Proc. Nati. Acad. Sci. USA Vol. 74, No. 12, pp. 5407-5410, December 1977 Defects of glycosyltransferase activities in human fibroblasts of pk and p group phenotypes (trihexosyl /globoside/net synthesis of P antigens/ 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

ABSTRACT We demonstrate that human fibroblasts of Present investigations were undertaken to determine the the rare pk phenotype lack globoside, which was identified as cause of genetic defects of the antigens in pk and p fibroblasts the blood group P antigen, and that p cells possess neither glo- by chemical analysis of , incorporation of radioactive boside nor trihexosyl ceramide, which was identified as pk an- tigen. Our investigations indicate also that these glyco- galactose into glycolipids, and assays of specific glycosyltrans- patterns are most likely caused by inherited pref- ferase and glycohydrolase activities. erential biosynthetic pathways in the abnormal phenotypes rather than by excess catabolism of the antigens. Evidence is MATERIALS AND METHODS presented that the fibroblasts of pk phenotype lack fl-N-acetyl- galactosaminyltransferase (globoside synthetase; UDP-N- Cell Lines and Culture. Fibroblasts from three donors whose acetylgalactosamine:trihexosylceramide fi-N-acetylgalactosa- phenotypes of P blood group were P2, Pi, and p were generously minyltransferase; EC 2.4.1.79) activity, and those of p are defi- donated by M. Fellous, Hopital Saint-Louis, Paris. Their anti- cient in a-galactosyltransferase (trihexosylceramide synthetase; genic and chemical properties are summarized in Table 1. The UDP galactose: a-galactosyltransferase) and fibroblasts, obtained from skin biopsy were cultured in possibly also in globoside synthetase. The diminished globoside (5),, synthetase activity in p cells, however, is not caused by the de- Eagle's minimum essential medium containing 20% fetal calf fect in the gene coding for this enzyme. It appears, rather, to be serum. The fibroblasts were labeled with [14Clgalactose as caused by a failure in gene expression because one-third of pk follows: The cells at the 17th or 18th passage were grown to X p hybrids became able to express P antigenicity with a time confluence on glass bottles of 4 X 10 cm, and the medium was lag of 3X4 days after cell fusion [Fellous, M., Gerbal, A., Nobillot, changed to 10 ml of fresh medium containing 5 ACi of [U- G. & Weils, J. (1977) Vox Sang. 32, 262-2681. 14C]galactose (Radiochemical Center, Amersham, 95 Ci/mol). After 10 days, the cells were washed five times with Dulbecco's The human blood group P system was demonstrated originally phosphate-buffered saline (pH 7.0) and harvested by use of on erythrocytes (1-4) and subsequently on fibroblasts and trypsin. For analysis of glycolipid contents, the cells'were har- lymphocytes (5). This blood group system, which consists of vested after trypsin treatment and pooled (10th to; 20th pas- three antigens and five phenotypes, has been immunologically sages). For enzymatic assays, the cells were scraped with a and genetically established (reviewed in ref. 6). Recently the rubber policeman and suspended in 0.32 M sucrose:containing chemical structures of these antigens have been identified as 14 mM 2-mercaptoethanol and 1 mM EDTA, disrupted by (7, 8) (Table 1). sonication for 20 sec (Kontes sonicator), and used for the assay Confirmative evidence has come from studies on erythro- without fractionation. cytes of the rare abnormal phenotypes (9): the pk erythrocytes Glycolipid Substrates. LacCer from equine spleen, Hex3Cer lack globoside and possess an increased amount of trihexosyl from equine kidney, and globoside from pig spleen were pre- ceramide (Hex3Cer), and p erythrocytes, which are virtually pared as described (13). Radioactive Hex3Cer (5.4 X 105 deficient in both globoside and Hex3Cer, accumulate lactosyl cpm/,gmol), supplied by S. Handa, University of Tokyo, and ceramide (LacCer), thus suggesting a genetic block in formation globoside (5.2 X 105 cpm/Amol) were prepared by labeling of globoside in pk individuals and of both Hex3Cer and glob- with tritium at their nonreducing terminal sugars by the oside in p persons. method of Suzuki and Suzuki (14), and used as substrates in the The biosynthesis of the carbohydrate moiety of glycolipids hydrolase studies. proceeds by the sequential addition of monosaccharide units Isolation and Analysis of Glycolipids. The fibroblasts were from their sugar nucleotide donors to growing acceptors by freeze-dried and extracted with a suitable volume of chloro- specific glycosyltransferases (10). The synthesis of Hex3Cer (11) form/methanol (2:1, vol/vol), and the solvent was removed by and globoside (12) occurs as follows: evaporation. The total were acetylated with 1 ml of pyridine/acetic anhydride (3:2, vol/vol) overnight at 600. The UDP Gal UDP acetylated glycolipids were fractionated on a column of Florisil LacCer Hex3Cer (pk antigen) (15). The acetylated glycolipids were deacetylated by incu- Ilex3Cer synthetase bation with 0.5 ml of 0.1 M methanolic NaOH for 3 hr at room UDP-Ga1NAc UDP temperature followed by neutralization with Dowex 50 (H+ form). Analytical thin-layer chromatography was carried out Hex 3Cer Globoside (P antigen) Globoside synthetase Abbreviations: GicCer, glucosyl ceramide; LacCer, lactosyl ceramide; Hex3Cer, trihexosyl ceramide; GM3 (hematoside), sialo- The costs of publication of this article were defrayed in part by the syl(a2 o 3)Gal(l1 - 4)Glc(#l - 1)ceramide; Hex3Cer synthetase, payment of page charges. This article must therefore be hereby marked UDPgalactose:lactosylceramide a-galactosyltransferase; globoside "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate synthetase, UDP-N-acetylgalactosamine:trihexosylceramide f3-N- this fact. acetylgalactosaminyltransferase (EC 2.4.1.79). 5407 Downloaded by guest on September 28, 2021 5408 Biochemistry: Kijimoto-Ochiai et al. Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Expression of blood group P antigens on erythrocytes, lymphocytes, and fibroblasts Frequency, Antigens on Antibodies Phenotype % cells in serum GLcCer - Pi 75 p1,p,pk* None P2 25 p, pk* Anti-P1 p Very rare None Anti-Pi, -P, pk Pk Very rare Pi, pk Anti-P Ik Very rare Anti-P Pk antigen (Hex3Cer), Gal(al - 4)Gal(ll - 4)Glc(f31 - 1)cer- amide; P antigen (globoside), GalNAc(B1 - 3)Gal(al - 4)Gal(fll 4)Glc(f1 - 1)ceramide; P1 antigen, Gal(al -* 4)Gal(#1 - 4)- GlcNAc(01 - 3)Gal(f1 - 4)Glc(,B1 - 1)ceramide. * k antigenic activity is detected only in cultured lymphocytes and fibroblasts. HexCerC

on coated Silica-gel H-60 plates (Merck) which were developed with chloroform/methanol/water (60:35:8, by volume) and :M<030 _ _ visualized with anthrone-sulfuric acid reagent. F:,Gw*. 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. SpSG 001. Glycosyltransferase Assays. The activities of Hex3Cer synthetase (UDP galactose:lactosylceramide a-galactosyl- transferase) (16) and globoside synthetase (UDP-N-acetylga- lactosamine:trihexosylceramide fl-N-acetylgalactosaminyl- transferase; 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 1 {2 3 4 Hex3Cer synthetase assay: 50 Mig of LacCer, 300. jAg of Triton X-100, 0.1 M Hepes (N-2-hydroxethylpiperazine-N'-2-etha- FIG. 1. Thin-layer chromatogram of glycolipids in fibroblasts nesulfonic buffer 6.55), 10 mM MnCl2, 70 of whole from P2 and Pk donors. Total glycolipids (approximately 50 1Ag each acid) (pH ,l as hexose) from P2 and pk cells were applied to lanes 3 and 4, respec- cell homogenates (112-395 Ag of protein), and 250 nCi of tively. Lanes 1 and 2, standard glycolipids. GlcCer, glucosyl ceramide; UDP[l-3HJgalactose (New England Nuclear; 25 Ci/mol for GLOBO, globoside; PG, paragloboside, Gal(j1- 4)GlcNAc(#1 - Exps. 1 and 2 and 5 Ci/mol for Exp. 3 in Table 2). For globoside 3)Gal(O1 - 4)Glc(01 - 1)ceramide; SPG, sialosyl paragloboside, synthetase assay: 50 jig of Hex3Cer, 300. Mg of sodium tauro- N-acetylneuraminyl (A2 - 3)Gal(O1 - 4)GlcNAc(f1 - 3)Gal(O1 cholate, 50 nCi of UDP-N-acetyl[l-'4C]galactosamine (New 4)Glc(j31 - 1)ceramide. England Nuclear, 51.2 Ci/mol), and- other components (buffer, MnCl2, and cell homogenates) as described for the Hex3Cer and globoside 3-N-acetylgalactosaminidase were determined synthetase assay. The mixture was incubated for 2 hr at 370, and with [3H]Hex3Cer and [3H]globoside as the substrates under the reaction was terminated by adding 2.5 ,mol of EDTA and the same conditions as the glycosyltransferase assays but without 5 ,mol of KCI in 20 Ml of water, followed by 0.6 ml of chloro- the labeled nucleotide sugars, respectively. After incubation form/methanol (2:1, vol/vol). After vigorous mixing and cen- for 2 hr at 370, the radioactive sugars released were determined trifugation, the lower layer was chromatographed on a thin- as described (17, 18). layer plate of Silica gel H (merck) in chloroform/methanol/ The a-galactosidase (EC 3.2.1.22) and fl-N-acetylgalactos- water (65:35:8, lower phase) and (58:35:8, by volume) for aminidase (EC 3.2.1.53) activities toward 4-methylumbellif- Hex3Cer and globoside synthetase assays, respectively. The eryl-a-galactoside (Seikagaku Kogyo) and 4-methylumbellif- radioactivity of the silica gel corresponding to each glycolipid eryl-,B-N-acetylgalactosaminide (Koch-Light Labs.), respec- was measured in toluene cocktail with a liquid scintillation tively, were determined as described (19, 20). spectrometer. In order to obtain the optimum conditions for the reactions, Hex3Cer and globoside-syntheses were examined with Pk and RESULTS P2 cells, respectively. Production of radioactive glycolipids Glycolipids in Fibroblasts. As shown in Fig. 1, pk fibroblasts increased with the time of incubation for 2 hr. Both enzymatic were completely lacking in globoside and the amounts of activities at 1-hr incubation were 75% of those at 2-hr incuba- Hex3Cer and ganglioside GM3 [hematoside; sialosyl(a2 -- 3)- tion. Triton X-100 was more effective for Hex3Cer synthetase Gal(,l3 - 4) Glc(3l - 1)ceramide] were increased compared activity than sodium taurocholate (84% of the activity with with those of P2 cells. Not enough p fibroblasts were available Triton X-100), but much- less effective (one-fourth that of for analysis of the constituent glycolipids because of the ex- taurocholate) for globoside synthetase. An incubation tem- tremely low growth rate of the cells. perature of 27° or 370 for the globoside synthetase reaction did When the uptake of radioactive galactose into each glycolipid not affect the activity, as was shown in hamster cells (unpub- was examined, we found that pk fibroblasts did not label glo- lished data). boside but contained increased radioactivity in Hex3Cer and Glycosidase Assays. Hex3Cer a-galactosidase (Ec 3.2.1.47) GM3 (Fig. 2). In the p fibroblasts, neither globoside nor Hex3Cer Downloaded by guest on September 28, 2021 Biochemistry: Kijimoto-Ochiai et al. Proc. Natl. Acad. Sci. USA 74 (1977) 5409

Table 2. Activities of Hex3Cer and globoside synthetases (pmol/ 1X3 mg of protein per 2 hr) in P2, P2, and p fibroblasts 9 2 Experiment 1 2 3 I 1 P2 I Hex3Cer synthetase 11 6.0* 17.0* Globoside synthetase 1.4 0.2* 1.0*

Pk2 Hex3Cer synthetase 4.8 11.4* 36.4 Globoside synthetase 0.04 0 0 p Hex3Cer synthetase 0.5 0.08 0 Globoside synthetase 0 0 0.1 All values were corrected for endogenous values. * Average value of duplicate determinations. increased activity of glycosidases, which degrade the glycolipids synthesized. We examined this possibility by determining the GIcCer activities of a-galactosidase and f3-N-acetylgalactosaminidase, Unknown which cleave terminal glycosidic linkages of Hex3Cer and ganglioside globoside, respectively, in the cells, using labeled glycolipids as substrates under the same conditions for the synthetase assays. However, both of the glycosidase activities for the glycolipids were low, and the differences among the three cell lines were within experimental errors. The possibility cited above, therefore, would be unlikely. On the other hand, some in- consistent results were obtained in the experiment using 4- methylumbelliferyl sugars as substrate: i.e., f3-N-acetylgalac- tosaminidase activity in p cells under the conditions for the glycosyltransferase assays was higher than that of pk and P2 cells (93, 33, and 27 nmol of 4-methylumbelliferon released per mg Glooside 1 of protein per hr for p, p14 and P2 cells, respectively), while little a-galactosidase activity was detected with the three cell Origin lines. FIG. 2. Radioautogram (Upper) and radioscanning (Lower) of On the basis of these results, therefore, it is most probable that a thin-layer chromatogram ofradioactive glycolipids from P2, Pk, and the faint trace of globoside in p4 cells, which results in the ab- p fibroblasts. Cells (approximately 2 X 106 cells) were incubated with sence of P specificity, and the marked defect of Hex3Cer and ["4C]galactose. Radioactive glycolipids from P2 (20,000 cpm), p (25,000 globoside in p cells, which are serologically missing pk and P cpm), and pk (40,000 cpm) fibroblasts were applied to lanes 1, 2, and 3, respectively. antigenic expression, are most likely due to the deficient activity of the glycolipid glycosyltransferases. However, we cannot became labeled, whereas LacCer and GM3 were heavily la- exclude completely that an elevated activity of ,B-N-acetylga- beled. No change in labeling of GlcCer and the other gan- lactosaminidase in p cells may, to some extent, be responsible gliosides was detected among these fibroblasts. Thus, the results for failure in the formation of globoside. of incorporation experiments were consistent with those of cell glycolipid analysis (Fig. 1). DISCUSSION Hex3Cer and Globoside Synthetase Activities. Three lines The human blood group P antigen system has been immu-

of fibroblasts from P2, 14, and p individuals were assayed for nologically and genetically studied in detail (reviewed in ref. Hex3Cer and globoside synthetase activities (Table 2). Hex3Cer 6). Elucidation of the structures of the antigens of the P system synthetase activity, although the absolute values fluctuated in (7, 8) provided studies on the relationships between the sero- triplicate determinations in each cell line, was present in P2 and logical expression on cells and the metabolic basis of the anti- cells, while negligible activity was detectable in the p cells. gens. On the other hand, almost no globoside synthetase activity was As has been postulated previously (21), there are two possible found in both p1 and p fibroblasts. P2 cells had appreciable reasons for the occurrence of p phenotype. (i) Lack of Hex3Cer globoside synthetase activity which was, in general, lower than (pk antigen, a precursor for globoside synthesis) is due to the that of Hex3Cer synthetase. absence of a-galactosyltransferase activity (Hex3Cer synthe- The activity of globoside synthetase in the mixed homoge- tase). This prevents the production of globoside (P antigen) in nates of p and P2 cells was comparable to that of P2 cells alone, spite of the presence of ,B-N-acetylgalactosaminyltransferase although the activity was low (data not shown). This suggests activity (globoside synthetase). (ii) The p cells lack both of the that the decreased activity observed in p cells was not caused synthetases. The former reason was supported in a previous by an inhibitor. paper (9). Glycosidase Activities. Defects in the formation of the an- The present investigations show clearly that the fibroblasts tigenic glycolipids found in fibroblasts might be caused by an from individuals with the rare phenotypes of the P system lack Downloaded by guest on September 28, 2021 5410 Biochemistry: Kijimoto-Ochiai et al. Proc. Natl. Acad. Sci. USA 74 (1977) globoside synthetase activity in pk cells and both globoside and a new GM3- was found to be caused by a defect Hex3Cer synthetase activities in p cells (Table 2). The conse- in ,-N-acetylgalactosaminyltransferase that adds N-acetyl- quence of defective enzyme activities in the cells was evident galactosamine onto GM3 (25). Patients with this disease suffer from the results of glycolipid patterns (Figs. 1 and 2). However, severe neurological dysfunction and death at childhood. At Fellous et al. (22) demonstrated very recently that 30-40% of present-there are no data to indicate whether the inherited polykaryon cells obtained by fusion of pk and p fibroblasts ex- defects of only particular glycosyltransferases cause "diseas- pressed P antigenicity 3-4 sdays after hybridization. On the es. other hand, approximately 60% of hybrids of P2 and p cells showed P reactivity immediately after cell fusion. These data We thank Dr. H. Katsuta, Institute of Medical Science, University suggest that either pk or p cells may possess the gene coding for of Tokyo, for help in maintenance of the cell lines. This work was globoside synthetase. Although we do not know the real reason supported in part by a Grant-in-Aid for Scientific Research from the for the discrepancy between the missing synthetase activities Ministry of Education, Science and Culture, Japan. found by us and the positive antigenic expression after cell 1. Landsteiner, K. & Levine, P. (1927) Proc. Soc. Exp. Biol. Med. fusion described by Fellous, we might venture the possibility 24,941-942. that globoside synthetase (activity) of pk X p hybrids may be- 2. Levine, P., Bobbitt, 0. B., Waller, R. K. & Kuhmichel, A. (1951) come "induced" by intracellular uptake of Hex3Cer from pk Proc. Soc. Exp. Biol. Med. 77,403-405. cells. This possibility would be consistent with the combined 3. Sanger, R. (1955) Nature 176, 1163-1164. findings. (i) There is a time lag (3-4 days) for expression of P 4. Matson, G. A., Swanson, J., Noades, J., Sanger, R. & Race, R. R. antigenicity (P2 phenotype) after pk X p cell hybridization, in (1959) Am. J. Hum. Genet. 11, 26-34. contrast to an immediate expression of P antigenicity by P2 X 5. Fellous, M., Gerbal, A., Tessier, C., Frezal, J., Dausset, J. & p cell fusion. This time lag might coincide with the period re- Salmon, C. (1974) Vox Sang. 26,518-536. synthetase. (if) There 6. Race, R. R. & Sanger, R. (1975) in Blood Group in Man (Backwell quired for the "induction" of globoside Scientific Publications, Oxford), 6th Ed., pp. 139-177. might also be a possible combination of the distribution in hy- 7. Naiki, M. & Marcus, D. M. (1974) Biochem. Biophys. Res. brids (pkpk, pkp, pp), i.e., one-third of the tetraploid hybrids Commun. 60, 1105-1111. of pk and p cells expressed P antigenicity. Similarly, two-thirds 8. Naiki, M., Fong, J., Ledeen, R. & Marcus, D. M. (1975) Bio- of P2 X p hybrids (P2P2, P2p, pp) showed P antigenicity. chemistry 14, 4831-4837. Therefore, the results of biochemical and cytological experi- 9. Marcus, D. M., Naiki, M. & Kundu, S. K. (1976) Proc. Natl. Acad. ments imply that pk cells lack the gene for globoside synthetase Sci. USA 73,3263-3267. and that p cells possess this gene but seem unable to express it. 10. Roseman, S. (1970) Chem. Phys. Lipids 5, 270-297. Globoside synthetase may be induced during intracellular in- 11. Hildebrand, J. & Hauser, G. (1969) J. Biol. Chem. 244, 5170- Hex3Cer after a prolonged posthybridization 5180. corporation of 12. Ishibashi, T., Kijimoto, S. & Makita, A. (1974) Biochim. Biophys. period. Acta 337, 92-106. The results of chemical analysis and galactose incorporation 13. Makita, A. (1964) J. Biochem. (Tokyo) 55,269-276. experiments of the glycolipids Of p2. p, and P2 fibroblasts were 14. Suzuki, Y. & Suzuki, K. (1972) J. Res. 13, 687-690. virtually similar to those of Marcus et al. on chemical analysis 15. Saito, T. & Hakomori, S. (1971) J. Lipid Res. 12, 257-259. in erythrocytes (9). The results of these biosynthetic blocks 16. Kijimoto, S. & Hakomori, S. (1971) Biochem. Biophys. Res. appears to be increased amounts of Hex3Cer and GM3 gan- Commun. 44,557-563. glioside in pk cells, and of LacCer and GM3 in p cells. There are, 17. Wenger, D. A., Okada, S. & O'Brien, J. S. (1972) Arch. Biochem. however, some variances in glycolipid composition between Biophys. 153, 116-129. In was a 18. Kano, I. & Yamakawa, T. (1974) J. Biochem. (Tokyo) 75, the twe cells types. normal erythrocytes, globoside 347-354. predominant component and Hex3Cer was present only in trace 19. Johnson, W. G. & Brady, R. 0. (1972) in Methods in Enzymology, amounts (9), as has been well recognized (23, 24), while the ratio ed. Ginsburg, V. (Academic Press, New York and London), Vol. of the two glycolipids was completely reversed in normal (P2) 28, pp. 849-856. fibroblasts (Figs. I and 2). These differences may, at least to 20. Kaback, M. (1972) in Methods in Enzymology, ed. Ginsburg; some extent, explain the absence of pk antigen on normal V. (Academic Press, New York and London), Vol. 28, pp. 862- erythrocytes and the positive expression on normal (P2) fibro- 867. blasts (5), and may reflect the metabolic specificity in bone 21. Naiki, M. & Marcus, D. M. (1975) Biochemistry 14, 4837- marrow cells and in skin fibroblasts producing the antigens. 4841. to be 22. Fellous, M., Gerbal, A., Nobillot, G. & Weils, J. (1977) Vox Sang. Low globoside content in normal fibroblasts appears 32,262-268. correlated with a lower level of globoside synthetase compared 23. Klenk, E. & Lauenstein, K. (1951) Hoppe-Seyler's Z. Physiol. with that of Hex3Cer synthetase (Table 2). Chem. 288, 220-228. The individuals with pk and p phenotypes appear to be 24. Yamakawa, T. & Suzuki, S. (1952) J. Biochem. (Tokyo) 39, physically normal and to have a normal life span despite their 393-402. glycosyltransferase defects, which result in abnormal glycolipid 25. Fishman, P. H., Max, S. R., Tallman, J. F., Brady, R. O., Maclaren, composition in erythrocytes and tissue cells. On the other hand, N. K. & Cornblath, M. (1975) Science 187,68-70. Downloaded by guest on September 28, 2021