Expression of ABH and X (Lex) antigens in various cells

Biochimie 70 (1988) 1613-1617 Soci6t6 ~le Chimie biologique/Elsevier, Paris

1613

Expression of ABH and X (Le x) antigens in various cells Jacques LE PENDU I, Thierry CAILLARD I, Rosella MOLLICONE 3, Philippe COUILLIN 2 and Rafael O R I O L 3

~INSERM U211, Nantes, 2INSERM U73, Paris, and 3CNRS UA622, 92296 Chdtenay-Malabry, France (Received 3-12-1987, accepted after revision 5-4-1988)

Summary m Using a panel of reagents specific to the various subtypes of ABH antigens, it could be demonstrated that platelets carry ABH type 2 monofucosylated determinants on intrinsic glycoproteins. The presence of these antigens is controlled by the H gene and correlates with the presence of Ot-2-Lfucosyitransferase and the absence of ot-3-L-fucosyltransferase. In contrast, intrinsic ABH antigens were not found on mononuclear cells, correlating with the absence of o~-2-L-fucosyltransferase on these cells. However, after transformation with the Epstein-Barr virus and stimulation with 12-O-tetradecanoylphorbol-13-O-acetate (TPA), B lymphocytes were found to express the H antigen under control of the H gene and not the Se gene. The iymphoblastoid cell lines also expressed the X and sialylated X antigens which are normally markers of the myeloid lineage. These antigens are also normally found in epithelial cells of the digestive tract, kidney proximal convoluted tubules and hepatocytes. The a-3-L-fucosyltransferase responsible for the synthesis of this antigen is present in the serum but we report the existence of two individuals, a mother and her daughter, who lack more than 90% of this serum enzyme. The young girl suffers from a congenital kidney anomaly: oligomeganephronic hypoplasia. Her kidney tubules are devoid of X antigen. However, she and her mother have the X antigen on their granulocytes and its sialylated form on their monocytes. It therefore appears that there are distinct genetic controls for the expression of antigen X in different body compartments. This would be quite similar to the H and Se gene controls in tissues of distinct embryological origins. ABH / X / antigens / genetic control

The ABH and X antigens can be considered as markers of cell differentiation [1]. However, it is not quite possible to relate the expression of any one of these antigens to a particular cell lineage during ontogeny, since a variety of cell types with various embryological origins can express the same antigenic pattern. We would like now to present some data in favor of a genetic model by ~,hich we try to reconcile the embryology and the expression of these antigens

[21.

In certain cells, ABH antigens are under the control of the secretor (Se) and Lewis (Le) genes. They are based on both type 1 and type 2 precursors. There are other cell types in which the expression of ABH antigens is independent

of the Se and Le genes and most likely under the control of the H gene in a variety of tissues which appear mainly to be based on Type 2 precursors. The Fig. 1 shows a summary of what we know about the tissue expression of these antigens. It seems roughly possible to relate the embryological origin of a cell type to the gene that controls its expression: Se and Le in tissues of endodermal origit, and H in tissues of ectodermal and mesodermai origins. There is now ample evidence that the o,-2-fucosyltransferases under the control of the H and Se genes have different enzymatic properties [3, 4]. We have shown that these two genes are closely linked on the short arm of chromosome 19 [5]. However, the expression of ABH antigens on

1614

J. Le Pendu et al. | ABR OF TYPE ] AND 2 [ CONTROLLED BY SE AND Z K

ABR OF TYPE 2 [ INDEPENDENT Og S K AND LE ECTODERM

I

[

[ NEUNOECTODERM~t%

ENDODERM ]

SALIVARY GLANDS

l

V

I :=°":.o !1"'°'"1

R.sP,R,TORY

I

DIGESTIVE AND [ RINAnY EPITREgIA[

V

I ENDOTHELIUM

PLATELETS

Fig. 1. Embryologicaloriginof tissuesexpressingABH antigens.

platelets and lymphocytes remains uncertain. The expression of an a-2-fucosyltransferase on lymphocytes has been repotted [6, 7] to be independent of the Se gene, although these cells apparently carry only adsorbed antigens "of endodermal origin. Another problem concerns the expression of ABH antigens in platelets, since it is still debated whether these antigens are only absorded from the plasma or whether they are also expressed independently of the Se and Le genes on intrinsic membrane components. We thus investigated by immunofluorescence the presence of ABH antigens on platelets, using a panel of well-defined reagents. The expression of these antigens was independent of the secretot and Lewis status but it was clearly under the control of the H gene, since H-deficient individuals were always negative. Furthermore, only those reagents reacting with monofucosylated type 2 ABH antigens were positive. Reagents specific for type 3 and 4 or difucosylated type 1 or 2 antigens did not stain platelets. The solubilized proteins of platelets were analyzed by Western blot after SDS-polyacrylamide gel electrophoresis (PAGE) under reducIng conditions. Anti-A, -B, and -H antibodies revealed a few specific bands. The major one comigrates with GpIb (Fig.2). Thus we conclude that platelets do carry intrinsic glycoproteins

substituted with ABH determinants, only recognized by reagents specific to monofucosylated type 2 structures and that they are under the control of the H gene. On peripheral lymphocytes, no ABH antigens can be detected by immunofluorescence, regardless of the fine specificities of the reagents [8] despite the reported presence of A, B and H glycosyltransferases in these cells. In fact, this discrepancy between the expression of antigens and glycosyltransferases could only be understood after preparation of lymphocytes with careful elimination of platelets. Indeed, lymphocytes prepared after collecting the blood on anticoagulant and separation on Ficoll or Percoll gradients are always contaminated by platelets. This can be easily seen by immunofluorescence using antibodies specific for platelet glycoproteins. In this case, the mononuclear cell preparations contain both the t~-2- and a-3-fucosyltransferase activities. On the other hand, when blood was collected on glass beads so as to aggregate platelets and the mononuclear cells were separated by Ficoll or Percoll density gradients, it was shown by immunofluorescence that they were totally free of platelets. In this last case, the t~-2-fucosyitransferase was absent, whereas the a-3-fucosyltransferase remained (Table I). Thus, there is a good correlation between the expression of the

A B H and X antigens

200.

=,.

116 92

D. ="

66

=,.

45

"

12

34

567

Fig. 2. Biochemicalcharacterization of the platelet glycoproteinscarryingABH determinants.Freshlyisolatedplatelets were solubilizedin 30 mM Tris-HCl, pH 7, 3 mM ethylenediaminetetraaeetic acid, 6% sodium dodecyl sulfate (SDS). After reduction in 5%/3-mercaptoethanol,the solubilizedplatelets were eleetrophoresedin 10% verticalpolyacrylamideslab gels. The Western blot method was used to transfer the platelet membrane proteins from SDS-polyaerylamidegels onto nitrocellulosemembranes. Bindingof purified monoclonal anti-A or -H antibodies was detected by immunoperoxidasestaining. The non-specificbinding wasquenchedusing5% bovineserum albuminin phosphate bufferedsaline. Lanes 1 and 2: Chinese-inkstainingof total proteins transferred onto nitr~-~,~!lu!ogefilter ~om blood groupO and A platelets,respectively,l.,anes3 and 4: immunostaining with anti-A (3-3A) of O and A platelet extracts. Lanes5, 6 and 7: immunostainingwith anti-H (IIIT4) of O, A and Oh(Bombay)platelets. Molecularweightmarkersare indicatedin kDa.

antigens and the glycosyltransferases detected. As expected, no a-3-fucosyltransferase was detected in the platelet preparations. We do not know as yet if the a-3-fu~:osyltransferase came from the lymphocytes or from the monocy.tes. Indeed the mononuclear cell preparations contain about 20% monocytes, which strongly express the sialylated X antigen, whereas the lymphocytes are devoid of X and sialylated X antigens. Although X and H antigens are lacking o n normal lymphocytes, they occur under abnormal conditions (Table II). We have transformed

1615

peripheral B !ymphocytes with the EpsteinBarr virus (EBV). These immortalized lymphoblastoid cell lines express the X antigen under classical culture conditions, although it is normally not present on lymphocytes but on granuIocytes. It seems that this expression decreases in the presence of 12-O-tertradecanoyl-phorbol13-O-acetate (TPA) an inducer of differentiation. TPA can also induce the expression of H antigen. This antigen is under the control of the H gene, since it appears only in individuals who express the H gene, irrespective of their secretor phenotype. Thus these cells of mesodermal origin, which normally do not express the H antigen, can express it in a pathological condition and this expression is under H gene control. In addition to granulocytes and monocytes, a large variety of cell types can express the X or sialylated X antigens. The kidney proximal convoluted tubules (PCT), various cell types ef the digestive tract and hepatocytes are the main sources of these antigens. While studying their expression in various non-cancerous conditions of the kidney, we found that a young patient had kidney PCT completely devoid of X and sialylated X antigens. However, her polymorphonuclear cells strongly expressed the X antigen and her monocytes strongly expressed the sia!ylated X antigen. This patient suffered from a rare congenital disease: oligomeganephroni¢ hypoplasia [9]. This disease is charaetized by an abnormal development of renal tissue. The kidneys are small (hypoplasia) and contain a small number of nephrons (oligonephronic), but the size of these nephrons is very large (meganephrons). It cannot be concluded at present that the absence of the antigen is responsible for this developmental anomaly, since the X antigen was normally present in the kidney of 5 other patients suffering from the same disease. However, the a-3-fucosyltransferase (a-3-FT) activity was measured in the first patient's serum and it appeared to be very low, whereas the o~-2-fucosyltransferase activity was normal. The patient's mother, who has no known disease, also had a very low a-3-FT (Table III). She also expressed the X and sialylated X antigens on her granulocytes and monocytes. Therefore~ the myeloid cells would contribute to less than 10% of the total serum a-3-fucosyltransferase. This dissociation between the expression of the X antigen on kidney cells and myeloid cells leads us to hypothesize that there are at least 2 gt:nes controlling the expression of the ot-3-fucosyitransferases. Interestingly, both were'Le(a-b-) non-

J. Le Pendu e t al.

1616

Table !. ct-2- and a-3-L-fucosyltransferases in mononuclear cells.

Method

Transferred [14C]fucose (cpm)

1 2 2 2

a-2-fucosyltransferase

c~-3-fucosyltransferase

14348 0 0 0

84504 122760 19851 33326

The reaction mixtures for a-2-fucosyltransferase contained in a total volume of 130 v,l: 1% Triton X-100 cell lysates ( ~ 5 x 106 cells); Tns-HCL, pH 7-2, 1.0/~mol; MgCI2 1.0/zmol; ATP 0.25/zmol; GDP [ C]fucose 620 pmoi; phenyl--/3-o-galactoslde 0.16 ~,mol. The reaction mixtures for a-3-L-fucosyitransferase were similar, except that MgCl2 was replaced by 1/zmol MnCl2 and phenyl-/3-n-galactoside by 0.23 v,mol N-acetyllactosamine. The mixtures were incubated for 64 h at 37°C and the products were separated by paper chromatography in ethyl acetate / pyridine / water (10:4:3) and characterized by their chromatographic mobilities. Mononuclear cells prepared after collection of the blood on anti-coagulant (method 1) were contaminated by platelets as revealed by immunofluorescence using anti-Gpb-IIIa platelet glycoprotein. Mononuclear cells prepared after collection of the blood on glass beads (method 2) were free of platelets. •

--

14

"

Table il. Immunofluorescence on E B V lymphoblastoid cell lines.

Cell line

Genotype

% positive cells without T P A

with T P A

X

Si-X

H

Ig

X

Si-X

H

lg

29930

H / H, Se / se

39 50 9 47

70 33 20 16

0 1 1 -

43 35 24 -

29 31 2 18

28 18 6 9

8 12 13 -

1 12 4 -

107977

H / H , se/se

12 7 25

46 76 -

0.5 2 0

49 20 43

0 0 7

30 44 -

8 15 10

6 3 1

107965

H / h , se/se

60

18

0

5

13

15

-

0

29932

h / h , se/se

68 57

21 23

0 -

37 21

27 48

21 28

-

39

19

0

13

13

11

107932

h / h , se/se

0

25 14

0

12

Cells were cultured in RPMI-1640 with 10% fetal calf serum (FCS); 12-O-tetradecanoylphorbol-13-O-acetate (TPA) was added (2/~g/ml) in some cultures and the immunofluorescence assays were performed after 2 days of culture. The X antigen was detected with antibody 80H5, its sialylated form (Si-X) with antibody CSLEX1, the H antigen with affinitypurifiedpolyclonal antibodies 115,1 and the surface immunoglohulins (lg) with a polyclonal anti-human immunoglobulin antiserum, uomparison ot the results for each experiment, with and without TPA, were performed by the paired t test: decrease of antigen X, p < 0.001 (n = 10); decrease of sialylated-X, p < 0.02 (n = 9); increase of antigen H, in H ~ H individuals, p < 0.001 (n = 5); decrease of surface immunoglobulins, p < 0.003 (n -- 10).

A B H and X antigens

1617

Table 111. Serum a-2- and a-3-L-fucosyltransferase activity in normal individuals, o~-3-L-fucosyltransferase defi-

cient child and mother and anephric patients.

Incorporation of [14C]fucose (pmol) a a-2-L-fucosyltransferaseb

a-3-L-fucosyltransferase

phenyi-/3-Gal

N-acetyllactosamine

N-acetyllactosamine

Normal (n = 19)d

60 - 21

33 --- 10

125 _ 20

Anephric (n =5)

30 +- 8

34 --- 11

87 - 27

Z.G. (child)

43

62

9

Z.J. (mother)

30

74

9

~Mean ± S.D. bEnzymc. ~Acceptor. din the case of phenyl-/3-galactopyranoside,n=7. The assayswere performed using 25 p.l of fresh serum; Tris-HCl, pH 7.2, 0.25/~mol;MnCI21.0/zmol;ATP 0.25 tzmol;GDP[t4C]fucose 620 pmol; N-acetyilactosamine 0.23 p.mol or phenyl-/3-o-galactopyranoside0.16 p.mol in a final volume of 50/~1. After 64 h of incubation at 37oC, the products were separated by paper chromatography in ethyl acetate/pyridine/water (1Q:4:3). The a-2-L-fucosyltransferaseproduct is on N-acetyllactosamine(Rlac= 1.0), or phenyl-/3-o-galatopyranoside(R~c= 1.5). The a-3-L-fucosyltransferaseproduct is on N-acetyllactosamine(Rla~= 0.75).

secretors. The existence of 2 other individuals with non-detectable serum ot-3-fucosyltransferase has been described and they were also L e ( a - b - ) [10]. The a-3-fucosyltransferase activity was also measured in anephric patients in order to determine the contribution of the kidney to the total serum enzyme (Table III). This activity was slightly decreased cc,m p a r e d to that of healthy individuals. This suggests that the kidney contributes to some of the, s e r u m a-3-fucosyltransferase but that othe~r cell types also contribute (under the same genetic control as the kidney cells). Some preliminary experiments were performed in order to d e t e r m i n e on which c h r o m o s o m e the a-3-fucosyltransferase gene expressed in leukocytes maps. For this, we constructed h u m a n mouse cell hybrids by fusing a h u m a n lymphoblastoid cell line, which expresses the a-3-fucosyltransferase, with the m o u s e m y e l o m a Sp20, which does not express this enzyme. These hybrids have lost various h u m a n c h r o m o s o m e s selectively and at r a n d o m . T h e cell line lacking the enzyme is the only o n e which also lacks the human c h r o m o s o m e X. It will be necessary to test more cell lines in order to confirm that this

oz-3-fucosyltransferase gene is indeed located on c h r o m o s o m e X. In conclusion, it appears that the h u m a n body is a mosaic with ,espect to the A B H , Lewis or X antigens. O n e single antigen may not result from the action of the same enzyme in different tissues. At least for the A B H antigens, the genetic control of their expression can be more or less related to the embryological origin of the histological structure which carries them. We also propose the hypothesis that the ct-3-fucosyltransferases are also encoded by a multigemc family, each of these genes being expressed in one type of cell, possibly in accordance with the embryological origin of these cells.

References 1 Feizi T. (1985) Nature 314, 53-57 2 0 r i o l R., Le Pendu J. & Mollicone R. (1986) Vox Sang. 51,161-171 3 Betteridge A. & Watkins W.M. (1985) Glycoconjugate Jr. 2, 61-78 4 Le Pendu J., Cartron J.P., Lemieux R.U. & Oriol R. (1985)Am. J. Hum. Genet. 37,749-760 5 0 r i o l R., Le Pendu J., Bernez L., Lambert F., Dalix A.M. & Hawkins B,R. (1984) Cytogenet. Cell Genet. 37,564

1618

J. Le Pendu et al.

6 Cartron J.P., Mulet C. & Salmon C. (1980) Blood Transfus. Immunohemat. 23, 271-282 7 GreenweU P., Ball M.G. & Watkins W.M. (1983) FEBS Lett. 164, 314-317 8 MoUicone R., CaiUard T., Le Pendu J., Francois A., Sansonetti N., Villaroya H. & Oriol R. (1988) Blood 71, 1113-1119

9 Caillard T., Le Pendu J., Ventura M., Mada M., Rault G., Mannoni P. & Oriol R. (1988) Exp. Clin. Immunogenet. 5, 15-23 10 Oreenwell P., Johnson P.H., Edwards J.M., Reed R.M., Moores P.P., Bird A., Graham H.A. & Wa'zkins W.M, (1986) Blood Transfus. immunohematol. 29, 233-249