Proc. Nati. Acad. Sci. USA Vol. 74, No. 12, pp. 5407-5410, December 1977 Biochemistry
Defects of glycosyltransferase activities in human fibroblasts of pk and p blood group phenotypes (trihexosyl ceramide/globoside/net synthesis of P antigens/glycolipid contents/glycosidases)
SHIGEKO KIJIMOTO-OCHIAI*, MASAHARU NAIKIt, AND AKIRA MAKITA* *
Biochemical Laboratory, Cancer Institute, School of Medicine, and t Department of Biochemistry, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060, Japan
Communicated by Herman M. Kalckar, September 21, 1977
We demonstrate that human fibroblasts of ABSTRACT the rare pk phenotype lack globoside, which was identified as the blood group P antigen, and that p cells possess neither globoside nor trihexosyl ceramide, which was identified as pk antigen. Our investigations indicate also that these glycosphingolipid patterns are most likely caused by inherited preferential biosynthetic pathways in the abnormal phenotypes rather than by excess catabolism of the antigens. Evidence is presented that the fibroblasts of pk phenotype lack fl-N-acetylgalactosaminyltransferase (globoside synthetase; UDP-Nacetylgalactosamine:trihexosylceramide fi-N-acetylgalactosaminyltransferase; EC 2.4.1.79) activity, and those of p are deficient in a-galactosyltransferase (trihexosylceramide synthetase; UDP galactose:lactosylceramide a-galactosyltransferase) and possibly also in globoside synthetase. The diminished globoside synthetase activity in p cells, however, is not caused by the defect in the gene coding for this enzyme. It appears, rather, to be caused by a failure in gene expression because one-third of pk X p hybrids became able to express P antigenicity with a time lag of 3X4 days after cell fusion [Fellous, M., Gerbal, A., Nobillot, G. & Weils, J. (1977) Vox Sang. 32, 262-2681.
The human blood group P system was demonstrated originally on erythrocytes (1-4) and subsequently on fibroblasts and lymphocytes (5). This blood group system, which consists of three antigens and five phenotypes, has been immunologically and genetically established (reviewed in ref. 6). Recently the chemical structures of these antigens have been identified as glycosphingolipids (7, 8) (Table 1). Confirmative evidence has come from studies on erythrocytes of the rare abnormal phenotypes (9): the pk erythrocytes lack globoside and possess an increased amount of trihexosyl ceramide (Hex3Cer), and p erythrocytes, which are virtually deficient in both globoside and Hex3Cer, accumulate lactosyl ceramide (LacCer), thus suggesting a genetic block in formation of globoside in pk individuals and of both Hex3Cer and globoside in p persons. The biosynthesis of the carbohydrate moiety of glycolipids proceeds by the sequential addition of monosaccharide units from their sugar nucleotide donors to growing acceptors by specific glycosyltransferases (10). The synthesis of Hex3Cer (11) and globoside (12) occurs as follows: UDP Gal LacCer
Ilex3Cer synthetase
UDP-Ga1NAc Hex 3Cer
UDP
Hex3Cer (pk antigen)
UDP
Globoside synthetase
Globoside (P antigen)
Present investigations were undertaken to determine the cause of genetic defects of the antigens in pk and p fibroblasts by chemical analysis of glycolipids, incorporation of radioactive galactose into glycolipids, and assays of specific glycosyltransferase and glycohydrolase activities.
MATERIALS AND METHODS Cell Lines and Culture. Fibroblasts from three donors whose phenotypes of P blood group were P2, Pi, and p were generously donated by M. Fellous, Hopital Saint-Louis, Paris. Their antigenic and chemical properties are summarized in Table 1. The fibroblasts, obtained from skin biopsy (5),, were cultured in Eagle's minimum essential medium containing 20% fetal calf serum. The fibroblasts were labeled with [14Clgalactose as follows: The cells at the 17th or 18th passage were grown to confluence on glass bottles of 4 X 10 cm, and the medium was changed to 10 ml of fresh medium containing 5 ACi of [U14C]galactose (Radiochemical Center, Amersham, 95 Ci/mol). After 10 days, the cells were washed five times with Dulbecco's phosphate-buffered saline (pH 7.0) and harvested by use of trypsin. For analysis of glycolipid contents, the cells'were harvested after trypsin treatment and pooled (10th to; 20th passages). For enzymatic assays, the cells were scraped with a rubber policeman and suspended in 0.32 M sucrose:containing 14 mM 2-mercaptoethanol and 1 mM EDTA, disrupted by sonication for 20 sec (Kontes sonicator), and used for the assay without fractionation. Glycolipid Substrates. LacCer from equine spleen, Hex3Cer from equine kidney, and globoside from pig spleen were prepared as described (13). Radioactive Hex3Cer (5.4 X 105 cpm/,gmol), supplied by S. Handa, University of Tokyo, and globoside (5.2 X 105 cpm/Amol) were prepared by labeling with tritium at their nonreducing terminal sugars by the method of Suzuki and Suzuki (14), and used as substrates in the hydrolase studies. Isolation and Analysis of Glycolipids. The fibroblasts were freeze-dried and extracted with a suitable volume of chloroform/methanol (2:1, vol/vol), and the solvent was removed by evaporation. The total lipids were acetylated with 1 ml of pyridine/acetic anhydride (3:2, vol/vol) overnight at 600. The acetylated glycolipids were fractionated on a column of Florisil (15). The acetylated glycolipids were deacetylated by incubation with 0.5 ml of 0.1 M methanolic NaOH for 3 hr at room temperature followed by neutralization with Dowex 50 (H+ form). Analytical thin-layer chromatography was carried out Abbreviations: GicCer, glucosyl ceramide; LacCer, lactosyl ceramide; Hex3Cer, trihexosyl ceramide; ganglioside GM3 (hematoside), sialo-
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
syl(a2 3)Gal(l1 - 4)Glc(#l - 1)ceramide; Hex3Cer synthetase, UDPgalactose:lactosylceramide a-galactosyltransferase; globoside synthetase, UDP-N-acetylgalactosamine:trihexosylceramide f3-Nacetylgalactosaminyltransferase (EC 2.4.1.79). o
this fact. 5407
Biochemistry: Kijimoto-Ochiai et al.
5408
Table 1. Expression of blood group P antigens on erythrocytes, lymphocytes, and fibroblasts Antibodies Frequency, Antigens on in serum cells Phenotype %
Pi P2 p
Pk Ik
75
25 Very rare Very rare
Very rare
p1,p,pk* p, pk*
None
None Pi, pk
Anti-Pi, -P, pk
Proc. Natl. Acad. Sci. USA 74 (1977)
GLcCer
-
Anti-P1 Anti-P Anti-P
4)Glc(f31
- 1)cerPk antigen (Hex3Cer), Gal(al - 4)Gal(ll P amide; antigen (globoside), GalNAc(B1 3)Gal(al 4)Gal(fll 4)Glc(f1 - 1)ceramide; P1 antigen, Gal(al -* 4)Gal(#1 - 4)- 3)Gal(f1 - 4)Glc(,B1 - 1)ceramide. GlcNAc(01 * k antigenic activity is detected only in cultured lymphocytes and fibroblasts.
on coated Silica-gel H-60 plates (Merck) which were developed with chloroform/methanol/water (60:35:8, by volume) and visualized with anthrone-sulfuric acid reagent. Gangliosides were detected with resorcinol-HCI reagent. Radioactivity was detected by a radio-thin-layer scanner (Aloka JTC-202B) and by autoradiography, i.e.,-exposing the plates on x-ray film (Fuji KX Safety Film) for 18 days. Glycosyltransferase Assays. The activities of Hex3Cer synthetase (UDP galactose:lactosylceramide a-galactosyltransferase) (16) and globoside synthetase (UDP-N-acetylgalactosamine:trihexosylceramide fl-N-acetylgalactosaminyltransferase; EC 2.4.1.7b) (12) were assayed as described, with some modifications. Complete incubation mixture contained the following components in a final volume of 100 gl. For Hex3Cer synthetase assay: 50 Mig of LacCer, 300. jAg of Triton X-100, 0.1 M Hepes (N-2-hydroxethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 6.55), 10 mM MnCl2, 70 ,l of whole cell homogenates (112-395 Ag of protein), and 250 nCi of UDP[l-3HJgalactose (New England Nuclear; 25 Ci/mol for Exps. 1 and 2 and 5 Ci/mol for Exp. 3 in Table 2). For globoside synthetase assay: 50 jig of Hex3Cer, 300. Mg of sodium taurocholate, 50 nCi of UDP-N-acetyl[l-'4C]galactosamine (New England Nuclear, 51.2 Ci/mol), and- other components (buffer, MnCl2, and cell homogenates) as described for the Hex3Cer synthetase assay. The mixture was incubated for 2 hr at 370, and the reaction was terminated by adding 2.5 ,mol of EDTA and 5 ,mol of KCI in 20 Ml of water, followed by 0.6 ml of chloroform/methanol (2:1, vol/vol). After vigorous mixing and centrifugation, the lower layer was chromatographed on a thinlayer plate of Silica gel H (merck) in chloroform/methanol/ water (65:35:8, lower phase) and (58:35:8, by volume) for Hex3Cer and globoside synthetase assays, respectively. The radioactivity of the silica gel corresponding to each glycolipid was measured in toluene cocktail with a liquid scintillation spectrometer. In order to obtain the optimum conditions for the reactions, Hex3Cer and globoside-syntheses were examined with Pk and P2 cells, respectively. Production of radioactive glycolipids increased with the time of incubation for 2 hr. Both enzymatic activities at 1-hr incubation were 75% of those at 2-hr incubation. Triton X-100 was more effective for Hex3Cer synthetase activity than sodium taurocholate (84% of the activity with Triton X-100), but much- less effective (one-fourth that of taurocholate) for globoside synthetase. An incubation temperature of 27° or 370 for the globoside synthetase reaction did not affect the activity, as was shown in hamster cells (unpublished data). Glycosidase Assays. Hex3Cer a-galactosidase (Ec 3.2.1.47)
C HexCer : M
Defects of glycosyltransferase activities in human fibroblasts of pk and p blood group phenotypes (trihexosyl ceramide/globoside/net synthesis of P antigens/glycolipid contents/glycosidases)
SHIGEKO KIJIMOTO-OCHIAI*, MASAHARU NAIKIt, AND AKIRA MAKITA* *
Biochemical Laboratory, Cancer Institute, School of Medicine, and t Department of Biochemistry, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060, Japan
Communicated by Herman M. Kalckar, September 21, 1977
We demonstrate that human fibroblasts of ABSTRACT the rare pk phenotype lack globoside, which was identified as the blood group P antigen, and that p cells possess neither globoside nor trihexosyl ceramide, which was identified as pk antigen. Our investigations indicate also that these glycosphingolipid patterns are most likely caused by inherited preferential biosynthetic pathways in the abnormal phenotypes rather than by excess catabolism of the antigens. Evidence is presented that the fibroblasts of pk phenotype lack fl-N-acetylgalactosaminyltransferase (globoside synthetase; UDP-Nacetylgalactosamine:trihexosylceramide fi-N-acetylgalactosaminyltransferase; EC 2.4.1.79) activity, and those of p are deficient in a-galactosyltransferase (trihexosylceramide synthetase; UDP galactose:lactosylceramide a-galactosyltransferase) and possibly also in globoside synthetase. The diminished globoside synthetase activity in p cells, however, is not caused by the defect in the gene coding for this enzyme. It appears, rather, to be caused by a failure in gene expression because one-third of pk X p hybrids became able to express P antigenicity with a time lag of 3X4 days after cell fusion [Fellous, M., Gerbal, A., Nobillot, G. & Weils, J. (1977) Vox Sang. 32, 262-2681.
The human blood group P system was demonstrated originally on erythrocytes (1-4) and subsequently on fibroblasts and lymphocytes (5). This blood group system, which consists of three antigens and five phenotypes, has been immunologically and genetically established (reviewed in ref. 6). Recently the chemical structures of these antigens have been identified as glycosphingolipids (7, 8) (Table 1). Confirmative evidence has come from studies on erythrocytes of the rare abnormal phenotypes (9): the pk erythrocytes lack globoside and possess an increased amount of trihexosyl ceramide (Hex3Cer), and p erythrocytes, which are virtually deficient in both globoside and Hex3Cer, accumulate lactosyl ceramide (LacCer), thus suggesting a genetic block in formation of globoside in pk individuals and of both Hex3Cer and globoside in p persons. The biosynthesis of the carbohydrate moiety of glycolipids proceeds by the sequential addition of monosaccharide units from their sugar nucleotide donors to growing acceptors by specific glycosyltransferases (10). The synthesis of Hex3Cer (11) and globoside (12) occurs as follows: UDP Gal LacCer
Ilex3Cer synthetase
UDP-Ga1NAc Hex 3Cer
UDP
Hex3Cer (pk antigen)
UDP
Globoside synthetase
Globoside (P antigen)
Present investigations were undertaken to determine the cause of genetic defects of the antigens in pk and p fibroblasts by chemical analysis of glycolipids, incorporation of radioactive galactose into glycolipids, and assays of specific glycosyltransferase and glycohydrolase activities.
MATERIALS AND METHODS Cell Lines and Culture. Fibroblasts from three donors whose phenotypes of P blood group were P2, Pi, and p were generously donated by M. Fellous, Hopital Saint-Louis, Paris. Their antigenic and chemical properties are summarized in Table 1. The fibroblasts, obtained from skin biopsy (5),, were cultured in Eagle's minimum essential medium containing 20% fetal calf serum. The fibroblasts were labeled with [14Clgalactose as follows: The cells at the 17th or 18th passage were grown to confluence on glass bottles of 4 X 10 cm, and the medium was changed to 10 ml of fresh medium containing 5 ACi of [U14C]galactose (Radiochemical Center, Amersham, 95 Ci/mol). After 10 days, the cells were washed five times with Dulbecco's phosphate-buffered saline (pH 7.0) and harvested by use of trypsin. For analysis of glycolipid contents, the cells'were harvested after trypsin treatment and pooled (10th to; 20th passages). For enzymatic assays, the cells were scraped with a rubber policeman and suspended in 0.32 M sucrose:containing 14 mM 2-mercaptoethanol and 1 mM EDTA, disrupted by sonication for 20 sec (Kontes sonicator), and used for the assay without fractionation. Glycolipid Substrates. LacCer from equine spleen, Hex3Cer from equine kidney, and globoside from pig spleen were prepared as described (13). Radioactive Hex3Cer (5.4 X 105 cpm/,gmol), supplied by S. Handa, University of Tokyo, and globoside (5.2 X 105 cpm/Amol) were prepared by labeling with tritium at their nonreducing terminal sugars by the method of Suzuki and Suzuki (14), and used as substrates in the hydrolase studies. Isolation and Analysis of Glycolipids. The fibroblasts were freeze-dried and extracted with a suitable volume of chloroform/methanol (2:1, vol/vol), and the solvent was removed by evaporation. The total lipids were acetylated with 1 ml of pyridine/acetic anhydride (3:2, vol/vol) overnight at 600. The acetylated glycolipids were fractionated on a column of Florisil (15). The acetylated glycolipids were deacetylated by incubation with 0.5 ml of 0.1 M methanolic NaOH for 3 hr at room temperature followed by neutralization with Dowex 50 (H+ form). Analytical thin-layer chromatography was carried out Abbreviations: GicCer, glucosyl ceramide; LacCer, lactosyl ceramide; Hex3Cer, trihexosyl ceramide; ganglioside GM3 (hematoside), sialo-
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
syl(a2 3)Gal(l1 - 4)Glc(#l - 1)ceramide; Hex3Cer synthetase, UDPgalactose:lactosylceramide a-galactosyltransferase; globoside synthetase, UDP-N-acetylgalactosamine:trihexosylceramide f3-Nacetylgalactosaminyltransferase (EC 2.4.1.79). o
this fact. 5407
Biochemistry: Kijimoto-Ochiai et al.
5408
Table 1. Expression of blood group P antigens on erythrocytes, lymphocytes, and fibroblasts Antibodies Frequency, Antigens on in serum cells Phenotype %
Pi P2 p
Pk Ik
75
25 Very rare Very rare
Very rare
p1,p,pk* p, pk*
None
None Pi, pk
Anti-Pi, -P, pk
Proc. Natl. Acad. Sci. USA 74 (1977)
GLcCer
-
Anti-P1 Anti-P Anti-P
4)Glc(f31
- 1)cerPk antigen (Hex3Cer), Gal(al - 4)Gal(ll P amide; antigen (globoside), GalNAc(B1 3)Gal(al 4)Gal(fll 4)Glc(f1 - 1)ceramide; P1 antigen, Gal(al -* 4)Gal(#1 - 4)- 3)Gal(f1 - 4)Glc(,B1 - 1)ceramide. GlcNAc(01 * k antigenic activity is detected only in cultured lymphocytes and fibroblasts.
on coated Silica-gel H-60 plates (Merck) which were developed with chloroform/methanol/water (60:35:8, by volume) and visualized with anthrone-sulfuric acid reagent. Gangliosides were detected with resorcinol-HCI reagent. Radioactivity was detected by a radio-thin-layer scanner (Aloka JTC-202B) and by autoradiography, i.e.,-exposing the plates on x-ray film (Fuji KX Safety Film) for 18 days. Glycosyltransferase Assays. The activities of Hex3Cer synthetase (UDP galactose:lactosylceramide a-galactosyltransferase) (16) and globoside synthetase (UDP-N-acetylgalactosamine:trihexosylceramide fl-N-acetylgalactosaminyltransferase; EC 2.4.1.7b) (12) were assayed as described, with some modifications. Complete incubation mixture contained the following components in a final volume of 100 gl. For Hex3Cer synthetase assay: 50 Mig of LacCer, 300. jAg of Triton X-100, 0.1 M Hepes (N-2-hydroxethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 6.55), 10 mM MnCl2, 70 ,l of whole cell homogenates (112-395 Ag of protein), and 250 nCi of UDP[l-3HJgalactose (New England Nuclear; 25 Ci/mol for Exps. 1 and 2 and 5 Ci/mol for Exp. 3 in Table 2). For globoside synthetase assay: 50 jig of Hex3Cer, 300. Mg of sodium taurocholate, 50 nCi of UDP-N-acetyl[l-'4C]galactosamine (New England Nuclear, 51.2 Ci/mol), and- other components (buffer, MnCl2, and cell homogenates) as described for the Hex3Cer synthetase assay. The mixture was incubated for 2 hr at 370, and the reaction was terminated by adding 2.5 ,mol of EDTA and 5 ,mol of KCI in 20 Ml of water, followed by 0.6 ml of chloroform/methanol (2:1, vol/vol). After vigorous mixing and centrifugation, the lower layer was chromatographed on a thinlayer plate of Silica gel H (merck) in chloroform/methanol/ water (65:35:8, lower phase) and (58:35:8, by volume) for Hex3Cer and globoside synthetase assays, respectively. The radioactivity of the silica gel corresponding to each glycolipid was measured in toluene cocktail with a liquid scintillation spectrometer. In order to obtain the optimum conditions for the reactions, Hex3Cer and globoside-syntheses were examined with Pk and P2 cells, respectively. Production of radioactive glycolipids increased with the time of incubation for 2 hr. Both enzymatic activities at 1-hr incubation were 75% of those at 2-hr incubation. Triton X-100 was more effective for Hex3Cer synthetase activity than sodium taurocholate (84% of the activity with Triton X-100), but much- less effective (one-fourth that of taurocholate) for globoside synthetase. An incubation temperature of 27° or 370 for the globoside synthetase reaction did not affect the activity, as was shown in hamster cells (unpublished data). Glycosidase Assays. Hex3Cer a-galactosidase (Ec 3.2.1.47)
C HexCer : M
