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Glycophorin Expression in Murine Erythroleukaemia Cells

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Glycophorin Expression in Murine Erythroleukaemia Cells Glycophorin expression in murine erythroleukaemia cells JEFFREY B. ULMER*, ELIZABETH D. DOLCI and GEORGE E. PALADE Department of Cell Biology, Yale University School of Medicine, P.O. Box 3333, Nezv Haven, CT 06510, USA •Author for correspondence. Summary We have identified mature and putative precursor synthesis was induced by dimethyl sulphoxide coin- forms of glycophorins expressed in a virus-trans- cidentally with that of gp-3 and gp-2. They do not formed murine erythroleukaemia (MEL) cell line appear to be glycoproteins, as evidenced by their and compared them with their normal erythroblast lack of incorporation of [3H]galactose, [3H]glucosa- counterparts. The following differences were mine or [3H]mannose. In contrast, gp-3 and gp-2 found: (1) the two major MEL cell glycophorins incorporated [3H]galactose and [3H]glucosamine 3 3 (apparent Mr values 29-30 and 43(xlO )) have but not [ H]mannose. Partial characterization of greater mobility on polyacrylamide gels than their the glycan moieties of MEL cell glycophorins indi- normal gp-3 and gp-2 counterparts, due at least in cates that they consist mostly of tri- and tetrasac- part to differences in their oligosaccharide side- charides, with no indication of any N-linked chains. chains; (2) MEL cell gp-3 consists of two discrete Hence, the glycans of MEL cell glycophorins are proteins; and (3) there are more potential glyco- mostly (if not all) O-linked. Furthermore, treatment phorin precursors in MEL cells than in normal with AT-glycanase did not change their electrophor- mouse erythroblasts. Four proteins, with apparent etic mobility on polyacrylamide gels. MEL cell 3 Mr values of 21, 23, 26 and 27(xlO ), have tenta- glycophorins were also shown to be modified by tively been identified as glycophorin precursors, phosphoryl and fatty acyl groups. based on the following findings: (1) they are immu- nologically related to the glycophorins; and (2) their Key words: glycophorin, erythroleukaemia cells. Introduction are fatty-acylated (Dolci & Palade, 1985). The Friend murine erythroleukaemia (MEL) cell is a The glycophorins are extensively studied, transmem- virus-transformed erythroid precursor cell that rapidly brane sialoglycoproteins of the plasmalemma of human proliferates in culture (Marks & Rifkind, 1978). Differen- red blood cells. Two of the proteins (glycophorins A and tiation along the erythrocyte pathway can be initiated by a B) are closely related to one another (Furthmayr, 1978; number of agents and results in decreased cell size, Siebert & Fukuda, 1986). The function of human glyco- changes in cell shape (Malik & Langzam, 1982), restric- phorins has not been established. It has been shown, ted proliferative capacity, expression of erythrocyte anti- however, that they may play a role in the maintenance of gens including glycophorins (Kasturi & Harrison, 1985), cell shape (Anstee et al. 1984) via interactions with band production of haemoglobin and, in some cases, extrusion 4.1 protein (Anderson & Lovrien, 1984) and polyphos- of nuclei to yield reticulocyte-like cells (Tsiftsoglou et al. phoinositides (Anderson & Marchesi, 1985). Other func- tions have not been excluded but remain to be defined. 1979; Volloch & Housman, 1982). Commitment of MEL The known amino acid sequences of human (Tomita et cells to differentiate appears to be a stochastic process al. 1978; Tomita & Marchesi, 1975), pig (Honma et al. (Gusella et al. 1976) but can be synchronized (Levenson 1980) and horse (Murayama et al. 1982) glycophorins are & Housman, 1979). Because of these characteristics, the known and their comparison shows that primary struc- MEL cell line appears to be an excellent system for the ture is not conserved to a high degree across species. Two study of glycophorin biosynthesis as affected by neoplas- murine glycophorin equivalents, termed gp-2 and gp-3 tic transformation and induced differentiation. We report (Sarris & Palade, 1979, 1982a,6; Dolci & Palade, 1985), here that two major mature forms of glycophorin, corre- have been identified in erythrocytes and erythroblasts. sponding to normal mouse erythroblast gp-2 and gp-3, Like their human counterparts, they are heavily sial- and four putative glycophorin precursors are expressed in ylated (Sarris & Palade, 1979, 1982a). In addition, they MEL cells. Journal of Cell Science 92, 163-171 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 163 Materials and methods To explore the fatty acylation of MEL cell glycophorins, 2xl07cellsmF' were biosynthetically labelled with [3H]palmi- Materials tate (200^CimF') for 60min at 37°C and then processed through immunoprecipitation, gel electrophoresis and fluor- Friend virus-transformed MEL cells (clone 745) were kindly ography. provided by Dr Edward Benz, Jr and endoglycosidase F was a For phosphorylation experiments, suspended MEL cells generous gift from Dr Steven Rosenzweig. All cell culture were incubated for 60 min at 37°C in 11-5 mM-A?-2-hydroxyeth- reagents, including RPMI 1640, foetal bovine serum, penicillin, ylpiperazine-Ar-2-ethanesulphonic acid (pH7-4), 136 mM- streptomycin and L-glutamine, were obtained from GIBCO NaCl, 5-8mM-KCl, l-5mM-CaCl , l-2mM-MgSO , 11-5 mM- Laboratories (Grand Island, NY). Dimethyl sulphoxide 2 4 glucose containing lOOjuCimF1 H332PO4 and then processed (Me2SO), hexamethylbisacetamide, all protease inhibitors and for autoradiography. Staphlococcus aureus V-8 protease were purchased from Sigma (St Louis, MO). iV-glycanase (peptide: iV-glycosidase F, pep- tide-;V4[/V-acetyl-/3-glucosaminyl]asparagine amidase) was ob- Immunoprecipitation tained from Genzyme Corporation (Boston, MA). [35S]methio- Glycophorins were immunoprecipitated from lysed MEL cells nine (1200Cimmol~ ) and NCSIM were obtained from either by the procedure of Dolci & Palade (1985) or (most often) Amersham (Arlington Heights, IL). [l,6-3H(yV)]glucosamine by a modification thereof, in which each batch of washed 1x10 hydrochloride (40-50 CimmoF1) and [9,10-3H(AO]palmitate to 2x 107 cells was treated for 15 min on ice with 1 ml of 1 % NP- (23-5 CimmoF ) were obtained from New England Nuclear 40 in 50mM-tris(hydroxymethyl)aminomethane (Tris-HCl), 32 1 3 (Boston, MA). H3 PO4 (285 Ci nig" ), [6- H]galactose pH7-5. The ensuing lysates were processed as described by (25 Ci mmoF ) and D-[2-3H]mannose (2-30CimmoF1) were Dolci & Palade (1985) up to immunoprecipitation, which was purchased from 1CN (Irvine, CA). carried out by adding 25 jA of polyclonal anti-mouse glyco- phorin antiserum (1:125 dilution) followed by vortexing and Cell culture incubation on ice for 15 min. Immune complexes were re- covered from these mixtures by incubation for 45 min at 4°C MEL cells were grown at 37 °C to densities ranging from 1X104 (with gentle agitation) with 10 mg of protein A-Sepharose CL- to 1 XlO6 cells ml"' in RPMI 1640 medium containing foetal 4B beads (Pharmacia). The beads were collected by centrifu- bovine serum (10%, v/v), penicillin (100 units ml"1), strepto- gation (10 min at 2000 g), washed three times according to mycin (lOO^tgmF1) and L-glutamine (0-3mgmF'), while Dolci & Palade (1985), then boiled in a water bath for 5 min in being maintained in a humidified atmosphere of 95 % air/5 % 15% sucrose, 5% SDS, 0-1% dithiothreitol and 0-1 M- CO2. Differentiation was initiated by seeding MEL cells at Tris-HCl, pH7-5 (to release the immune complexes). 5Xl04mF' ingrowth medium containing Me2SO (2%, v/v) or 5mM-hexamethylbisacetamide. At day 2 after the addition of In some experiments, the lysis (1% NP-40 in 50IYIM- the inducer, the cell culture was diluted 1: 1 with fresh growth Tris-HCl) and solubilization (4% SDS in 20mM-dithiothrei- medium and at day 4 the cells were harvested for experimen- tol, 50mM-Tris-HCl) buffers contained the following protease inhibitors (final concentration): antipain (0'ljUgmF ), aproti- tation. Differentiation of MEL cells was monitored by follow- 1 1 ing haemoglobin production via benzidine staining. To this nin (0-lmgmF ), benzamidine (01 jUgmF ), chymostatin intent, 1X106 cells were suspended in 05ml phosphate- (0-1 jUgmF ), diisopropyl fluorophosphate (O'l HIM), ethylene- glycol-bis-(/3-aminoethyl ether)-A',Ar'-tetraacetic acid (0'5ITIM) buffered saline containing 2 mM-sodium azide and bovine serum 1 albumin (002%, w/v); 50^1 of 1% benzidine in methanol and pepstatin (O'l jUgmF ). (w/v) and 10;«l of 30% hydrogen peroxide were added to the suspension and mixed by inversion. The presence of haemo- Alkaline borohydride treatment globin in MEL cells was indicated by a bluish green reaction Alkali-labile ohgosacchandes were cleaved according to Carlson product observed after lOmin by light microscopy. Cell vi- (1968) and separated by gel filtration according to Cummingse/ ability was monitored by Trypan Blue exclusion. Cell number al. (1983). [3H]galactose labelled immunoprecipitates from and percentage of cells positive for benzidine or Trypan Blue splenic erythroblasts and MEL cells were eluted from the 5. were calculated by counting cells in a haemocytometer by light aureus complexes with 0-5 % sodium dodecyl sulphate (SDS). microscopy. An equal volume of 2M-sodium borohydride in lOOmM-NaOH was added and the SDS concentration was further reduced to Metabolic labelling of MEL cells 01% by the addition of 50mM-NaOH, 1 M-sodium boro- hydride. The mixture was incubated for 14-20 h at 45 °C, Cells, collected by centrifugation (10 min at 2000g) and washed placed on ice and diluted with 2 vols of distilled water. The pH three times with methionine-free RPMI medium (by repeated was adjusted to 50 by the addition of 1 M-acetic acid and resuspension and sedimentation), were incubated in the same borohydride was removed by rotary evaporation in methanol. medium with the following reagents: 200 /.id ml" [3sS]methio- 1 1 The dried samples were resuspended in 0-1% SDS, 50mM- nine, 100 units mF penicillin, lOOjUgmF streptomycin, NH4HCO3, pH80, and the alkali-labile material was size- 0-3mgmF' L-glutamine, and under the following conditions: 7 1 fractionated on a column of Biogel P-6 (1-6 cm X 97 cm) IXW to 2xl0 cellsmF , 37°C in an atmosphere of 95% (BioRad).
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