The nature of ABH blood group antigens in human gastric secretion

278

Biochimica et Biophysica Acta, 540 (1978) 278--284 © Elsevier/North-Holland Biomedical Press

BBA 28499 THE N A T U R E OF ABH BLOOD G R O U P ANTIGENS IN HUMAN GASTRIC SECRETION

AMALIA SLOMIANY, BRONISLAW L. SLOMIANY and GEORGE B.J. GLASS Gastroenterology Research Laboratory, Departments of Medicine and Biochemistry, New York Medical College, New York, N.Y. 10029 (U.S.A.)

(Received September 23rd, 1977)

Summary The origin of blood group ABH activity in human gastric content was investigated. Dialyzed and lyophilized samples of ten individual gastric secretions were assayed for ABH antigen under various conditions. The native activity persisted in delipidated residue of the respective secretions, b u t was completely missing in the lipid extracts of the analyzed samples. The alkaline degradation of the native and delipidated samples led to total loss of blood group activity of the analyzed materials, b u t no effect on A-active glycosphingolipid was evolved. Purified glycolipid portion of the lipid extract was lacking ABH activity and was shown to have distinct composition. This fraction contained only glyceroglucolipids and neither sphingosine nor other carbohydrates were present. On the basis of blood group activity assays of the native, delipidated, alkaline degraded samples and also on glycolipid analysis it was established that the ABH blood group activity of stomach secretion originated entirely from the glycoprotein portion of these samples.

Introduction The occurrence and nature of blood-specific antigens in tissue and gastric secretion has been studied b y a number of investigators [1--7], who established that water- and alcohol-soluble antigens are of quite distinct character, b u t in spite of that, they exhibit same serological activity. It has been suggested [6,7] that gastric secretion contains exclusively water-soluble substances, whereas red cells and most of the other tissues contain only the alcohol-soluble antigens. Over the past years it has been shown that alcohol-soluble blood group substances of the red cells [8--12], gastrointestinal mucosa [13--15], pancreas [16] are of glycosphingolipid nature, and their coexistence with blood group active glycoproteins on erythrocyte membrane and other tissues has been accepted [17--20]. Although much work has been done on the tissue antigens,

279 the nature of blood group substances of the secretions (stomach, saliva) has not been sufficiently clarified. Until now, it was commonly assumed that gastric secretion represents a mixture of blood group active glycoproteins and glycosphingolipids [17]. Our recent studies on lipids of gastric secretion [21--24] and saliva [25] showed that these secretions contain glycerogiucolipids without a trace of glycosphingolipids. In this paper, evidence that blood group antigens of human gastric secretion are exclusively of glycoprotein origin is presented. Experimental procedure

Materials. Pentagastrin stimulated human gastric secretion was obtained from ten individuals by gastric intubation. Human red cells A,, A2, B, O types, human blood grouping serum anti-A, anti-B, and anti-H (Ulex europeous extract) were from Biological Corporation of America (Port Reading, N.J.). Blood group A-active glycosphingolipids were prepared from hog gastric mucosa [26]. Standard long-chain bases were purchased from Miles Laboratories Inc. (Elkhart, Ind.). Preparation of gastric secretion samples for ABH blood group activity assay. Human gastric secretion of ten individuals was utilized for the preparation of six sets of samples. Each sample was derived originally from 15 ml of gastric secretion, which, prior to any treatment, was dialyzed against distilled water, lyophilized and weighed. The prepared samples consisted of (1) native gastric secretion 4 mg/ml saline; (2) delipidated gastric secretion 8 mg/ml; (3) lipid extract of gastric secretion 8 mg/ml saline; (4) and (5) delipidated and native gastric content after alkaline degradation treatment; (6) purified glycolipids fraction of gastric secretion 8 mg/ml saline. To five randomly-chosen samples of lipid extract from gastric secretion (group 3), of the delipidated and native residue of gastric secretion (groups 4 and 5), and to purified glycolipids of stomach secretion (group 6), 200 pg of blood group A-active glycosphingolipids [26] were added. The addition was made before alkaline degradation of the delipidated and native samples of gastric secretion (groups 4 and 5) and to saline suspensions, before sonication of total lipid extract and of purified glycolipids of gastric secretion (groups 3 and 6). Lipid extraction and preparation of glycolipids. Aliquots of individual human gastric secretions (15 ml) were dialyzed extensively, lyophilized and weighed. The lyophilizates were extracted twice, each time for 24 h with 100 ml chloroform/methanol, 2 : 1, and filtered. The lipids contained in the combined filtrates were concentrated, dissolved in a small volume of chloroform and applied to silicic acid columns [21]. The columns were eluted with chloroform, acetone, acetone/methanol and methanol. Each fraction was analyzed by thin-layer chromatography for glycolipids [24]. The major fraction of glycolipid, eluted from silicic acid column with acetone was evaporated to dryness, suspended in saline with aid of sonication at a concentration of 8 mg/ml and utilized for ABH blood group activity assay (group 6). Alkaline degradation. The lyophilized native and delipidated residues of stomach contents were subjected to degradation in 0.5 N NaOH at room temperature for 60 h. Five samples from each group of ten contained 200 ~g of

280 purified blood group A-active glycosphingolipids [26]. The alkaline treatment was followed by extensive dialysis, lyophilization and weighing. All these steps were necessary for preparation of the samples for ABH blood group assay (group 4 and 5). Glycolipid fractions composition. Aliquots of acetone fractions from silicic acid columns (major glycolipids fraction) were subjected to acid methanolysis. Methyl glycosides, fatty acid methyl esters and glyceryl ethers were obtained as previously described [22,23]. Portions of acid methanolysates were dried and treated with BC13 [27]. Fatty acids and alkyl chlorides were then removed with hexane. The methanolic phases were neutralized with silver carbonate, dried under reduced pressure at 4°C and the residues analyzed for glycerol [28]. Long-chain base analysis. The glycolipids of the acetone fraction from the silicic acid column were hydrolyzed in 1.0 M HC1 in aqueous methanol [29] and examined on thin-layer plates for long-chain bases in the presence of authentic sphingosine standards [ 30 ]. Hemagglutination and hemagglutination-inhibition assay. Assays for ABH blood group activity were performed with Takatsy microtiter (Cooke Engineering Co., Alexandria, Va.) using 0.025-ml loops and 2% suspensions A, B and H positive human red cells. The anti-A, anti-B serums used were diluted to 4 U/0.025 ml; the anti-H lectin (Ulex europaeus extract) was used undiluted. Other procedures. Glycolipids were visualized on thin-layer plates with orcinol, ninhydrin, ammonium bisulfate and iodine vapors [31] and also classified on the basis of benzidine reaction [32]. Gas-liquid chromatography analysis. Analyses of trimethylsilyl derivatives of glycerol and methyl glycosides were performed on columns (180 × 0.2 cm) packed with 3% SE-30 on chromosorb, W, AW, DMCS (80--100 mesh) programmed at 2°C/min from 100--210°C. The same columns were used for analyses of fatty acid methyl esters and trimethylsilyl derivatives of glyceryl ethers programmed (170--270°C at 2°C/min for fatty acids and 190--270°C at 2°C/min for alkyl ethers. Results

Hemagglutination-inhibition assay Results of ABH hemagglutination inhibition assays of native and delipidated residues of ten human gastric secretions are given in Table I. The blood group activity of the individual gastric samples ranged from 0.8--0.4pg residue against 4 units of anti-ABH serums or lectin. The activities of delipidated stomach contents were not diminished, but a 2-fold increase in some of the samples was found. The lipid extracts of gastric secretions and their giycolipid portions (acetone fraction from silicic acid column) were inactive in ABH blood group system. At high concentration from 8 mg/ml down to 2 mg/ml these lipids displayed a lytic property towards all types of red cells. Unaltered activity of A-active glycosphingolipids was obtained in the presence of human gastric secretion lipids or glycolipids. The ABH activity was completely abolished in alkali degraded samples. The

281 alkaline degradation showed no effect on A-active glycosphingolipids which were added to five alkali-treated samples. In each case activity of glycosphingolipids was identical for the samples both before and after alkali treatment.

Nature of glycolipids of human gastric secretion Lipid portion of human gastric secretion varied from one individual to the next and ranged from 16.2 to 31.5% of dry gastric secretion lyophilisates. The glycolipid portion eluted from silicic acid with acetone and acetone/methanol (9 : 1) a m o u n t to about 42% of total lipid extract. Thin-layer chromatography in chloroform/methanol/H20, 65 : 35 : 8, revealed the presence of three major and several minor glycolipid components, all of which were orcinol stain positive but undetectable with benzidine spray [22--24]. Analyses of the methanolyses products of glycolipid fraction eluted with acetone from silicic acid column showed the presence of glucose, alkyl ethers and fatty acids. The other hexoses and hexosamines were absent in methanolysates of the glycolipids fractions. Sphingosine was not detected in thin-layer chromatographed hydrolysates of these glycolipids. Neither ninhydrin nor benzidine stains revealed any traces of long-chain base compounds. Treatment of the methanolysates with BC13 resulted in liberation of glycerol. Discussion Distribution of ABH and Lewis antigens is limited to blood cells and serum, internal coat of the cardiovascular system, columnar, goblet, mucous acini of mucous membranes and their secretions and to some non-mucous glands [33]. In the stomach of ABH secretors these substances are located in the cytoplasm of the superficial and foveolar epithelium of the corpus mucosa and the superficial and glandular epithelium of the pyloric mucosa [34]. In nonsecretors, these antigens persist in the deeper parts of gastric glands, from where TABLE ABH

I

BLOOD

Sample (initials)

L.A. R.O.

GROUP

ACTIVITY

B l o o d group type

BH AH

AND

POTENCY

OF HUMAN

Hemagglutaination

GASTRIC

inhibition

SECRETION

assay *

Native

Delipidated

Lipid p o r t i o n

(4 mg/ml)

(S mg/ml)

(8 mg/ml)

I : 128 1 : 128

1 : 512 1 : 512

1 ** --*** 1 --

K.A.

H

1 : 64

1 : 218

1

--

V.E. D.E. R.S. S.P.

AH AB AH H

1 I 1 1

1 1 1 I

1 1 1

-----

C.Z. T.R.

BH H

I : 256 I : 128

1 : 1024 I : 256

1

---

S.B.

BH

1 : 128

1 : 512

I

--

: : : :

64 128 256 64

: : : :

256 512 512 256

* R e p r e s e n t s dilution o f samples at w h i c h t h e i n h i b i t i o n o f h e m a g g l u t i n a t i o n a g a i n s t

sera occurred. * * D e n o t e s l y s i s (1) o f t h e e r y t h r o c y t e s . * * * Indicates h e m a g g l u t i n a t i o n ( - - ) .

4 units o f anti-

282 they are extractable by ethanol b u t not by water. The cellular location of blood group substances thus corresponds with cell types that by histochemical methods contain and secrete glycoproteins [33,35]. Which form of blood group substance, glycoprotein or glycolipid, is distributed in various tissues and cells mentioned above is not well known. The conclusion derived from the vast evidence was that both blood group active glycosphingolipids and glycoproteins coexist in many organs and tissues [10,13,14,16,33,34,36--45]. In view of our findings presented here the concept of glycoprotein and glylipids coexistence is incorrectly simplified and as a result leads to partially wrong interpretation of data. The true picture was overlooked since no data on glycolipids of secretion were available until now. Recently, in our laboratory we obtained evidence indicating that the stomach secretion [21--24] and saliva [25] do n o t contain glycosphingolipids; instead, glyceroglucolipids were found. Also, secretory glands of gastric mucosa treated with 2 M NaC1, which is known for its capacity to extract the content of the secretory cell without damaging the cell membrane [46], are depleted of material of which the lipid portion contains only glucoglycerolipids. The glycosphingolipids were not detectable in these glandular extracts (unpublished data). These findings led us to the concept that the secretory ABH antigens are solely represented b y glycoproteins, whereas membrane-attached antigens are of glycosphingolipid nature. Our data clearly indicate that ABH activity of the stomach secretions are not represented by a lipid portion. The lipid extracts of the analyzed samples at concentration as high as 8 mg/ml were totally inactive in inhibition of hemagglutination in the ABH system. The same lack of inhibition of hemagglutination was observed when purified glycolipid fractions were utilized in the serological tests. Chemical analyses of these glycolipids showed the presence of only glucose, accompanied with glycerol ethers and fatty acids. Sphingosine, galactose, fucose or hexosamines were not detected. This analysis further proved, that blood group active glycosphingolipids are not secreted, and also that none of the other glycosphingolipids comprised the secretory products. To determine the nature of blood group antigens, the native and delipidated samples of stomach secretion were treated with alkali, which is known to destroy blood group active glycoprotein b u t is completely ineffective in degradation of glycosphingolipids. This treatment resulted in complete loss of native blood group activity, whereas the activity of added blood group A-active glycosphingolipids was n o t affected. Neither alkali nor the presence of large amounts of lipids were capable to suppress antigenic properties of these glycosphingolipids. Removal of the lipids from .lyophilized residue of stomach secretion did not abolish native activity by denaturation of the protein portion of these compounds, b u t to the contrary, a slight increase in potency per mg of residue was noted. The data interpreted above clearly indicate a rigorous division of blood group active antigens between gastric mucosa and its secretory products. Glycoprotein antigens belong to the secretion, whereas glycosphingolipids thus far isolated from gastric mucosa represent antigens which are an integral part of the mucosa cell membranes. This distinctive feature of gastric mucosa versus its secretion apparently does

283

not apply to erythrocytes, where antigenic activity resides in glycosphingolipids components and may be in glycoprotein. Here, the dual origin of ABH antigens is still disputable [19,20,47]. It is possible that the orderly separation of blood group active glycoproteins and glycosphingolipids between tissue and secretion is not applicable in the case of erythrocytes, which represent an entirely unusual type of tissue. Acknowledgements This investigation was supported N.I.A.M.D.D., N.I.H., P.H.S.

by Grant No.

AM-00068-25

from

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