.2 23.3 27.4 30.4

23.4

19.6 19.1 24.1 27.7

Galactosamine

Peptide

8.8 8.6 8.7 7.7 7.3

3.4 4.1 4.3 4.8 4.4

3.1 5.4 8.0

9.1

7.8 4.3 4.6 5.7

14.4

17.7

19.3

19.1

17.7 15.8

24.1 27.4

15.0 17.8 22.8 22.8

5.6 5.0 5.2 3.0

17.2 18.5 17.0 14.4

18.3 20.0 20.4 15.7

17.0 17.3 14.5 11.2

5.1 6.9 10.5 7.2

7.5 5.6 4.7 7.4

6.4

5.6

21.6

27.6

23.2

11.5

4.3

8.6 6.9 8.8 8.7

2.8 3.4 3.3 3.7

15.0 18.0

13.2 14.3

12.0 12.1 8.5 7.4

4.6 3.8 4.4 7.3

7.6 6.0 7.8 7.6

9.1 6.1

2.0 0.7 3.8

10.0

2.4 1.6 5.4 1.6 5.6 3.1

8.3 5.4 4.7 4.5 6.7 4.9

6.5 5.0 7.7 5.9

1.1 2.3 2.8

15.9

12.4

18.0

14.0

12.7 14.7 18.0 10.7 15.3 13.4

10.4 8.9 22.5 6.6 12.6 12.3

8.2 20.3 5.5 8.8 6.4

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 Nacetylglucosamine. 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 precipitation 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 TABLE

515

GLYCOPROTEINS

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 Total N

All fractions Low High Most active B fractions Low High Largest fractions Low High mIndicated

Methylpentose (fucose)

(weight)

Hexose (galactose)

Hexosamine

N-acetylhexosamine

Galactosamine

Peptide N

4.4 8.3

0.7 11.3

10.7 28.4

5.5 27.7

6.6 30.4

1.6 14.3

2.7 8.3

4.4 8.9

5.0 10.6

17.0 28.4

12.5 26.8

17.1 27.6

5.1 11.7

2.7 7.5

5.1 8.8

3.3 11.3

14.0 26.2

11.2 23.4

14.8 27.6

4.0 11.7

3.4 7.6

in Table Ia by an asterisk

(*).

other, non-B-active, includes materials in Table Ia with a dagger (t). Their properties are described more fully in the following 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 human standard, JS phenol insoluble. Hemagglutination inhibition data, Table II, on the individual fractions from Table 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 mucosa, individual fractions showed some variation 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. Mucosae with A. or B activity also showed weak H activity, except for strongly Bactive mucosa 14, which was also strongly H-active. The weak A activity of mucosa 8 was found only in pepsin-digested fractions. Pronase-digested mucosa 7 showed weak A activity. Certain fractions from pepsin and Pronase digestions had high serological activities (Tables II and III). The main fractions

of Pronase-digested mucosa 8 (phenol insoluble and 10% 2 x 1were less active in inhibition of B hemagglutination than the pepsin fractions (10% 2x and 20% from 2nd 10%) while, in inhibition of H hemagglutination, the most active fractions from the Pronase-digested portion of mucosa 11 were as potent as those from pepsin treatment. In phenol/ethanol fractionation, Bactive mucosae 7, 8, 9 and 13 (Tables Ia and II) showed a distribution pattern that differed somewhat depending on the enzyme 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 uniformly distributed. Table Ia demonstrates the wide range of calorimetric values for the 50 fractions analyzed. 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 highest yield regardless of their serological activity. The ranges of values for hexosamine, N-acetylhexosamine and galactosamine were substantially narrower both for

.- ._

- ,. ,._

- ._ - -

Horse 7 (Pronase) Phenol insoluble 10% 2x 10% from 1st 20% Horse 8 (pepsin) 10% 2x 20% from 2nd 10% 20% 2x 25% Horse 8 (Pronase) Phenol insoluble 10% 2x 20% from 2nd 10% Horse 8 (pepsin-Pronase) Phenol insoluble Horse 9 (Pronase) Phenol insoluble 10% 2x 20% from 2nd 10% 10% from 1st 20% 20% 2x Horse 10 (Pronase) Phenol insoluble 10% phenol insol. 10% 2x 20% from 2nd 10% 10% from 1st 20% 20% 2x Horse 11 (pepsin) 10% 2x 20% from 2nd 10% 20% 2x 25% ~1080 >1070 >1400 >1160 >1190 >1370 >1390 >1260 >1140 >1040 >1180 >1140 >1150 >1030 >1250 >1290

17 16.7 1.4 19.1 18.6 43 >1390 >1260 >1140 >1040 >1180 >1140 >1150 >1030 >1250 >1290

34 16.7 5.5 18.1 74 43 >1390 >1260 >1140 >1040 >1180 >1140

8.4 2.7

>1730

31 37 62

18 33 54

2.1 2.1 10 18

19 8.2 54

>960 285 159

6.8

1.2 2.5 13.7 41

>1150 >1030 >1250 21290

1.5

2.4 2.5 6.8 41

30 4.5 5

6.3

7.5 4.5 5

3.1

15 4.5 20

6.3

Beach phenol insoluble

>1280

>1230 523

151 108 219 >1310

1.6

303 158 109 655

0.8

>1230 >1045

MSM

>1170

133 131 318 580

0.8

9 16 20

9 9 16 20 81

20

>1390

>1390 630 570 >1040 >1180 >1140

>1390 >1260 570 >1040 >1180 > 1140

62 73 62

88 73 149 171

62

33 33 40 73

12.0

6.3 120 143 317

Hog 33B

Hog 25

268 88 145 297 172

68

62

33 33 79 73

240 36 159

Hog 25 __-~_ 6.3

TABLE II HEMAGGLUTINATION INHIBITION ASSAYS OF HORSE BLQOD GROUP FRACTIONS FOR A, B, H, Le” AND Leh ACTIVITIES Minimum concentration @g/ml) giving complete inhibition of hemagglutination” Standard B A H

--&se

-

19 146 133 72 >1210 ~1260 >1360 > 1290 >1240 >340

>1210 >1260 11360 >1290 > 1240 >940

12.3

16 41 43 168

38 73 265 144

6.1

16 10.3 43

> 1000 >1040 >1350 >1090 > 1000

6.1

8 41 43 167 6.1

8 10 11 84

Le”

Le”

H

-~__---

1.9 19.0 0.093 6.2

0.70 0.95 1.3 -

25 38 73 575 152 630 85 >1280 > 1240 >340

12 38 73 133 575 152 630 85 >1290 >1240 >340

111 130 110 95

Concentration (~glml)

64 83 343

64 83 171 >1340

o Several experiments were performed; activities are expressed relative to the endpoint for the standard blood group substance assayed at the same time, since twofold serial dilution values may differ by a factor of 2. b Numbers represent milliliters of blood group substance needed for 50% inhibition of hemagglutination by 1 ml of antibody. Anti-Lea diluted 1:300 for use; anti-Leb diluted 1:lOO for use; Uler extract diluted 1:30 for use. Dash (-) indicates no activity seen. Goat anti-Lea and -Leb were kindly supplied by Dr. Donald Marcus (52).

>1210 >1260 >1360 >1290 >1240 1340

305 73 265 >1150

>1570

>1020 >1320 >1370 >1340

34 45 23 36

34 22 23 18

,108o >1430 >1450 >1160

: 1080 > 1430 >1450 >1160

> 1080 >1430 > 1450 >1160

16 8 11 17 31

16 16 5 3 16

>lOOO >1040 >1350 >1090 11000

>lOOO >1040 >1350 >1090 > 1000

Le”, Leh and H activities Horse non-B-active blood group substane (pool) Horse B-active blood group substance (pool) Standard human HLe” substance, JS phenol insoluble 2.2h Standard human Lea substance, N-l Lea 20% from 2nd 10% 0.062

11 (Pronase) Phenol insoluble 10% 2x 20% from 2nd 10% 10% from 1st 20% 25% Horse 12 (pepsin) Phenol insoluble 10% 2x 20% from 2nd 10% 20% 2x Horse 13 (pepsin) Phenol insoluble 10% 2x 20% from 2nd 10% 20% 2x Horse 14 (pepsin) 10% 2x Horse 15 (pepsin) Phenol insoluble 10% 2x 20% 2x 25% Horse 16 (pepsin) Phenol insoluble 10% Phenol insoluble 10% 2x 20% from 2nd 10% 20% 2x 25%

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 Pronase digested

Pepsin digested Yield (mg)

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

3.7 5.7 38 153 6 19.5 12 238

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 3 28 117 2 84 44 281

Methylpentose

(o/o)

11.7 0.6 6.5 5.8 2.7 4.0 5.1

Methylpentose

(mg)

0.43 0.03 2.50 8.90 0.16 0.78 0.61 13.41

Hemagglutination inhibition”

>1230 >1140 2.4 2.5 303 6.8 41

Yield

(mg)

132 46 47 17 4 9 7 262

Methylpentose (%)

10.8 0.8 9.1 11.3 2.7 4.0 5.8

Methylpentose (mg)

14.25 0.37 4.30 1.92 0.11 0.40 0.41 21.76

0.10 0.07 2.32 8.42 0.09 6.30 3.04 20.34

the most active B materials and for those fractions obtained in highest yield. B-active fractions generally had higher fucose and gala&se as compared with non-B-active 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 peptide nitrogen. Strong serological activity correlated with the higher proportion of carbohydrate and lower peptide content independently of method of digestion even though the Pronase-digested materials tended to have lower peptide nitrogen

95 12 19 53 24 2 39 244

3.3 2.7 7.2 6.8 5.8 7.4 4.8

6.3d

-

n Minimum concentration @g/ml) giving complete b Standard B substance, Beach phenol insoluble. c Not done. d Standard H substance, hog 25.

n.d.’ n.d. 9 16 n.d. 20 81

inhibition

19 139 8.2 54 >1360 211 670 6.3*

6.3b

3.4 2.3 8.3 7.2 4.4 7.5 6.9

Hemagglutination inhibition”

3.14 0.32 1.37 3.60 1.39 0.15 1.87 11.84

16 32 16 5 9 nd. 16 6.3d

of hemagglutination.

than the pepsin-digested fractions. The weak A activity of mucosae 7,8 and 15 was not associated with higher amounts of galactosamine than found in non-A-active materials. Table III and Figs. 2 and 3 compare fractions prepared by pepsin and Pronase digestion from portions of the same mucosa. 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 pepsin or Pronase digestions were about the same. Methylpentose, the residue most likely to be hydrolyzed under the conditions of peptic digestion is essentially unaffected, and no methylpentose was detected

HORSE 200~1

2

0

4

BLOOD

HUMAN

6

8

GROUP-ACTIVE

ANTI-B

SERUM,SUBJECT

10 12 14 16 0

2

4

6

8

MlCRclGRAMS

10

0

519

GLYCOPROTEINS

2

nNT,GEN

3103

4

6

8

Total Volumr~3OOpl

IO 0

2

4

6

8

,o

12

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 3

LOTUS HORSE

ECTIN II

PEPSIN

6.2pg

N

CYGESTED

Total VolumeHORSE II

225)d PROMSE

‘VGESTED

f FIG. 3. Quantitative precipitin curves of the lectin from Lotus tetrugonolobus with fractions from pepsin- or Pronase-digested portions of strongly Hactive 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 hemagglutination. H-active blood group substances of equal potency were obtained from both portions of mucosa 11. The most active B fractions in inhibition of hemagglutination, designated by an asterisk (*) 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 precipitated by the Beach standard is indicated for each curve by an arrow and a number. Fractions from both digestions are as active as the standard. The somewhat weaker inhibition of B hemagglutination by Pronase-digested fractions from mucosa 8 as compared with pepsin-digested portions is associated with precipitation of somewhat less total nitrogen at the maximum, although. no difference was noted in the quantities needed to precipitate 50% of the total specific precipitate nitrogen. The phenol-insoluble fraction from Pronase digestion of mucosa 3 (Fig. 24 was unchanged in precipitating ability by subsequent peptic digestion (Fig. 2d). The non-B-active material (Fig. 2e) precipitated a small amount of antibody nitrogen but much less than any of the B-active substances. Figure 3 shows results of the precipitation of Lotus lectin by pepsin- and Pronase-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 Pronase-digested portions tended to go into the inhibition zone more readily. Table IV demonstrates the effect of pepsin digestion on the phenol-insoluble fraction of the Pronase-digested portion of mu-

520

NEWMAN

AND

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 inhibiting (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 fractions are the most potent (Figs. 4a, d, f, j and 1). There is no association between Con A reactivity and blood group specificity. Some fractions, most frequently the phenol insoluble (Figs. 4a, c and d) and 25% (Figs. 4f, k and 1) showed negligible or

KABAT

low Con A activity. No consistent association was observed between Con A reactivity and either hexosamine or N-acetylhexosamine content. DISCUSSION

The findings confirm earlier studies (1, 16) showing that some horse gastric mucosae are a good source of blood group Bactive substances roughly similar in their chemical and immunochemical properties to human saliva (1) and ovarian cyst blood group B substances (17, 40, 41). Other mucosae yielded strongly H-active, weakly Aor H-active and inactive products. No Lewis activities were found. All precipitated with Con A and thus are similar to blood group substances from hog gastric mucosae (32, 42). H activity in horse mucosae was not detected in an earlier study (11, but the reagent used was goat antiShiga serum absorbed with A,B cells and not the Ulen europeus orLotus tetragonolobus 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

Phenol insoluble

10% Phenol insoluble

104 2x

20% from 2nd 10%

65.2

2.8

7.8

0.3

Pronase digested Pepsin + Pronase digested Minimum

concentration

(pg/ml)

5.1 4.4

Peptide N

1.9

3.6 2.7

Pronase digested Pepsin + Pronase digested Standard B substance, A substance, H substance,

11.3 10.6

of each phenol insoluble hemagglutination

Activity

II Standard DStandard f Standard

Methylpentose

Beach phenol insoluble. MSM. hog 25.

material

20% 2x

10% from 1st 20%

Percent composition Total N

(starting

Total

2.1

fraction

N-acety1hexosamine

19.4 22.5

22.8 26.8

required

64

fractions

Hexosamine

26.2 27.6

Recoverv (VI) ”

80.1

of phenol-insoluble

Hexose (galactose)

125 mgl

for complete

Galactosamine

7.7 8.3 inhibition

B

A

H

19 17 6.3"

>I230 >1280 1.6"

68 68 6.3'

of

HORSE

BLOOD

CONCANAVALIN

A

nOmE 7 PRONASE DIGESTED

HORSE PEPSIN

GROUP-ACTIVE

8.INg

N

8

DIGESTED

521

GLYCOPROTEINS

Total Volume = 260~11 HORSE 8 PRONASE OIGESlEO

HORSE 9 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 activity. 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 contents, especially methylpentose and galactose. In the previous investigation (1) only one horse B fraction of 30 mg had a methylpentose content of 9.3%, the next highest value being 6.9%. Table Ia shows that at least six H-active fractions from three mucosae 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 humans (43-46). Moreover, no evidence of fucose being split off was found either by examination of dialysates from digestions or by differences in methylpentose contents of the pepsin- and Pronase-treated purified substances from individual mucosae. Pepsin digestion appears satisfac-

tory, but Pronase digestion has been used effectively with porcine submaxillary mutin (471, ovarian cyst and other glycoproteins (48, 49) and is as satisfactory as pepsin 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 materials 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 narrower than that for horse mucosae. It should be emphasized that a substantial contribution to the wide range in composition of different mucosae is made by the different A, B, H and Con A specificities, each of which is associated with different sugars. The most active B materials are comparable 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

tination with the Ulex lectin. The most active B materials were isolated most often in the phenol-insoluble and 10% 2x fractions from Pronase digests and from the 10% 2x and 20% from 2nd 10% fractions 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 Pronase-digested B materials tended to show somewhat lower peptide nitrogen. Mucosae with strong B and/or H activites contain significantly higher amounts of carbohydrate and lower amounts of peptide 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 observations on its reactivity with hog stomach A and H materials (32, 421, human gastric aspirates, saliva (50) and group 0 erythrocyte membrane glycoprotein (51) and its failure to react with human ovarian cyst blood group substances (50). Most horse fractions reacted with Con A, probably indicating, as for other blood group substances (32, 421, the presence of terminal nonreducing a-linked DG~cNAc; heterogeneity of fractions with respect to this parameter is present since the 10% 2x fractions 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 remained phenol insoluble. Pepsin digestion of crude blood group substance will usually solubilize phenol-insoluble material (17). Purified blood group substance that is already 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-

AND

KABAT

rial was isolated from peptic horse gastric mucosae.

digests

of

ACKNOWLEDGMENT The expert technical assistance of Mr. Jerry is gratefully acknowledged.

Liao

REFERENCES 1. BAER, H., KABAT, E. A., AND KNAUB, V. (1950) J. Exp. Med. 91, 105-114. 2. SCHIFFMAN, G., KABAT, E. A., AND THOMPSON, W. (1964) Biochemistry 3, 113-120. 3. IYER, R. N., AND CARLSON, D. M. (1971) Arch. Biochem. Biophys. 142, 101-105. J. AND BENMAMAN, J. D. (1967) 4. LUDOWIEG, Anal. Biochem. 19. 80-88. 5. GINSBURG, V. (1972) in Methods in Enzymology (Ginsburg, V., ed.), Vol. 38, Part B, pp. 3-201, Academic Press, New York. 6. CLAMP, J., BHATTI, T., AND CHAMBERS, R. (1972) in Glycoproteins (Gottschalk, A., ed.), 2nd ed., Part A, pp. 300-321, Elsevier, Amsterdam. 7. ROVIS, L., ANDERSON, B., KABAT, E. A., GRUEZO, F., AND LIAO, J. (1973) Biochemistry 12, 53405354. 8. LONNGREN, J., AND SVENSSON, S. (1974) in Advances in Carbohydrate Chemistry (Tipson, R. S., and Horton, D., eds.), Vol. 29, pp. 42106, Academic Press, New York. 9. STELLNER, K., AND HAKAMORI, S. (1974) in Methodologie de la Structure et du Metabolisme des Glycoconjugues, pp. 95-109, Centre National de la Recherche Scientifique, Paris. 10. SCOTT, C. D. (1974) Science 186, 226-233. 11. SCOTT, C. D., JOLLEY, R. L., PITT, W. W., AND JOHNSON, W. F. (1970) Amer. J. Ctin. Pathol. 53, 701-712. 12. KATZ, S., DINSMORE, S. R., AND PITT, W. W. (1971) Clin. Chem. 17, 731-743. 13. BELUE, G. P., AND MCGINNIS, G. D. (1974) J. Chromatogr. 97, 25-31. 14. WITEBSKY, E., KLENDSHOJ, N. C., AND MCNEIL, C. (1944) Proc. Sot. Exp. Biol. Med. 55, 167170. 15. WITEBSKY, E. (1946) Ann. N. Y. Acad. Sci. 46, 887-898. 16. KAZAL,

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YOUNG, M., AND ARNOW, L. E. (1947)Arch. B&hem. 13, 329-342. 17. KABAT, E. A. (1956) Blood Group Substances, Their Chemistry and Immunochemistry, Academic Press, New York. (Gotts18. WATKINS, W. M. (1972) in Glycoproteins chalk, A., ed.), 2nd ed., Part B, pp. 830-891, Elsevier, Amsterdam. 19. EYQUEM, A., PODLIACHOUK, L., AND MILLOT, P. (1962)Ann. N. Y. Acad. Sci. 97, 320-328.

HORSE

BLOOD

GROUP-ACTIVE

20. SKORYNA, S. C., AND WALDRON-EDWARD, D. (1967)
Chem. Scund. 25, 1663-1678. 22. SPIRO, R. G. (1966) in Methods in Enzymology (Neufeld, E. F., and Ginsburg, V., eds.), Vol. 8, pp. ‘29-30, Academic Press, New York. 23. KABAT, 15. A. (1961) Kabat and Mayer’s Experimental Immunochemistry, 2nd ed., C. C Thomas, Springfield, Ill. 24. MORGAN, W. T. J., AND KING, H. K. (1943) Biothem. J. 37, 640-651. 25. ALLEN, F’. Z., AND KABAT, E. A. (1959)5. Immunol. 82, 340-357. 26. MORENO, C., AND KABAT, E. A. (1969) J. Exp. Med. 129, 871-896. 27. BERKMAN, E., NUSBACHER, J., KOCHWA, S., AND ROSENFIELD, R. E. (1971) Transfusion 11,317332. S., BEN-DAVID, A., NUSBACHER, J., 28. BAR-SHANY, SZYMANSKI, I., KOCHWA, S., AND ROSENFIELD, R. E. (1970) Ann. N. Y. Acad. Sci. 169, 217224. 29. AGRAWAL, B. B. L., AND GOLDSTEIN, I. J. (1967) B&him. Biophys. Acta 147, 262-271. C. A., 30. JEANES, A., HAYNES, W. C., WILHAM, RANKIN, J. C., MELVIN, E. H., AUSTIN, M. J., CLUSKEY, J. E., FISHER, B. E., TSUCHIYA, H. M., APED RIST, C. E. (1954) J. Amer. Chem. Sot. 76, 5041-5052. 31. So, L. L., AND GOLDSTEIN, I. J. (1467) J. Biol. Chem. 242. 1617-1622. 32. LU)YD, K. O., KABAT, E. A., AND BEYCHOK, S. (1969) ,J. Immunol. 102, 1354-1362. 33. PEREIRA., M. E. A., AND KABAT, E. A. (1974) Biochemistry 13, 3184-3192. 34. WARREN, L. (1959) J. Biol. Chem. 234, 19711975. 35. SCHIFFMAN, G., KABAT, E. A., AND LESKOWITZ, S. (1960) J. Amer. Chem. Sot. 82, 1122-1127.

GLYCOPROTEINS

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36. BENDICH, A., KABAT, E. A., AND BEZER, A. E. (1947) J. Amer. Chem. Sot. 69, 2163-2167. 37. KABAT, E. A., AND LESKOWITZ, S. (1955) J. Amer. Chem. Sot. 77, 515995164. 38. LU)YD, K. O., KABAT, E. A., AND LICERIO, E. (1968) Biochemistry 7, 2976-2990. 39. NEWMAN, W., AND KABAT, E. A. (1976) Arch. Biochem. Biophys. 172, 524-534. 40. WATKINS, W. M., AND MORGAN, W. T. J. (1962) VOX Sung. 7, 129-150. 41. PUSZTAI, A., AND MORGAN, W. T. J. (1961) Biothem. J. 80, 107-121. 42. ETZLER, M. E., ANDERSON, B., BEYCHOK, S., GRUEZO, F., LLOYD, K. O., RICHARDSON, N. G., AND KABAT, E. A. (1970) Arch. Biochem. Biophys. 141, 588-601. 43. CEPPELLINI, R. (1959)in Ciba Foundation Symposium Biochemistry of Human Genetics (Wolstenholme, G. E. W., and O’Connor, C. M., eds.), pp. 242-263, Churchill, London. 44. WATKINS, W. M., AND MORGAN, W. T. J. (1959) VOX Sang. 4.97-119. 45. WATKINS, W. M. (1970) in Blood and Tissue Antigens (Aminoff, D., ed.), pp. 441-459, Academic Press, New York. 46. GINSBURG, V. (1972) in Advances in Enzymology (Meister, A., ed.), Vol. 36, pp. 131-149, Wiley, New York. 47. PAYZA, N., RIZVI, S., AND PIGMAN, W. (1969) Arch. Biochem. Biophys. 129, 68-74. 48. LAMPORT, D. T. A. (1969) Biochemistry 8, 11551163. 49. DONALD, A. S. R. (1973) B&him. Biophys. Acta 317,420-436. 50. CLARKE, A. E., AND DENBOROUGH, M. A. (1971) B&hem. J. 121, 811-816. 51. FUKUDA, M., AND OSAWA, T. (1973) J. Biol. Chem. 248, 5100-5105. 52. MARCUS, D. M., AND GROLLMAN, A. P. (1966) J. Immunol. 97, 867-875.