Differentiation of Human Erythroid Cells Is Associated with Increased O

Proc. Natl. Acad. Sci. USA Vol. 81, pp. 6752-6756, November 1984 Cell Biology

Differentiation of human erythroid cells is associated with increased O-glycosylation otthe major sialoglycoprotein, glycophorin A (erythrocyte differentiation/mabria/erythroleukemia) CARL G. GAHMBERG*, MARJA EKBLOM,t AND LEIF C. ANDERSSONt *Department of Biochemistry, University of Helsinki, 00170 Helsinki 17, Finland; and tDepartment of Pathology and Transplantation Laboratory, University of Helsinki, 00290 Helsinki 29, Finland Communicated by Robert L. Hill, July 13, 1984

ABSTRACT Glycophorin A, the major human erythro- tionation according to cell size was accomplished using 1 x g cyte sialoglycoprotein, is found exclusively on cells of the ery- velocity sedimentation (13). Blood from neonatal patients throid lineage. The amino acid sequence is known, and glyco- with erythroblastosis fetalis was obtained from the Depart- phorin A isolated from mature erythrocytes contains a single ment of Pediatrics, Helsinki University Hospital, and the N-glycosidic and 15 O-glycosidic oligosaecharides. Monoclonal mononuclear cells were isolated. K562 cells were obtained antibodies against erythrocyte glycophorin A reacted weakly from G. Klein, Karolinska Institute, Stockholm, and HEL with erythroid precursors while a monospecific rabbit antise- cells from E. Papayannopoulou, University of Washington, rum reacted strongly with immature and mature red cells. Seattle. Glycophorin A was isolated from cells representing various Induction of Differentiation in K562 and HEL Cells. K562 stAges of erythropoiesis in normal bone miarirow, from blood cells and HEL cells were grown in RPMI 1640 medium con- cells of neonates with erythroblastosis fetalis, and from the taining 10% fetal calf serum. Cells were induced to differenti- erythroleukemic cell lines K562 and HEL before and after in- ate for 1-6 days with 1.5 mM sodium butyrate, 0.1 ,uM retin- duced differentiation. Analysis of the oligosaccharides showed oic acid, 10 nM phorbol i2-myristate 13-acetate (PMA), or less O-glycosylation of glycophorin A in erythroid precursors. 25-50 ,uM hemin as described (14-16). The degree of differ- The degree of glycosylation increased concomitantly with dif- entiation was estimated from May-Grunwald-Giemsa- ferentiation. stained smears, and cultures containing differentiated cells were harvested for further studies. The major sialoglycoprotein of human erythrocytes, glyco- Antisera. Rabbit anti-GPA antisera were produced by im- phorin A (GPA), consists of 131 amino acids distributed in munizing with purified GPA (17). The antisera were ad- three separate domains: at the cell surface, within the lipid sorbed with En(a-) red cell membranes, which lack GPA bilayer, and in the cytoplasm (1). The external NH2-terminal (18-20), as described (5). Monoclonal anti-GPA antisera R10 portion is highly glycosylated, containing one N-glycosidic and R18, and VIE-G4 were obtained from P. A. W. Edwards oligosaccharide at Asn-26 and 15 O-glycosidic oligosaccha- (21) and W. Knapp (22), respectively. rides. The structure of the N-glycosidic oligosaccharide has Binding of Protein A-Containing Staphylococci to Anti- been determined (2). Most O-glycosidic oligosaccharides GPA-Treated Bone Marrow Cells. The presence of GPA in have the structure Neu5Aca2-3Gall31-3(Neu5Aca2-6)Gal- bone marrow cells was assessed with a quantitative staphy- NAc (ref. 3; Neu5Ac, N-acetylneuraminic acid; see ref. 4 for lococcal rosetting assay of anti-GPA antiserum-treated cells the condensed symbolism for oligosaccharide chains). (5). It was shown that GPA is confined to the erythroid cell Radioactive Labeling. Cell surface glycoconjugates were lineage and appears at the basophilic normoblast stage of radioactively labeled using the periodate/NaB3H4 technique erythropoiesis (5, 6). GPA is also found on the erythroleuke- (23). Radiolabeled red cell membranes were isolated as de- mia cell lines K562 (7) and HEL (8, 9). The biosynthesis of scribed (24). Labeled nucleated cells and red cell membranes the protein has been extensively studied in K562 cells and its were solubilized in 1% Triton X-100/0.01 M sodium phos- N- and O-glycosylations have been elucidated (10-12). phate/0.15 M NaCl, pH 7.4, at 0°C and centrifuged at 5000 x We have now isolated GPA from normal bone marrow g for 10 min, and the supernatants were recovered. For la- precursor cells and from the K562 and HEL cell lines before beling with [35S]methionine and 3H, 3 x 107 uninduced cells and after induction of differentiation, and we have studied its or K562 cells induced with hemin for 3 days were incubated oligosaccharides. Our results show that, during differentia- for 90 min with [35S]methionine (10), washed, and labeled by tion of red cells, the GPA molecules become increasingly 0- the periodate/NaB3H4 method. After solubilizatioti in deter- glycosylated. This change in structure of a major membrane gent, the extracts were passed through lentil lectin-Sepha- molecule may be important to the understanding of cellular rose columns and the radioactive glycoproteins were eluted interactions and of the relationship between cellular differen- with a-methylmannoside (10, 11). tiation and membrane protein glycosylation. Immunoprecipitation. Labeled cell extracts were subjected to immune precipitation using the staphylococcal protein MATERIALS AND METHODS A technique (25). When monoclonal antibodies were used, Cells. Normal and En(a-) erythrocytes were obtained rabbit anti-mouse IgG antiserun (Dako, Copenhagen) was from the Red Cross Blood Transfusion Service, Helsinki. used as a second antibody. Bone marrow was recovered from pieces of ribs, which were Polyacrylamide Slab Gel Electrophoresis. Polyacrylamide resected during open thorax surgery at the Helsinki Univer- slab gel electrophoresis in the presence of sodium dodecyl sity Hospital. The cells were subjected to Ficoll-Isopaque sulfate was done using 8% acrylamide gels (26). The gels centrifugation, and the interphase cells were collected. Frac- were fixed with 5% sulfosalicyclic acid and treated for fluo- rography (27). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: GPA, glycophorin A; PMA, phorbol 12-myristate 13- in accordance with 18 U.S.C. §1734 solely to indicate this fact. acetate. 6752 Downloaded by guest on September 29, 2021 Cell Biology: Gahmberg et aL Proc. Natl. Acad Sci USA 81 (1984) 6753

Table 1. Binding of protein A-containing Staphylococcus aureus cells to anti-GPA-treated bone marrow cells Rabbit mAb R10 mAb control Rabbit anti-GPA preimmune IgG Cell type N Binding N Binding N Binding N Binding Pronormoblasts 64 1.03 ± 3.98 56 0.13 ± 0.81 76 7.99 ± 10.39 50 2.88 + 7.9% Basophilic normoblasts 66 2.30 ± 4.43 50 0.06 ± 0.31 76 15.43 ± 10.99 51 1.47 ± 2.16 Polychromatic normoblasts 53 11.83 ± 10.14 50 0.04 ± 0.20 63 21.60 ± 11.24 53 3.92 ± 7.73 Orthochromatic normoblasts 59 17.80 ± 12.54 50 0.04 ± 0.20 63 21.27 ± 10.52 61 2.46 ± 2.71 Mature erythrocytes 76 16.84 ± 10.91 50 0 0 50 22.30 ± 8.99 50 1.48 ± 1.84 Binding of staphylococci to bone marrow cells that had been treated with the indicated antisera was determined by a rosetting assay (5). Values are given as the mean and SD of the number of bacteria bound per bone marrow cell. N, number of marrow cells examined; mAb R10, a monoclonal antibody specific for GPA; mAb control, a monoclonal antibody of unrelated specificity.

Preparation and Analysis of Glycopeptides/Oligosaccha- jected to immune precipitation using the R10 monoclonal rides. 3H-labeled GPAs isolated by immune precipitation anti-GPA antibody. Fractionation by polyacrylamide gel were treated with 5 mg of Streptomyces griseus protease electrophoresis revealed heavily labeled GPA monomer (Pronase, Sigma) per ml of 0.15 M NaCl/0.01 M sodium (GPA-M) and dimer bands (GPA-D) (Fig. 1, lane A). A simi- phosphate, pH 7.4/0.1% sodium dodecyl sulfate at 60°C for lar pattern was obtained using the rabbit antiserum (Fig. 1, 24 hr. After lyophilization, the samples were dissolved in lane B). 0.25 ml of 0.05 M NaOH/1 M NaBH4 and incubated at 45°C The surface glycoprotein patterns of K562 cells before and for 16 hr to liberate O-glycosidic oligosaccharides (28). One after hemin-induction are shown in Fig. 2 (lanes C and D). drop of glacial acetic acid was then added and the samples There was a relative increase in radioactivity in the position were lyophilized. The samples were dissolved in 0.1 M of GPA from induced cells. Rabbit antiserum precipitated NH4HCO3/0. 1% sodium dodecyl sulfate and applied to a 1 GPA molecules from uninduced cells (Fig. 2, lane E) but no x 80 cm Bio-Gel P-6 column equilibrated in the same buffer. precipitate was seen with the R10 antibody (Fig. 2, lane F). The void volume was determined each time using Blue Dex- However, after hemin-induced differentiation, the monoclo- tran 2000 (Pharmacia). Radioactivity in eluate fractions was nal antibody also precipitated GPA (Fig. 2, lane H). Flow measured in a Triton X-114-based scintillation fluid using an cytometry (FACS IV, Becton Dickinson) gave similar re- LKB-Wallac 1210 Ultrobeta counter. sults: K562 and HEL cells showed increased reactivity with the monoclonal antibodies R10 and R18 after induced differ- RESULTS entiation (results not shown). Reactivity of GPA from Normal Erythroid Cells and K562 Polyacrylamide Slab Gel Electrophoresis Patterns of GPA Cells with Monoclonal Anti-GPA-Antibodies and Heteroanti- from Bone Marrow Cells and Blasts from Patients with Eryth- serum. Results obtained using the staphylococcal rosetting roblastosis Fetalis. Erythroid cells from bone marrow were assay to detect reactivity with monoclonal (R10) and hetero- size-fractionated and surface-radiolabeled, and GPA was anti-GPA antiserum in normal bone marrow cells are shown isolated by immunoprecipitation with rabbit antiserum. The in Table 1. The monoclonal antibody reacted poorly with pronormoblasts and basophilic normoblasts whereas poly- A B C D E F G H chromatic normoblasts and cells at later stages ofdifferentia- tion showed a strong reaction. In contrast, rabbit anti-GPA M- antiserum showed a strong reactivity even with pronormoblasts and basophilic normoblasts. PHb- "I SW1 Surface-labeled blood erythrocyte membranes were sub- GPA-D- k - BS q A B OA - *Er GPA-M-- m * - GPA-M CA GPA-D- I -. GPB- FIG. 2. Fluorogram of a polyacrylamide slab gel after electro- GPA-M phoresis of extracts of surface-labeled uninduced and induced K562 cells and of immunoprecipitates obtained with anti-GPA antisera. Lane A: "4C-labeled standard proteins (M, myosin; PHb, phosphory- lase b; BSA, bovine serum albumin; OA, ovalbumin; CA, carbonic anhydrase). Lanes B-D: extracts of surface-labeled erythrocytes (B), uninduced K562 cells (C), and hemin-induced K562 cells (D). Lanes E-H: immunoprecipitates obtained using rabbit anti-GPA FIG. 1. Fluorogram of a polyacrylamide slab gel after electro- antiserum and uninduced cells (E), monoclonal antibody R10 and phoresis of immunoprecipitates obtained from equal numbers of sur- uninduced cells (F), rabbit anti-GPA antiserum and induced cells face-labeled erythrocytes using monoclonal antibody R1O (lane A) (G), and monoclonal antibody R10 and induced cells (H). The cells or rabbit anti-GPA antiserum (lane B). GPA-D, GPA dimer; GPA- were allowed to differentiate for 3 days, and similar amounts of ra- M, GPA monomer. dioactivity were used for the immunoprecipitations. Downloaded by guest on September 29, 2021 6754 Cell Biology: Gahmberg et al. Proc. NatL Acad Sci. USA 81 (1984) Table 2. Erythroid cell composition of bone marrow fractions from the most immature bone marrow cells (fraction 1, see % of total cells Table 2), there was more label in the O-glycosidic tetrasaccharide and some in the trisaccharide region (Fig. SB). GPA Pro- Basophilic Poly- Ortho- from fraction 2 was highly O-glycosylated (Fig. 5C) and had Frac- normo- normo- chromatic chromatic Erythro- a labeling pattern similar to that of GPA from fraction 3, the tion* blasts blasts normoblasts normoblasts cytes fraction which contained late normoblasts and erythrocytes 1 20 45 20 15 0 (Fig. 5D). 2 5 15 17 63 0 GPA isolated from uninduced K562 cells contained a rela- 3 3 2 5 55 35 tively small amount of O-glycosidic oligosaccharides, and the tetrasaccharide/trisaccharide ratio was lower than in *Cells were fractionated according to size by unit-gravity velocity GPA from bone marrow cells (Fig. 6A). A very small amount sedimentation (13). of radioactivity was precipitated with the monoclonal anti- erythroid cell compositions of the three cell fractions isolat- body R10 (Fig. 6B) After differentiation induced by hemin ed are shown in Table 2. Fraction 1 was enriched in the early the relative amount of tetrasaccharide increased (Fig. 6C). precursor cells, fraction 2 consisted of a mixed cell popula- The GPA molecules from induced cells reacted with the tion, and fraction 3 contained erythrocytes and late normo- monoclonal antibodies to give a glycopeptide/oligosacchar- blasts. From fraction 1, two weakly labeled bands were ob- ide pattern (Fig. 6D) similar to that obtained using the rabbit served after polyacrylamide gel electrophoresis, one in the antiserum. Treatment with sodium butyrate gave a small rel- position of the GPA monomer and the other, designated GP- ative increase in tetrasaccharides (results not shown). 26, with an apparent molecular weight of 26,000 (Fig. 3, lane To get a semiquantitative value for the change in glycosy- B). The GPA monomer was the major species precipitated lation, uninduced and hemin-induced K562 cells were la- from fraction 2 cells, but GP-26 was also obtained (Fig. 3, beled with both [35S]methionine and periodate/NaB3H4. The lane C). Only the species corresponding to GPA monomers GPA molecules were isolated and the 3H/35S ratios were de- and dimers were precipitated from fraction 3 (Fig. 3, lane D). termined. The ratios were 1.51 for uninduced cells and 2.01 GPA-M and GP-26 were both recovered from nucleated for induced cells. blood cells of patients with erythroblastosis fetalis (Fig. 3, The glycosylation of HEL cell GPA molecules also lane G). changed after treatment with inducing agents. After cultiva- Electrophoretic Mobilities of GPA Molecules Obtained from tion in the presence of retinoic acid, the relative level of tet- K562 and HEL Cells Before and After Induction. Induction of rasaccharides increased (Fig. 7B); PMA treatment had the differentiation of K562 cells with sodium butyrate or hemin opposite effect (Fig. 7C). decreased the electrophoretic mobilities of the GPA molecules (Fig. 4, lanes A, C, and E and lanes G-J, respectively). DISCUSSION Treatment of HEL cells with retinoic acid or PMA did not There are few examples of polypeptides whose carbohydrate result in any major change in the apparent molecular weights structures vary depending on the tissue localization or the of the GPA molecules (results not shown). developmental stage of the cells of origin. Best known are Analysis of Glycopeptides/Oligosaccharides of GPA Mole- the ABO and Ii blood-group antigens, which in the red cell cules. Gel filtration of 3H-labeled Pronase/alkaline borohy- are associated predominantly with the band 3 and band 4.5 dride-treated GPA molecules obtained by immune precipita- proteins (30, 31). Fetal cells contain simple, essentially un- tion was used to determine relative degrees of glycosylation. branched i-active oligosaccharides whereas erythrocytes We know (29) that the N-glycosidic glycopeptide appears in from adults contain high molecular weight branched I-active the void volume (peak 1) of Bio-Gel P-6 columns, followed oligosaccharides (32). The rodent Thy-1 glycoproteins from by the O-glycosidic tetrasaccharide (peak 2) and the O-gly- brain, thymocytes, and T-lymphocytes are also differently cosidic trisaccharides (peak 3). Fig. 5A shows that GPA from glycosylated (33-35). blasts of patients with erythroblastosis fetalis were labeled mainly in the N-glycosidic oligosaccharide (peak 1). In GPA A B C D E F G H I J 9. A B C D E F G ,:--ssGP 100-- GPA-D -" .. GPA-D-- 3, GPA-D--6 sb M.-.

- GPA-M - 4' GPA-M -- GPA-M b*- GPA-MJ- -GPA-M GPA-26:!!- ; - GPA-26 GPB* I GPB-* l1 FIG. 4. Fluorograms of polyacrylamide slab gels after electrophoresis of immunoprecipitates obtained using rabbit anti-GPA antiserum and K562 cells before and after induction of differentiation FIG. 3. Fluorograms of polyacrylamide slab gels after electro- with sodium butyrate or hemin. Lanes A-F: patterns obtained from phoresis of extracts of surface-labeled erythrocytes (lanes A and E) uniriduced cells with antiserum (A) and preimmune serum (B); pat- and of immunoprecipitates obtained using rabbit anti-GPA antise- terns obtained from cells induced with sodium butyrate for 3 days rum and bone marrow blast cells (fraction 1) (lane B), bone marrow with antiserum (C) and preimmune serum (D); patterns obtained cells (fraction 2) (lane C), bone marrow cells (fraction 3) (lane D), from cells induced with sodium butyrate for 6 days with antiserum and nucleated precursor cells from a patient with erythroblastosis (E) and preimmune serum (F). Note the decreased mobility of the fetalis (lane G). Also shown is the extract of the surface-labeled cells GPA monomer from induced cells. Lanes G-J: patterns obtained of the patient (lane F). GPA-26, GPA molecule with an apparent with antiserum and cells that were uninduced (G) or grown in the molecular weight of 26 000; GPB, glycophorin B; GP-100, glycopro- presence of hemin for 1 day (H), 3 days (I), or 6 days (J). The mobil- tein with an apparent molecular weight of 100,000. The GPA-26 ities of the GPA monomer (GPA-M) and dimer (GPA-D) bands show bands are barely seen in lanes B and G. a decrease with increased time of induction. Downloaded by guest on September 29, 2021 Cell Biology: Gahmberg et aL Proc. Natl. Acad. Sci. USA 81 (1984) 6755

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FIG. 5. Bio-Gel P-6 chromatography of 3H-labeled glycopeptides/oligosaccharides from GPA molecules immunoprecipi- _ tated with rabbit anti-GPA antiserum. (A) Pattern obtained from GPA of erythroid precursor cells from a patient with erythroblastosis fetalis. (B) Pattern from GPA of bone marrow cells (fraction 1, Table 2). (C) Pattern from GPA of bone marrow cells (fraction 2). (D) Pattern from GPA of bone marrow cells (fraction 3). Peak 1 cor- responds to the N-glycosidic glycopeptide, peak 2 to the O-glycosidic tetrasaccharide, and peak 3 to the O-glycosidic trisaccharide. All cells were surface-labeled 80 using periodate/NaB3H4. Arrow indicates Fraction the void volume.

The type of variation in the glycosylation of GPA is quite show any signs of erythrocyte malfunction. On the other different. GPA acquires an increased number of O-glycosid- hand, it is possible that GPA is needed at earlier stages of ic chains when the erythroid cells differentiate. This was erythrocyte differentiation. GPA and its incompletely glyco- true for GPA both from normal precursor cells and from sylated precursor molecules could function as receptors in erythroleukemia cell lines induced to differentiate. The cellular recognition; f3-galactosyl-binding lectins have been changes in glycosylation were detected after labeling cell found in several vertebrates (36). surface sialoglycoconjugates by the periodate/NaB3H4 GPA has recently been shown to act as a receptor for the method, which is specific for sialic acids (23). The results malarial parasite Plasmodium falciparum (37, 38). The dif- were essentially the same for cells labeled using the galac- ferentiation-related structural changes in the O-glycosidic tose oxidase/NaB3H4 technique (24) to detect terminal ga- oligosaccharide composition of GPA reported here could ex- lactose/N-acetyl galactosaminyl residues. The increased plain the well-known restriction in infectibility of the P. fal- 3H/35S ratio of [3 S]methionine/periodate/NaB3H4 labeled ciparum merozoites to mature red cells (39) and the inhibi- GPA molecules from hemin-induced cells also indicates that tion of merozoite binding by carbohydrate (40). the number of O-glycosidic oligosaccharides increased dur- The change in GPA structure during erythroid differentia- ing differentiation. The GP-26 band seen in some precursor tion is also reflected in its reaction with monoclonal anti- cell preparations apparently represents GPA molecules with GPA antibodies. The R10 antibody reacts with an epitope in a very low level of O-glycosylation because a similar mole- the middle part of the polypeptide chain, the R18 antibody cule was obtained when erythrocyte GPA was partially de- reacts with a region close to the lipid bilayer (21), and the glycosylated with endo-N-acetylgalactosaminidase (12). VIE-G4 antibody needs sialic acid for reactivity (22). All of GPA is not important for the mature red cell because these antibodies reacted weakly with the GPA molecules En(a-) individuals, lacking glycophorin A (18-20), do not from immature cells. This indicates that the carbohydrate

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FIG. 6. Bio-Gel P-6 chromatography patterns of 3H-labeled glycopeptides/oligosaccharides of immunoprecipitated GPA from K562 cells. Shown are results obtained using uninduced cells and rabbit anti-GPA antiserum (A), uninduced cells and monoclonal antibody R10 (B), cells treated with hemin for 2 days and rabbit anti-GPA antiserum (C), and cells treated with hemin for 2 days and monoclonal antibody R10 (D). Arrow indicates the Fraction void volume. Downloaded by guest on September 29, 2021 6756 Cell Biology: Gahmberg et al. Proc. NatL Acad Sci. USA 81 (1984) 6. Robinson, J., Sieff, C., Delia, D., Edwards, P. A. W. & Greaves, M. (1981) Nature (London) 289, 68-71. 7. Gahmberg, C. G., Jokinen, M. & Andersson, L. C. (1979) J. Biol. Chem. 254, 7442-7448. 8. Martin, P. & Papayannopoulou, T. (1982) Science 216, 1233- 1234. 9. Papayannopoulou, T., Yohochi, T., Nakamoto, B. & Martin, P. (1983) Globin Gene Expression and Hematopoietic Differen- tiation (Liss, New York), pp. 277-292. 10. 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G., Myllyla, G., Leikola, J., Pirkola, A. & Nordling, S. (1976) J. Biol. Chem. 251, 6108-6116. 19. Dahr, W., Uhlenbruck, G., Leikola, J., Wagstaff, W. & Land- fried, K. (1976) J. Immunogenet. 3, 329-346. 20. Tanner, M. J. A. & Anstee, D. J. (1976) Biochem. J. 155, 701- 703. 21. Anstee, D. J. & Edwards, P. A. W. (1982) Eur. J. Immunol. 12, 228-232. Fraction 22. Liszka, K., Majdic, O., Bettelheim, P. & Knapp, W. (1983) Am. J. Hematol. 15, 219-226. FIG. 7. Bio-Gel P-6 chromatography patterns of 3H-labeled gly- 23. Gahmberg, C. G. & Andersson, L. C. (1977) J. Biol. Chem. copeptides/oligosaccharides of GPA molecules isolated from HEL 252, 5888-5894. cells using rabbit anti-GPA antiserum. Patterns were obtained from 24. Gahmberg, C. G. & Hakomori, S. (1973) J. Biol. Chem. 248, digests of GPA from uninduced cells (A), from cells treated with 4311-4317. retinoic acid for 3 days (B), and from cells treated with PMA for 3 25. Gahmberg, C. G. & Andersson, L. C. (1978) J. Exp. Med. 148, days (C). 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