Loss of mannosyl phosphoryl polyisoprenol synthesis upon conversion of reticulocytes to erythrocytes

475

Biochimica et Biophysics Acta, 528 (1978) 475-482 0 ElsevierlNorth-Holland Biomedical Press

BBA 57147

LOSS OF MANNOSYL PHOSPHORYL POLYISOPRENOL SYNTHESIS UPON CONVERSION OF RETICULOCYTES TO ERYTHRO~YTES

JOHN J. LUCAS and CHRISTINA

NEVAR

Department of Biochemistry, State University of New York, Upstate Medical Center, Syracuse, N. Y. 13210 (U.S.A.) (Received

August 19th, 1977)

Summa~ Reticulocyte membranes transfer [ 14C]mannose from GDP[ 14C]mannose to an endogenous lipid forming a compound that has the characteristics of p-mannosylphosphoryldolichol. The lipid co-chromatographs with hen oviduct mannosylphosphoryldolichol, is stable to mild alkali and labile to mild acid, and strong base releases mannose phosphate. Formation of the [ 14C]mannosyl lipid by reticulocyte membranes is stimulated by the addition of exogenous dolichylphosphate. The transfer of mannose from GDPmannose to the lipid fraction is reversible upon the addition of GDP, but not GMP. The apparent I&, for GDPmannose, in the formation of the mannosyl lipid, is 0.56 ,uM. Erythrocyte membranes transfer less than 5% as much mannose from GDPmannose to endogenous acceptors as do assays of reticulocyte membranes. However, assays in the presence of exogenous dolichylphosphate reveal the presence of a mannosyltransferase capable of forming a [14C]mannosyl lipid that co-chromatographs in two solvent systems, on SG-81 paper, with hen oviduct and reticulocyte mannosylphosphoryldolichol. The mannosyltransferase activities from reticulocyte and erythrocyte membranes exhibit similar behavior toward divalent cations, Ammonyx detergent and dolichylphosphate. Thus, the enzymes may be identical, and the apparent absence of mannosyl phosphoryl polyisoprenol synthesis in erythrocyte membranes may be accounted for by the absence of endogenous acceptor molecules.

Introduction Many eukaryotic tissues glycosylate membrane-bound glycoproteins via lipid intermediates [l-3]. The lipid carrier in most cases is dolichylphosphate. This pathway appears to operate in the glycosylation of proteins having an N-acetylglucos~inyl-~p~a~ne linkage region. The major glycoprotein on the erythrocyte surface contains the blood group activities and one of its oligosaccharides

476

is linked through this type of group [4]. We have begun studying rabbit erythrocyte and reticulocyte membranes to determine whether any of the red cell membrane glycoproteins are glycosylated via the lipid intermediate pathway. Specifically, we have studied mannose incorporation via mannosylphosphoryldolichol. We have found transfer of [‘4C]mannose from GDP[14C]mannose to form [ “C]mannosylphosphoryldolichol in reticulocyte membranes. Erythrocyte membranes, however, do not exhibit this reaction when assayed with the endogenous acceptor. Using exogenous dolichylphosphate as an acceptor, erythrocyte membrane preparations can be shown to possess a mannosyltransferase capable of transferring mannose from GDPmannose to form mannosylphosphoryldolichol. Thus, it appears that erythrocyte membranes do not form mannosylphosphoryldolichol because they lack the endogenous acceptor rather than an absence of the mannosyltransferase(s). jackals

and Methods

GDP[‘4C]mannose (246 Ci/mol) was purchased from New England Nuclear Corporation. [ 14~~Mannosylphosphoryldolichol was prepared and purified by incubating mature hen oviduct membrane preparations with GDP[ 14C]mannose as previously described [ 51. Doli~hylphosphate was purchased from Sigma Chemical Co ., St. Louis, MO. Hydromix liquid scintillation fluid was purchased from Yorktown Research, South Hackensack, N.J. Ammonyx detergent was the gift of Dr. Michael Gold, the Rockefeller University. All other chemicals were reagent grade and from commercial sources, unless otherwise noted. Female white New Zealand rabbits were purchased from Camm Research, N.J. Radiochromato~ams were scanned on a Packard Model 7201 Strip Scanner. Radioactivity was measured in Hydromix scintillation fluid using a Searle Analytic Model Delta 300 Liquid Scintillation Counter. Protein was determined by the method of Lowry et al. [6] and lipid phosphorus by the method of Bartlett

[71. Rabbits were made anemic by 5-daily subcutaneous injections of 2.5% phenylhydrazine, pH 7.4 (6 mg/kg body weight). Blood was collected from an ear vein into heparinized tubes 48-72 h after the last injection. A reticulocyte count of 65-80% was obtained as determined by methylene blue staining. Erythrocytes were obtained from normal animals bled from the ear. A reticulocyte count of l--3% was obtained. Reticulocyte and erythrocyte cell membranes were prepared by the method of Hanahan and Ekholm 181 and stored at -20°C. Transfer of mannose from GDP[i4C]mannose into the endogenous acceptor is lost on repeated freezing and thawing. The assay for the transfer of mannose from GDPmannose to form mannosyl lipid was as follows. Incubation mixtures contained 10 ~1 MnCl, (50 m&I), 50 r.ll GDP[‘4C]mannose (3 PM, 440 cpm/pmol) and 50 r_ll enzyme suspension (approx. l-2 mg protein). Incubations were for indicated times at 37°C and terminated by the addition of 2 ml CHCl,/CH,OH (2 : 1, v/v). The suspensions were centrifuged in a desk-top centrifuge, the supernatants were removed and the pellets were re-extracted with 2 ml CHC1&H30H (2 : 1, v/v). The samples were again centrifuged and the supernatants combined, washed once with 1 ml

0.9% NaCl followed by 1 ml 0.9% NaC1/CH30H (1 : 1, v/v). The lower phase was dried with air in a scintillation vial and counted with 15 ml scintillation fluid. When exogenous dolichylphosphate was added as an acceptor of [14C]mannose from GDP[14C]mannose the reaction mixtures contained, in addition to the above, 10 ~1 dolichylphosphate dispersed (by sonication) in 0.5% Ammonyx. Radioactive lipids were chromato~aphed on SG-81 paper treated with EDTA [9]. The solvents employed were (A) CHCl~~CH~OH/H~O (65 : 25 : 4, v/v), (B) di-isobutylketone/glacial acetic acid/H,0 (60 : 45 : 6, v/v), (C) CHC13/ CH,OH/conc. NH40H (36 : 13 : 3, v/v). Radioactive zones were detected by autoradiography using Kodak No-Screen X-ray film. Paper chromatography was performed on Whatman No. 3 MM using solvent systems (D) isobutyric acid/NH~OH~H*O (57 : 4 : 39, v/v) and (E) ethylaceta~~ pyridine/H,O (12 : 5 : 4, v/v). When exogenous dolichylphosphate was added as an acceptor of [14C]mannose from GDP[14C]mannose, the unlabeled sugars were detected by dipping the chromatograms in 0.1 M NaIO,/acetone (1 : 19, v/v) followed by an aniline~diphenylamine reagent [lo]. Results and Discussion Reticulocyte membranes incubated with GDP[ 14C]mannose transfer [ 14C]mannose to a lipid fraction. The time course of the transfer is shown in Fig. 1. Transfer of [ “C]mannose into the lipid fraction is dependent on the presence of a divalent cation with the order of effectiveness being Mn” > Co*‘> Ca2+. The mannosyltransferase activity has a broad pH optimum: pH 6-7.5. The

10

t

, 0

#

# 20

I A0

TIME

I

I 60

IMINI

Fig. 1. Time course of transfer of mannose from GDPmannose to endogenous acceptors. Reticulocyte membranes were incubated as described in Materials and Methods. Reactions were terminated with CHC13/CH3OH3 (2 : 1, vfvf at the indicated times and assayed for incorporation into the lipid fraction.

478

apparent K, for GDPmannose in the transfer of mannose into the mannosyl iipid is 0.56 PM. Formation of the mannosyl lipid is reversible upon the addition of GDP (Fig. 2). Incubation of membranes, prelabeled by GDP[ 14C]mannose with GDP and Mn” results in a loss of the [ 14C]mannosyl lipid (RF = 1) and the appearance of GDP[ 14C]mannose (Fig. 2, upper trace). Incubation of the labeled membranes with GMP and Mn*’ or Mn*’ alone shows no change in the profile (Fig. 2, lower and middle traces). Quantitation of the peak areas indicates that all the radioactivity that disappears from the mannosyl lipid can be accounted for by an increase in the GDPmannose peak. The scans indicate the presence of a small amount of GDP[‘4C]mannose and [‘4C]mannose not removed by the washing procedure. In addition, a compound with an RF of approx. 0.9 is observed. This compound appears not to be involved in the lipid intermediate pathway since it partitions in the aqueous phase of a Folch extraction, is sensitive to mild alkali, and its synthesis is not stimulated by dolichylphosphate. The reversibility of the formation of the mannosyl lipid by GDP, not by GMP, indicates that a mannosyl unit and not mannose l-phosphate is transferred. In form and thus the mannosyl addition, the mannosyl unit is in an “activated” lipid may act as a donor of the mannosyl unit. The mannosyl lipid is sensitive to mild acid hydrolysis. The tr,s of hydroly-

MIlClN

FRONT

Fig. 2. Reversibility of [14C]mannosyl lipid formation. Reticulocyte membranes were incubated with GDP[14Clmannose for 30 min. The entire reaction mixture was centrifuged at 39 000 X g for 10 min. washed three times with 1.0 ml buffer and finally suspended in buffer containing 1 mM GDP and 10 mM MnCl2 (upper trace), 1 mM GMP and 10 mM MnCl2 (middle trace). and 10 mM MnClz (lower trace).

479

sis in 0.2 M HCl/propan-l-01 (1 : 1, v/v) at 50°C is 11 min. The product of mild acid hydrolysis co-chromatographs with authentic mannose in solvent systems D and E. Mild alkaline methanolysis (0.1 M KOH in CH,OH/toluene (3 : 1, v/v) 60 min, 0°C) had no effect on the lipid; these are conditions which deacylate glycerophosphatides. Strong alkaline treatment (0.1 M NaOH in 90% ethanol, 85”C, 30 min) releases two radioactive water-soluble compounds that co-chromatograph with mannose-1-P and mannose on solvent system D. Treatment of water-soluble products with bacterial alkaline phosphatase converts the peak co-chromatographing with mannose-1-p to a product co-chromatographing with mannose. The mannosyl lipid is eluted from a DEAE-cellulose column equilibrated in CHC1&HJOH (7 : 3, v/v), with 5 mM ammonium acetate in CH30H. [ 14C]mannosylphosphoryldolichol from hen oviduct elutes in the same fashion. Autoradiographs of chromatograms of the [14C]mannosyl lipid from reticulocytes chromatographed on SG-81 paper in solvent systems A, B, and C with hen oviduct [’ 4C]mannosylphosphoryldolichol indicate that the reticulocyte mannosyl lipid is chromatographically identical to oviduct mannosylphosphoryldolichol. The chemical and chromatographic data are consistent with the [ 14C]mannosyl lipid being /3-mannosylphosphoryldolichol. Erythrocyte membranes transfer less than 5% as much [ 14C]mannose from GDP[‘4C]mannose to endogenous acceptor as do reticulocyte membranes. All of the GDP[14C]mannose remains intact during the incubation, as judged by chromatography, and therefore removal of the substrate by competing reactions is not responsible for the absence of transfer. The addition of Triton X-100, deoxycholate or sodium dodecyl sulfate (SDS, at various concentrations, does not stimulate the transfer of [14C]mannose. However, [14C]mannose transfer into the lipid fraction is observed when exogenous dolichylphosphate is included in the reaction mixtures. Chromatography of the [ L4C]mannosy1 lipid formed in the presence of exogenous dolichylphosphate on solvent systems A and B (Fig. 3) indicates that the compound co-migrates with both reticulocyte mannosyl lipid and hen oviduct mannosylphosphoryldolichol. In light of these results, the question arises whether the enzyme catalyzing the transfer of mannose from GDPmannose to dolichylphosphate is the same in erythrocyte membranes as that in reticulocyte membranes. Several experiments suggest that the enzymes are identical. Fig. 4 shows the effect of exogenous dolichylphosphate concentration on the formation of [ 14C]mannosyl lipid. In the case of both reticulocyte and erythrocyte membranes, the incorporation shows a distinct maximum at 50 nmol of dolichylphosphate in each incubation mixture. The cause of the inhibition of transfer at higher concentrations is unknown but may be due to a detergent effect at the relatively high concentrations. Nevertheless, both reticulocyte and erythrocyte membranes respond in a similar fashion. The data shown in Fig. 4 also suggest that although the mannosyltransferase activity is present in erythrocyte membranes, it is about 40% the level found in reticulocyte membranes. Reticulocyte and erythrocyte membranes have similar metal ion requirements for catalyzing the transfer of mannose from GDPmannose to dolichylphosphate. Table I shows that EDTA abolishes the reaction while Mn” and

480

FRONT _

ORIGIN

1

2

3

4

5

Fig. 3. Chromatography of the product formed by incubation of erythrocyte membranes with dolichylphosphate and GDP[ 14C]mannose. Erytbrocyte membranes were incubated with dolichylphosphate and GDP[14C]mannose as described in Materials and Methods. The [I4 C]mannosyl lipid was chromatographed with hen oviduct mannosylphosphoryldolichol on solvent system B and the chromatogram was subjected to autoradiography. Lane: 1, reticulocyte mannosyl lipid. 2100 cpm: 2, reticulocyte mannosyl lipid, 2100 cpm and hen oviduct mannosylphosphoryldolichol, 3000 cpm; 3. hen oviduct mannosylphosphoryldolichol, 3000 cpm; 4, erythrocyte mannosyl lipid, 2200 cpm and hen oviduct mannosylphosphoryldolichol. 3000 cpm; 5. erythrocyte mannosyl lipid, 2200 cpm.

Co’+ are equally efficacious in fulfilling the divalent cation requirement. Ca”‘is less effective. Exogenous dolichylphosphate is dispersed in Ammonyx detergent when used as an acceptor of mannosyl units. Table II shows the effects of varying the Ammonyx concentration on the reaction. Erythrocyte and reticulocyte membranes behave similarly and both exhibit an optimum at 0.05% Ammonyx. The removal of dolichylphosphate from the biosynthetic apparatus of the red blood cell upon conversion of the reticulocyte to the erythrocyte is probably a reflection of the mature red blood cell’s relative quiescence compared to its precursors. Presumably because of the lack of protein synthesis in the erythrocyte, dolichylphosphate is no longer required to act as an intermediate in the glycosylation of proteins. The mechanism by which available dolichylphosphate is removed from the membrane when the reticulocyte is converted to an erythrocyte is unknown. Dolichol exists in several forms [l] and thus several possibilities exist for the removal of dolichylphosphate: it may be dephosphorylated and acylated; it may be metabolized and destroyed; it may be

481

50

100 DOLICYLPHOSPHATE

150

200

250

inMOLES

Fig 4. Dependency of mannosyl lipid formation on exogenous dolichylphosphate. Erythrocyte (~-----CJ) and reticulocyte (a---+) membranes were incubated for 30 min with varying amounts of dolichylphosphate dispersed in Ammonyx detergent as described in Materials and Methods, 1 mg of protein was used in each case.

TABLE I EFFECT OF DIVALENT CATIONS ON TRANSFER NOSE TO EXOGENOUS DOLICHYLPHOSPHATE

OF r14ClMANNOSE

FROM GDP[‘4CJMAN-

Erytbrocyte and reticulocyte membranes were incubated for 30 min as described in Materials and Methods. Mixtures contained 50 nmol dolichylphosphate in 0.05% Ammonyx. Activity expressed as percent of maximum. Addition

Erythrocyte

Reticulocyte

5 mM 10 mM H2G 10 mM 10 mM

100 0 3 98 31

100 0 2 102 24

MnCl2 EDTA CoCl2 CaCl2

TABLE II EFFECT OF AMMONYX CONCENTRATION ON TRANSFER MANNOSE TO EXOGENOUS DOLICHYLPHOSPHATE

OF I14C1MANNOSE

FROM GDPl’4Cl-

Erythrocyte and reticulocyte membranes were incubated for 30 min as described in Materials and Methods. 25 nmol dolichylphosphate were dispersed by sonication in varying concentrations of Ammonyx. Ammonyx concentration is the final concentration in the incubation mixtures. Ammonyx concentration

Erythyrocyte

Reticulocyte

0.1% 0.05% 0.01% 0.005%

69 100 45 24

89 100 54 43

482

dephospho~lated to free dolichol; or it may be glycosylat~ and unable to discharge the glycosyl residue because no protein acceptors are available in erythrocyte membranes. The loss of dolichylphosphate from the membrane may be analogous to the loss of “excess” phospholipid and cholesterol reported by Shattil and Cooper [ 111. In any event, the lipid intermediate pathway seems to be another example of a process that is lost during the maturation process of reticulocyte to erythrocyte. After this paper was submitted for publication a report appeared [12] indicating that erythrocyte membranes transfer mannose from GDPmannose to exogenous dolichylphosphate but virtually no transfer to endogenous dolichylphosphate was observed.

This work was supported by N.I.H. Grant HD09046 and General Research Support Grant RR05402 from the General Research Branch, Division of Research Resources, N.I.H. References 1 Hemming, F.W. (1974) in Biochemistry of Lipids, Biochemistry Series One (Goodwin, T.W., ed.), Vol. 4, pp. 39-98, University Park Press, Baltimore 2 Lucas, J.J. and Waechter, C.J. (1976) Mol. Cell. Biochem. 11.67-78 3 Waechter, C.J. and Lennarz, W.J. (1976) Annu. Rev. Biochem. 45.95-112 4 Marchesi, V.T., Furthmayr, H. and Tomita, M. (1976) Annu. Rev. Biochem. 45.667-698 5 Waechter, C.J., Lucas, J.J. and Lennarz. W.J. (1973) J. Biol. Chem. 248, 7570-7579 6 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 7 Bartlett, G.R. (1969) J. Biol. Chem. 234.466468 8 Han&ran, D.J. and Ekholm, J.E. (1974) Methods Enzymol. 31, Part A, 168-172 9 Steiner, S. and Lester, R.L. (1972) J. Bacterial. 109, 81-88 10 Dawson. R.M.C., Elliott, D.C., Elliott, W.H. and Jones, K.M. (1969) Data for Biochemical Research, 2nd edn., p. 542, Oxford University Press. New York 11 Shattil, S.J. and Cooper. R.H. (1972) J. Lab. Clin. Med. 79, 215-227 12 Martin-Barrientos, J. and Parodi, A.J. (1977) Mol. Cell. Biochem. 16,111-117