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Identification of Leb antigens in meconium of a phenotypically a Le(a+b−) non-secretor individual
Biochimica et Biophysica Acta, 760 (1983) 381-383
381
Elsevier BBA21595
IDENTIFICATION OF Leb ANTIGENS IN M E C O N I U M O F A PHENOTYPICALLY A Le(a+b -) N O N - S E C R E T O R INDIVIDUAL GORAN LARSON
Department of Medical Biochemistry, University of G6teborg~ Box 33031, S- 400 33 G6teborg (Sweden) (Received July llth, 1983)
Key words: Blood group; Secretor status," Fucolipid," Monoclonal antibody; Chromatogram binding assa)7 (Human meconium)
Blood group active fucolipids of human meconium have been shown to correlate to the A B H and Lewis blood groups and to the secretor status of the corresponding children. Using a monoclonal anti-Le b antibody and an antibody to chromatogram binding assay the presence of Le b antigens in meconium of a phenotypically A Le(a+b -) non-secretor individual is here demonstrated. Pbenotype was determined on cord blood and saliva obtained 2 years after birth.
Introduction
Human meconium, the first sterile stools of the newborn, contains large amounts of blood group active glycosphingolipids [1,2]. The individual patterns have been shown to correlate to the blood group and secretor status of the corresponding children [2-5]. However, the composition in individual meconia of minor blood group fucolipids have not been fully elucidated. In this paper an anti-Le b monoclonal antibody [6] and a technique of binding antibodies directly to thin-layer chromatograms [6,7] have been used to specifically analyse the presence of Le b antigens in a phenotypically A Le(a+b-) non-secretor individual. The result is briefly discussed in view of existing theories on the genetics of blood group antigens. Materials and Methods
Total non-acid glycosphingolipids of meconium of a single individual was obtained as described [2]. The techniques for subfractionation of the acetylated derivatives by silicic acid column chromatography and characterization of the major sphingolipids by mass spectrometry, proton nmr0304-4165/83/$03.00 ~' 1983 Elsevier Science Publishers B.V.
spectroscopy and immunology have already been reported [3-5]. Hospital routine testing of cord blood established the blood group type of the child. Saliva, obtained 2 years after birth, was assayed for A, B, H, Le a and Le b activity in a hemagglutination-inhibition system [8]. The monoclonal antibody NS10 was a kind gift from Dr. H. Koprowski, Wistar Institute, Philadelphia, USA. The specificity of this anti-Le b reagent has already been described [6] as well as the experimental conditions for the chromatogram binding assay [7]. Results and Discussion
Erythrocytes of cord blood of this individual were typed as belonging to the blood group A. Saliva contained no inhibitory A, B, H or Le b activity but strong Le a activity (inhibition of a 4 + hemagglutination). Thus, phenotypically this was an A Le(a+b -) non-secretor individual [9]. Apart from the mono- to tetraglycosylceramides characteristically found in this tissue [2-5], a Le a active pentaglycosylceramide (see Fig. 1) was the only major fucolipid of this individual's meconium (Larson et al., unpublished data). This
382
~'~
Le b
T
2
3
4
. . . . .
Le b
T
2
3
4
Fig. 1. Thin-layer chromatograms, run identically in chlorof o r m s / m e t h a n o l / w a t e r ( 6 0 : 3 5 : 8 by vol). with 1 ttg of a Le b active hexcaglycosylceramide (Leb: [14]), 68 t~g of total non-acid glycosphingolipids (T) of this single meconium and 9. 23 and 21 ttg, respectively of the three subfractions (2, 3 and 4) isolated therefrom. The left chromatogram was visualized with the anisaldehyde reagent and the right autoradiographed (48 h) after antibody labeling. Figures to the left indicate number of sugars in the glycolipid carbohydrate chains.
was in contrast to the complex fucolipid patterns found in meconiurn of secretor positive individuals [4,5] but paralleled the immunological findings in saliva. Mass spectrometrically, evidence for a difucosyl hexaglycosylceramide with a Leb-like structure was found in subfraction 3 of this meconium (compare Fig. 1). Difucosyl determinants were also found for compex glycolipids of subfraction 4.
TABLE I BLOOD G R O U P Le b ACTIVITIES O F S U B F R A C T I O N S 2,
However, as these were minor components of the respective subfractions, type 1 (Gal/31---, 3GIcNAc) or type 2 (Galfll---, 4GIcNAc) chain differences could not be settled for these structures. When using polyclonal anti-Le b antisera subfractions 3 and 4 showed both inhibitory activities (see Table I), indicating the possible presence of minor amounts of Le h antigens in these fractions. The binding of the monoclonal antibody NS 10 to specific glycosphingolipids of this meconium (see Fig. 1) clearly established the presence of Le h antigens in this tissue. However, as shown in Fig. 1, when comparing the chemical and immunological detections, Le°-active glycolipids were only minor components of the total glycolipid content. The gross pattern of glycosphingolipids in meconium thus correlates well with the saliva phenotype while minor components may show a divergent appearance. Using immunofluorescence techniques similar results have been reported for water soluble glycoconjugates of the gastrointestinal tract [10]. Thus, regardless of whether the secretory gene is regulatory [9,11] or structural [12,13] its variable expression in different tissues still requires an explanation.
Acknowledgements Drs. K.-A. Karlsson and B.E. Samuelsson are gratefully acknowledged for stimulating discussions and help with the immunological characterizations. I thank Dr. H. Koprowski for providing us with the anti-Le b monoclonal antibody. The work was supported by grants from The Swedish Medical Research Council (No. 3967).
3 A N D 4 O F THIS S I N G L E M E C O N I U M
The assay was done as inhibition of hemagglutination and graded from - (no agglutination) to 4 (full agglutination). Antigens were tested as liposomes of 50 p,g glycolipid, 50 ~tg hydrogenated phosphatidylcholine and 25 #g cholesterol [8]. The Leb-hexaglycosylceramide reference (Leb-6) was isolated from h u m a n small intestine [14]. Antigens
Subfraction 2 Subfraction 3 Subfraction 4 Leb_6 Saline
Anti-Le b antiserum with dilution factors 2
4
8
16
32
4 4 4 . 4
4 3 3
3 -
1 -
-
3
2
l
.
. 4
.
64
References 1 Karlssom K.-A. and Larson, G. (1978) FEBS Lett. 87, 283-287 2 Karlsson, K.-A. and Larson, G. (1981) J. Biol. Chem. 256, 3512-3524 3 Karlsson, K.-A. and Larson, G. (1981) FEBS Lett. 128, 71-74 4 ,~ngstrrm, J., Falk, K.-E., Karlsson, K.-A. and Larson, G. (1982) Biochim. Biophys. Acta 710, 428-436 5 ~,ngstrrm, J., Falk, K.-E., Karlsson, K.-A. and Larson, G. (1982) Biochim. Biophys. Acta 712, 274-282 6 Brockhaus, M., Magnani, J.L., Blaszczyk, M., Steplewski, Z., Koprowski, H., Karlsson, K.-A., Larson, G. and Ginsburg, V. (1981) J. Biol. Chem. 256, 13223-13225
383 7 Hansson, G.C., Karlsson, K.-A., Larson, G., McKibbin, J.M., Blaszczyk, M., Herlyn. M., Steplewski, Z. and Koprowski, H. (1983) J. Biol. Chem. 258, 4091-4097 8 Dahlgren, K.-E., Emilsson, P.M., Hansson, G.C. and Samuelsson, B.E. (1981) J. lmmunol. Methods 44, 223-234 9 Race, R.R. and Sanger, R. (eds.) (1975) Blood Groups in Man, 6th Edn, pp. 311-349, Blackwell Scientific Publications, London 10 Szulman,. A.E. (1977) in Human Blood Groups (Mohn, J.,
11 12 13 14
Plunkett, R.W., Cunningham, R.K. and Lambert. R.M., eds.), pp. 426-436, Karger, Basel Watkins, W.M. (1978) Proc. R. Soc. Lond. B 202, 31-53 Oriol, R., Danilows, J. and Hawkins, B.R. (1981) Am. J. Hum. Genet. 33. 421-431 Le Pendu, J., Lemieux, R.U., Lambert, F., Dalix, A.-M. and Oriol, R. (1982) Am. J. Human Genet. 34, 402-415 McKibbin, J.M., Spencer, W.A.. Smith, E.L., M~msson, J.-E., Karlsson, K.-A., Samuelsson, B.E., Li, Y.-T. and Li, S.-C. (1982) J. Biol. Chem. 257, 755-760
381
Elsevier BBA21595
IDENTIFICATION OF Leb ANTIGENS IN M E C O N I U M O F A PHENOTYPICALLY A Le(a+b -) N O N - S E C R E T O R INDIVIDUAL GORAN LARSON
Department of Medical Biochemistry, University of G6teborg~ Box 33031, S- 400 33 G6teborg (Sweden) (Received July llth, 1983)
Key words: Blood group; Secretor status," Fucolipid," Monoclonal antibody; Chromatogram binding assa)7 (Human meconium)
Blood group active fucolipids of human meconium have been shown to correlate to the A B H and Lewis blood groups and to the secretor status of the corresponding children. Using a monoclonal anti-Le b antibody and an antibody to chromatogram binding assay the presence of Le b antigens in meconium of a phenotypically A Le(a+b -) non-secretor individual is here demonstrated. Pbenotype was determined on cord blood and saliva obtained 2 years after birth.
Introduction
Human meconium, the first sterile stools of the newborn, contains large amounts of blood group active glycosphingolipids [1,2]. The individual patterns have been shown to correlate to the blood group and secretor status of the corresponding children [2-5]. However, the composition in individual meconia of minor blood group fucolipids have not been fully elucidated. In this paper an anti-Le b monoclonal antibody [6] and a technique of binding antibodies directly to thin-layer chromatograms [6,7] have been used to specifically analyse the presence of Le b antigens in a phenotypically A Le(a+b-) non-secretor individual. The result is briefly discussed in view of existing theories on the genetics of blood group antigens. Materials and Methods
Total non-acid glycosphingolipids of meconium of a single individual was obtained as described [2]. The techniques for subfractionation of the acetylated derivatives by silicic acid column chromatography and characterization of the major sphingolipids by mass spectrometry, proton nmr0304-4165/83/$03.00 ~' 1983 Elsevier Science Publishers B.V.
spectroscopy and immunology have already been reported [3-5]. Hospital routine testing of cord blood established the blood group type of the child. Saliva, obtained 2 years after birth, was assayed for A, B, H, Le a and Le b activity in a hemagglutination-inhibition system [8]. The monoclonal antibody NS10 was a kind gift from Dr. H. Koprowski, Wistar Institute, Philadelphia, USA. The specificity of this anti-Le b reagent has already been described [6] as well as the experimental conditions for the chromatogram binding assay [7]. Results and Discussion
Erythrocytes of cord blood of this individual were typed as belonging to the blood group A. Saliva contained no inhibitory A, B, H or Le b activity but strong Le a activity (inhibition of a 4 + hemagglutination). Thus, phenotypically this was an A Le(a+b -) non-secretor individual [9]. Apart from the mono- to tetraglycosylceramides characteristically found in this tissue [2-5], a Le a active pentaglycosylceramide (see Fig. 1) was the only major fucolipid of this individual's meconium (Larson et al., unpublished data). This
382
~'~
Le b
T
2
3
4
. . . . .
Le b
T
2
3
4
Fig. 1. Thin-layer chromatograms, run identically in chlorof o r m s / m e t h a n o l / w a t e r ( 6 0 : 3 5 : 8 by vol). with 1 ttg of a Le b active hexcaglycosylceramide (Leb: [14]), 68 t~g of total non-acid glycosphingolipids (T) of this single meconium and 9. 23 and 21 ttg, respectively of the three subfractions (2, 3 and 4) isolated therefrom. The left chromatogram was visualized with the anisaldehyde reagent and the right autoradiographed (48 h) after antibody labeling. Figures to the left indicate number of sugars in the glycolipid carbohydrate chains.
was in contrast to the complex fucolipid patterns found in meconiurn of secretor positive individuals [4,5] but paralleled the immunological findings in saliva. Mass spectrometrically, evidence for a difucosyl hexaglycosylceramide with a Leb-like structure was found in subfraction 3 of this meconium (compare Fig. 1). Difucosyl determinants were also found for compex glycolipids of subfraction 4.
TABLE I BLOOD G R O U P Le b ACTIVITIES O F S U B F R A C T I O N S 2,
However, as these were minor components of the respective subfractions, type 1 (Gal/31---, 3GIcNAc) or type 2 (Galfll---, 4GIcNAc) chain differences could not be settled for these structures. When using polyclonal anti-Le b antisera subfractions 3 and 4 showed both inhibitory activities (see Table I), indicating the possible presence of minor amounts of Le h antigens in these fractions. The binding of the monoclonal antibody NS 10 to specific glycosphingolipids of this meconium (see Fig. 1) clearly established the presence of Le h antigens in this tissue. However, as shown in Fig. 1, when comparing the chemical and immunological detections, Le°-active glycolipids were only minor components of the total glycolipid content. The gross pattern of glycosphingolipids in meconium thus correlates well with the saliva phenotype while minor components may show a divergent appearance. Using immunofluorescence techniques similar results have been reported for water soluble glycoconjugates of the gastrointestinal tract [10]. Thus, regardless of whether the secretory gene is regulatory [9,11] or structural [12,13] its variable expression in different tissues still requires an explanation.
Acknowledgements Drs. K.-A. Karlsson and B.E. Samuelsson are gratefully acknowledged for stimulating discussions and help with the immunological characterizations. I thank Dr. H. Koprowski for providing us with the anti-Le b monoclonal antibody. The work was supported by grants from The Swedish Medical Research Council (No. 3967).
3 A N D 4 O F THIS S I N G L E M E C O N I U M
The assay was done as inhibition of hemagglutination and graded from - (no agglutination) to 4 (full agglutination). Antigens were tested as liposomes of 50 p,g glycolipid, 50 ~tg hydrogenated phosphatidylcholine and 25 #g cholesterol [8]. The Leb-hexaglycosylceramide reference (Leb-6) was isolated from h u m a n small intestine [14]. Antigens
Subfraction 2 Subfraction 3 Subfraction 4 Leb_6 Saline
Anti-Le b antiserum with dilution factors 2
4
8
16
32
4 4 4 . 4
4 3 3
3 -
1 -
-
3
2
l
.
. 4
.
64
References 1 Karlssom K.-A. and Larson, G. (1978) FEBS Lett. 87, 283-287 2 Karlsson, K.-A. and Larson, G. (1981) J. Biol. Chem. 256, 3512-3524 3 Karlsson, K.-A. and Larson, G. (1981) FEBS Lett. 128, 71-74 4 ,~ngstrrm, J., Falk, K.-E., Karlsson, K.-A. and Larson, G. (1982) Biochim. Biophys. Acta 710, 428-436 5 ~,ngstrrm, J., Falk, K.-E., Karlsson, K.-A. and Larson, G. (1982) Biochim. Biophys. Acta 712, 274-282 6 Brockhaus, M., Magnani, J.L., Blaszczyk, M., Steplewski, Z., Koprowski, H., Karlsson, K.-A., Larson, G. and Ginsburg, V. (1981) J. Biol. Chem. 256, 13223-13225
383 7 Hansson, G.C., Karlsson, K.-A., Larson, G., McKibbin, J.M., Blaszczyk, M., Herlyn. M., Steplewski, Z. and Koprowski, H. (1983) J. Biol. Chem. 258, 4091-4097 8 Dahlgren, K.-E., Emilsson, P.M., Hansson, G.C. and Samuelsson, B.E. (1981) J. lmmunol. Methods 44, 223-234 9 Race, R.R. and Sanger, R. (eds.) (1975) Blood Groups in Man, 6th Edn, pp. 311-349, Blackwell Scientific Publications, London 10 Szulman,. A.E. (1977) in Human Blood Groups (Mohn, J.,
11 12 13 14
Plunkett, R.W., Cunningham, R.K. and Lambert. R.M., eds.), pp. 426-436, Karger, Basel Watkins, W.M. (1978) Proc. R. Soc. Lond. B 202, 31-53 Oriol, R., Danilows, J. and Hawkins, B.R. (1981) Am. J. Hum. Genet. 33. 421-431 Le Pendu, J., Lemieux, R.U., Lambert, F., Dalix, A.-M. and Oriol, R. (1982) Am. J. Human Genet. 34, 402-415 McKibbin, J.M., Spencer, W.A.. Smith, E.L., M~msson, J.-E., Karlsson, K.-A., Samuelsson, B.E., Li, Y.-T. and Li, S.-C. (1982) J. Biol. Chem. 257, 755-760