Partial characterization of an erythropoiesis inhibitory factor

BIOCHEMICAL

MEDICINE

33,

8-16

(198%

Partial Characterization

of an Erythropoiesis

Inhibitory Factor’

W. A. NEAL, F. A. GARVER, J. P. LEWIS,’ E. GARDNER,

JR..

ANI)

c. L. L,UT(.tIER

It has become apparent that several agents modulate erythropoiesis. Although more emphasis has been placed upon erythropoietin (EP).? the relative importance of inhibitors of erythropoiesis (EIFs) has grown (l-7). Our laboratory described a basic protein urinary fraction associated with (x,-acid glycoprotein and EIF. and more recently it was demonstrated that prostaglandin FZcr(PGF,,,) could be extracted from the complex (8,9); the protein-PGF,,, complex was isolated by several techniques. The purpose of this report is to further characterize the EIF described in this laboratory. MATERIALS

AND METHODS

Urine from normal laboratory personnel was collected, frozen immediately. and processed later by chromatography on DEAE-cellulose as previously reported (10). The last fraction (III-A) to leave the column was collected. dialyzed thoroughly with pretested membranes, and lyophilized. The porosity of the membranes was tested at Union Carbide by the pressure required to pass water, and we checked the dialysate for activity after dialysis with EP. a hormone with relatively 101~ molecular weight. The dried protein fraction (100 mgl was dissolved in Tris-borate buffer at pH 8.1 and applied to a column electrophoresis apparatus as previously described (4). The EIF fraction was collected as reported (4). dialyzed with pretested membranes, and lyophilized. The lyophilized EIF (75 mg) wa\ isolated further by gel filtration on Sephadex G-100 as applied to t:P (1 I I. A Sephadex G-100 column (I .O x 75 cm) wa\ equilibrated with Tris-maleate buffel (0.005 M. pH 6.8, 18.3 mg% EDTA). The protein (75 mg) was applied, and Z-ml aliquots were eluted with Tris-maleate, pH 6.X. buffer. The total volume of’ each ’ Supported

by

the

Medical

Reseal-ch

Service

of the

Vcic~-,ms

.\Jminlstratiun

College of Georgia (NIH Grants HLH-15158. FK-5365. and W-0(161 I. ’ To whom correspondence zhnuld he addresd: Medial Research Sr~-\~cc Center. Augusta, (>a. 30910. ’ Abbreviations used: et-ythropoiesi\ regulatory factor\ I izKk+s~. cs.cthropotetln stimulating roxysmal

factor nocturnal

(ESF). erythropoieqis hemoglobinuria

inhibitory (PNH). sodium

factor t FIF). prostaglandin~ dodec~l \olfate (SIX)

x 0006-2944/85

$3.00

‘[rid / Ii! (LPI tPGFI,.

the

Llrd~~,d

I. \’ ,\ lle~i~~;!i c‘i yihropolev~, PGF.).

pz

ERYTHROPOIESIS

INHIBITORY

FACTOR

9

fraction was equal to the volume circumscribed by the optical density readings of the 2-ml tubes. The collected fractions were dialyzed against distilled water (10 x vol with three changes during 24 hr and with constant stirring) in selected, pretested tubing (Union Carbide g in. flat width), and lyophilized. The lyophilized fraction (last fraction to leave the column) was again subjected to gel filtration through Sephadex G-100. The eluted fraction was dialyzed and lyophilized. The described procedure was repeated until 90 mg of the fraction was obtained, which was applied in triplicates to hydroxylapatite4 suspended in columns (I .O x 20 cm) with 0.01 M maleate buffer at pH 6.8 and eluted with the same buffer. dialyzed, and lyophilized. Countercurrent distribution of the EIF fraction obtained from hydroxylapatite was done in a 30-tube transfer apparatus.’ The upper phase was isobutanolpropanol (1:l) and the lower phase 1 M NaCl. All of the cells in the lower phase contained 10 ml of salt solution except the first, which contained 10 mg of the EIF fraction in 10 ml of 1 M NaCl. The upper phase (10 ml) was added to the first cell, equilibrated, and run through the train. the fresh upper phase being added to the first cell at each transfer. After completion of 30 transfers, the upper phase was recycled through the entire train. The recycling attachment was then removed, and the two phases were separated and collected. Each tube was read at 280 A. The contents of the tubes including the fractions were dialyzed as previously described. The fractions were then lyophilized prior to assay in the exhypoxic polycythemic mouse in combination with EP, and EP was assayed as a control. Protein nitrogen determinations were done by the micro Kjeldahl nitrogen method. The hydroxylapatite eluate (2 mg) was also applied to a polyacrylamide gel (0.5 x 7.5-cm column) for electrophoresis (PAGE) as described (12). The neutral pH-SDS-PAGE system was used. The gels were 7.5% acrylamide, 0.2% N,N’methylenebisacrylamide, 0.1 M Na,P04 buffer, pH 7.2,O. 1% SDS, 0.5% N, N,N’tetramethylenediamine, and 0.1% ammonium persulfate. The acrylamide solution polymerized in 0.5 x IO-cm tubes. The running buffers were 0.1 M phosphate, pH 7.2. and 0.1% SDS. Samples of EIF and standards, 2 mg of each, were applied to the top of the gels in 50% sucrose solution with and without bromphenol blue. Electrophoresis was done at 7.5 mA per tube. After electrophoresis the gels were stained with 0.25% Coomassie blue for 5 hr and destained with 7%’ acetic acid. The same technique was also applied to the protein in the lower phase and in the upper phase obtained during countercurrent distribution. The movements of the stained proteins were scanned spectrophotometrically (Gilford 2400) and measured with a centimeter rule. The proteins ribonuclease A (MW 13,500). chymotrypsinogen A (MW 25,000), ovalbumin (MW 45,000). phosphorylase B (MW 94,000), and aldolase (MW 158,000) were used as standards. With a semilog plot of the MW of the standards versus the distance migrated. the MW of the unknown protein(s) was determined. The gels were sliced into 5-mm pieces, and the EIF was tested for inhibition of erythropoiesis by incubating ’ Bio-Rad Laboratories, Richmond. Calif. ’ Constructed by H. Post, 6822 60th Road,

Maspeth.

N.Y

10

NEAL El-AL.

it with EP and comparing the activity of the incubated mixture with the activity of the EP alone. Thin-layer chromatography of benzene extracts from EIF fractions was done as described ( 13). Antisera to EIF were produced in New Zealand white rabbits by intradermal injections of 10 mg into the footpads at 3-day intervals for 2 weeks as described (14). The antigen was first emulsified in complete Freund’s adjuvant. Blood was collected 5-7 days after the last injection. The EIF antigen was obtained by column electrophoresis and selective membrane ultraliltration (4,8). The most antigenic EIF fraction was in the retentate after ultrafiltration through a membrane with a cutoff at a MW of 10,000. Immunoelectrophoresis indicated one protein contaminant, a y globulin. Anti-Lu,-acid glycoprotein was prepared in a similar way except that 3 mg of the pure antigen (Calbiochem, Los Angeles) was injected. The antiserum was placed in slots between the ElF and the cu,-acid gIycoprotein, which were placed in wells, the EIF in the inner wells and &,-acid glycoprotein in the outer wells. Immunoelectrophoresis was done by the method of Scheidegger (15). Strain AKR female mice made polycythemic by exposure ( 13h hr) to a simulated altitude of 7320 m were used for the bioassay for ESF activity (16). The mice were intermittently kept in glass enclosures in an atmosphere of 8% oxygen and 92% nitrogen. The packed cell volumes were determined by a microhematocrit technique. Mice injected with 0.9% saline were used as controls for all groups. The values for the control groups injected with saline solution were subtracted from the EP activity values. The results were recorded as the mean of the percentage of 5yFe incorporated into red blood cells and as international standard B units of EP activity, which were determined from a log dose-log response curve (17). The ELF activity was measured as the decrease in EP activity observed after incubation of EIF with an EP control. Ten mice were used for each assay. Standards were interspersed between the unknowns. RESULTS Two fractions were observed during gel filtration of 75 mg of the urinary EIF fraction obtained by column electrophoresis. Repeated gel filtration with Sephadex G-100 of the last fraction to leave the column also yielded two components as indicated immunoelectrophoretically (Fig. Il. one in the cr,-acid glycoprotcin region and one in the y-globulin region, whereas anti-human serum reacted with multiple serum proteins as indicated in Fig. I. The yields and inhibitory activities of these fractions are reported as the means and SEM of three or more determinations (Table 1). The fraction indicated as the last fraction to leave the column during gel filtration inactivated EP as indicated by the loss of activity from the EP control. Repeated gel filtration increased the specific inhibitory activity by one unit and removed all traces of inhibitory activity from the first fraction to leave the column. The active EIF fraction. obtained by gel filtration, was next passed through hydroxylapatite, collecting 2-ml aliquots (the EIF fraction was in tubes I?-- 14). prior to application on acrylamide gel electrophoresis and countercurrent distribution. Both electrophoresis and countercurrent distribution separated the EIF

FIG. 1. Immunoelectrophoresis of the EIF fraction obtained by gel filtration with Sephadex G100. The anti-EIF and anti-human (0.05 ml of each) sera were in tracks I and 2. respectively, and the EIF and normal human serum were in the wells I and 2 respectively (1.0 ~1 containing 70 pg).

fraction into two components, one with a MW of 185,000 (globulin) and one of 9300. We were unable to detect any inhibitory activity in the y-globulin or the cY,-acid glycoprotein after removing PGF?, . A spectrophotometric scan of the components and protein standards is depicted in Fig. 2; the y-globulin moved slower and the EIF faster than any standard. The PGFb and the inhibitory activity were retained with the smaller component, a fragment of a,-acid glycoprotein, as indicated by immunoelectrophoresis with anti-a,-acid glycoprotein serum. The fragment moved more slowly than the pure protein (Fig. 3). During countercurrent distribution the EIF went into the upper organic phase while the y-globulin remained in the lower phase (Fig. 4). PGF?, was detectable by thin-layer chromatography of a benzene extract of the EIF obtained by countercurrent distribution and acrylamide gel electrophoresis as previously described (9). The protein was 4.0 2 O.l%, and the remainder was PGF,,, . Inhibitory activity was found only in the prostaglandin F?,, which was carried by a,-acid glycoprotein. Immunoelectrophoresis of the fraction in the upper phase after countercurrent distribution indicated the reaction line with anti-EIF serum to be extended. (Fig. 5). DISCUSiION

The two protein components that we find associated with the EIF fraction, one in the &,-acid glycoprotein region and one in the y-globulin region (Fig. I), have been implicated before, (14,18-20). The inhibitory effect of the fragment of cY,-acid glycoprotein, demonstrated in this work, is due to the association with PGFzu which inhibits erythropoiesis in small amounts (9). However, previously it was observed that pure cu,-acid glycoprotein potentiated erythropoiesis (21). For some time cY,-acid glycoprotein has been known to bind drugs such as dipyridamole, imipramine, and quinidine: diseased-induced changes in the concentrations of a,-acid glycoprotein can influence the binding of propranolol and chlopromazine (22). This acidic protein has the ability to bind many compounds. Whether it remains neutral, potentiates, or inhibits a biological process depends on the type of compounds that occupies the majority of the binding sites.

NEAL

12

c E

ET Al>

ERYTHROPOIESIS

INHIBITORY

FACTOR

13

Globulin

1

, 1

, 2 Electrophoretic

FIG. 2.

Spectrophotometric

3

4

5

Migration.

6

i

Centimeters

scan of EIF

and standards.

The fact that PGF?, is found in urine bound to a fragment of cY,-acid glycoprotein could be due to the presence of proteolytic enzymes in urine. It is probable that PGF,, will readily bind with the whole protein molecule in blood plasma. Since the PGF?, can be extracted with benzene, it would appear that the binding is noncovalent. The relative amount of protein compared to prostaglandin is small, and the molecular weight could be in error and dependent on various amounts

Well

1 -

Track

1 -

we11 2 -

Track

2 -

we11 3 -

Track

3 -

FIG. 3. Immunoelectrophoresis of the EIF fraction indicated in Fig. 2. The anti-a,-acid glycoprotem sera were in tracks 1-3 (0.05 ml of each); pure @,-acid glycoprotein was in wells 1 and 4 and EIF was in wells :! and 3. The pure ol,-acid glycoprotein moved faster than EIF.

NEAL ET 241

14 A260 563 52. 48. 44 40. 36. 3226. 24 20 16 12 06 04 0

0

2

4

6

6

IO 12

14

16

16 20 22

24 26

26 30

FRACTION NUMBER FK. 4. EIF obtained from hydroxylapatite recycled by countercurrent distribution as dcscrtbed under Mate&s and Methods. EIF is indicated by open circles in the upper phase. and the dark circles indicate the y-globulin in the lower phase.

of PGF?,. Although Lindeman reported the MW to be 5000 for an ELF from urine, the EIF at 9300 MW could be the same or a similar fragment and differ in the amount of bound PGF?,, (5). The dual solubility of EIF in water or organic solvents could be explained by the dual exposure of protein and lipid groups to the solvent (23). The slower immunoelectrophoretic movement of the fragment of cu,-acid glycoprotein (Fig. 3) as compared to the pure protein could be explained by relatively fewer sialic

Track

we11 .3Track .24-

F:m. 5, lmmunoelectrophoresis of the EIF before and after countercurrent distribution. I’he EII z serum was in the tracks and EIF in the wells (well I after column electrophoresis. well ?. gel filtration, well 3 after countercurrent distribution. top layer. well 4 after hydroxylapdtite)

ERYTHROPOIESIS

INHIBITORY

FACTOR

15

acid groups remaining on the fragment and/or the amount of PGFzu bound, and this could also partially explain the extended indication of the reaction of EIF with anti-EIF as seen in Fig. 5. The simplest version of the EIF described herein would be a combination of PGFzu with a fragment of cw,-acid glycoprotein. Three extractions of EIF with organic solvents removes some PGF2, as indicated by thin-layer chromatography (14). The remaining EIF goes into solution during the fourth extraction, and PGFzu can still be detected. The tromethamine salt derivative of PGF*, (1 mg) inactivates 7.5 IU of EP as determined by the bioassay for EP. Equated to pure PGFz, the inactivation of EP by 1 mg of PGF?, would be 10 IU of EP. It is of interest to compare the production of EP by PGE, with the inactivation of EP by PGF*,. PGE2 (I mg) produces 9.5 IU of EP (14); the same work suggests that EIF is a granulopoietic factor and thus inhibits erythropoiesis indirectly. According to the bioassay PGF?,, inactivates EP to the same extent as PGEz produces EP within 5%. Our EP dose-response curve has the same slope as the PGE? percentage inhibition curve (17) and the picogram quantity of PGE2 is easily converted to units of erythropoiesis activity. Welch et al. obtained data to suggest that EP has a lipid functional group (24), and Grenett et al. demonstrated concurrent loss of PGE? with EP activity (25). If the lipid functional group is PGE2, this would explain the competitive inhibition of EP by PGFz, (9, 25, 26). Lipid, protein, and spermine inhibitors of erythropoiesis have been described (1, 18, 19, 27, 28). SUMMARY

An inhibitory factor of erythropoiesis, obtained from normal human urine, is indicated to be a complex of a fragment of cw,-acid glycoprotein and prostaglandin F?, . Immunoelectrophoresis reveals two protein components in the EIF complex which separate during acrylamide gel electrophoresis. A y-globulin (MW 185,000) is a carrier of the complex. A fragment of cw,-acid glycoprotein (MW 9300) retains the inhibitory factor, PGF2,. Noncovalent forces bind the PGFzu to the protein, and PGF?, can be extracted with benzene. ACKNOWLEDGMENTS The authors thank Dr. Robert D. Lange for editorial assistance, Betty Williams and Carol Cope for technical assistance. and Dr. John E. Pike and Dr. K. T. Kirton (The Upjohn Company) for the prostaglandins.

REFERENCES I. 2. 3. 4.

Erslev. A. L., Kazal, L. A., and Miller. 0. P.. Proc. Sot. Exp. Biol. Med. 138, 1025 (1971). Kiulaakso, E.. and Rytomaa. T., Cell Tiss. Kiner. 4, I (1971). Krzmowska, H., Acfa Physiol. Polon. 17, 1 (1966). Lewis, J. P.. Neal, W. A., Moores. R. R., Gardner, E.. Jr., Alford. D. A.. Smith, L. L.. Wright. C.-S., and Welch, E. T., J. Lab. C/in. Med. 74, 608 (1969). 5. Lindeman, R., in “Erythropoiesis Regulatory Mechanisms and Developmental Aspects” (Y. Matoth. Ed.), pp. 185-189. Academic Press, Tel Aviv, 1976. 6. Whitcomb, W. H., in “Kidney Hormones” (J. W. Fisher. Ed.), pp. 461-484. Academic Press. New York/London. 1971.

16

NEAL

El’ AI

7. Whitcomb. W. H., and Moore, M. Z.. J. Luh. (‘lit]. Med. 66, 641 ( lY6h). 8. Neal, W. A., Lewis, J. P., Welch, E. T.. Lutcher. C. L.. Moores. K. R.. and Wright. < .-$.. Amer. .J. Vet. Rex. 40, 493 (19791. 9. Neal, W. A.. Lewis. J. P., Garver. F. A.. and Lutchet-. ( L.. Biod7z111. Med. 23, 35 ( 19X01. IO. Lewis, J. P., Gallagher. N. I.. Carmody. S. E.. and Lange. R. D.. Bioc.hi/x. Biophvs itc’trc 104. 218 (1965). I I. Espada, J., Langton, A. A., and Dordda, M., B!oc./I~u. Biop/!v.\. Atrcc 285, 427 ( 197’1, 12. Shapiro, A. L.. Vinuela. E.. and Maizel. J. V.. Jr. 1 Hioc~hr~,~. Biophv,~. Rr.\. C‘ott~tt~utt. 28. Xii (1967). 13. Green. K.. and Samuelsson, B.. J. Lipid RCA. 5. I I7 i IYh4). 14. Garver, F. A.. Neal. W. A.. Lewis. J. P.. Grenett. H. E.. L.utcher. C. IL.. O/awu. ‘I‘.. Moore\. R. R.. and Wright. C.-S.. B&hen!. Med. 25, I74 (1981) 15. Scheidegger, J. J.. Iw?. Arch. A//er~~~ 7, I03 (1955). 16. Lewis. J. P.. Neal, W. A.. Alford. D. A.. Moores. R. R.. Gardner. E.. Jr.. Welch. E. I . Wrtght. C.-S., and McWhirter. J. D.. Amer-. J. I/e/. Res. 31, XY1 (1970). 17. DuBose. C.. Jr.. Welch. E. T.. Lewis, J. P.. Neal. W. A.. und Lutchcr. C‘. L.. Uioc,i~c,,rt Lf~~~l 17, 310 (1977). 18. Browman. G. P., Freedman. M. H.. Blajchman. M. A.. and McBride. J. .A.. .+\/u~I.. ./. !Ifc*lt 61, 572 (1976). 19. Krantz. S. B.. Moore. W. H.. and Zaentz. S. I>.. .I. (‘/i/~. I,II’(~\I. 52, 324 (1073)” 20. Moore, M.. and Whitcomb. W. H.. .I. Ltrh. CIiu. .‘Mcc/. 89, Y3 (1977). 21. Gardner. E.. Wright. C.-S.. Lewis. J. P.. and Moorer. R. R.. Hril. .I. lftrcttwr~u 13. 317 ( IYo~I 22. Editorial, Lorrwt 1. 368 (1979). 23. Lewis. J. P.. Welch. E. T.. Neal. W. A.. DuBoye. ( M.. Jr.. Lewl\ W. G.. 111. Wrtghi. C.-S.. and Smith. L. L.. L)ioc,hen~. Mrd. 10, 374 ( lY741. 24. Welch, E. T.. Neal, W. A., Lewis. J. P.. Dunn. (‘. I). K.. and Lange, K. U.. Hlrxi~c,~n. %,I:,:/. 23, 373 ( 1980). 25. Grenett. H. E.. Garver. F. ,4.. l.cw~s. J. P.. Neal. WI;. .I.. Ora~va. I and 1.111~hcr. t I Bioc~hetu. Med. 27. 26 ( 19%). 26. Lewis. J. P.. Moores, R. R., Neal. W. A.. Garver. F. .I l.im3lrt. (’ I Lucai. _I. I< md Mirand. E. A.. &p. Hemrfol. 9. 540 (I981 1. 27. Radtke. H.. Bartos. D.. Bartos, F.. Rege. ‘4. B.. C’ampbell. K.. and FI$her. J. W.. L-\/I. Ilc~~rii~~/. 8, 198 (1980). 28. Zucker. S.. Beck. G. Y. P.. Lysik. R. M.. and DiStef’ano. S. f:.. ~..~~~. H~~~/~tr~~~i. 9. !CO (I9lii I