Biochem. J. (1977) 164, 469-472Printed in Great Britain

Self-Digestion ofIluman Erythrocyte MembranesROLE OF ADENOSINE TRIPHOSPHATE AND GLUTATHIONE

By AUGUSTA BROVELLI, MOHAMMAD SUHAIL,* GIOSUi PALLAVICINI,FABIOLA SINIGAGLIA and CESARE BALDUINI

Institute ofBiological Chemistry, Faculty ofSciences, University ofPavia, 27100 Pavia, Italy

(Received 21 February 1977)

Intact human erythrocytes incubated at 37°C, pH 7.4, release a sialoglycopeptide similarin its chemical composition, immunological and aggregation properties to the glycopeptidereleased by isolated 'ghost' membranes. The presence of ATP or reduced glutathione atphysiological concentrations in the incubation medium of 'ghost' membranes inhibitsthis self-digestion process.

Sialic acid residues are responsible for thenegative charge at the cell surface in humanerythrocytes (Eylar et al., 1962), and are involved inM- and N-group specificity (Ebert & Jurgen, 1972).The sialic acid content decreases as erythrocytesage (Balduini et al., 1974; Baxter & Beeley, 1975),and its role as a determinant of the erythrocyte life-span is described in several reports (Jancik & Schauer,1974; Durocher et al., 1975; Gattegno et al., 1975).Human erythrocyte 'ghost' membranes can digesttheir own sialoglycoproteins and release into theincubation medium a glycopeptide containing sialicacid, glucosamine, galactosamine, galactose andmainly polar amino acids (Brovelli et al., 1976).If glycoprotein breakdown has a physiologicalrole in erythrocyte aging, it must also take placein intact cells; therefore we have tested intact humanerythrocytes for the ability to digest in vitro theirmembrane glycoproteins. Moreover, it seems reason-able that a mechanism must exist by which thisprocess is inhibited in the young cell. On the basis ofmetabolic considerations, the ability of ATP andglutathione to modulate membrane self-digestionwas hypothesized, and the effect of these metaboliteson sialoglycopeptide release was studied, by evaluat-ing linked sialic acid released by human erythrocyte'ghost' membranes in the presence of ATP orglutathione. Membrane glycoprotein patterns ob-tained on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after incubation of 'ghost' mem-branes with ATP or glutathione at physiologicalconcentrations were also investigated.

ExperimentalExperiments on self-digestion of intact erythrocytes

Fresh human blood (40-60 ml) of the O-A-B-ABRhesus-positive group was collected for each

* Permanent address: Department of Biochemistryand Home Science, University of Allahabad, 211002Uttar Pradesh, India.

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experiment from the veins of donors, with the use of3.8 % (w/v) sodium citrate as anti-coagulant. Plasmaand buffy coat (leucocytes) were carefully removedafter centrifugation at 40C at 1000g for 15min.For each sample 4-6ml of cells was suspended invol. of 0.9% NaCl or Krebs-Ringer phosphate

buffer [lOOvol. of 0.9% NaCl, 4vol. of 1.15% (w/v)KCI, 3 vol. of 1.22% (w/v) CaCl2, 1 vol. of3.82% (w/v)MgSO4,7H20, 20vol. of 0.1 M-sodium phosphatebuffer, pH 7.4], put into a dialysis bag and immersedin 200vol. of the same solution. The samples wereincubated for increasing periods at 37°C, withshaking. Control experiments were performed byincubating cells at 4°C in the same buffer for the sameperiod. All the experiments were carried out withsterile material. The addition of ampicillin (50,ug/mlof Krebs-Ringer solution) to some samples resultedin the absence of bacterial contamination at theend of 30h incubation. No difference in the autolyticprocess was observed in the presence of antibiotic.After incubation, the cell suspension was centrifugedat 1000g for 5min; the supernatant was then filteredon Dowex 2 (X8; acetate form) for the isolation ofthe sialic acid-containing material, which was thenpurified by gel filtration on Bio-Gel P-30 or P-150as previously described (Brovelli et al., 1976).Sedimented erythrocytes were lysed and washed asdescribed by Marchesi & Palade (1967): 'ghost'membranes were used for sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Experiments on self-digestion ofisolatedmembranes'Ghost' membranes, prepared as previously

described (Brovelli et al., 1976), were suspendedin 0.5vol. of 0.05M-Tris/HCI buffer, pH7.4, andincubated for 4h at 37°C, with shaking. In someexperiments, ATP, ADP, AMP, NAD+, NADH,NADP+, NADPH, GSHt or GSSG was added, at

t Abbreviations: GSH and GSSG, reduced andoxidized glutathione.

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physiological concentrations (Pennell, 1974). Theeffect of GSH concentrations two and four timesthe physiological ones was also investigated. Afterincubation, the samples were centrifuged at 25000gfor 15min; the supernatant was tested for linkedsialic acid as described by Svennerholm (1958), afterthe sialic acid-containing material had been isolatedon Dowex 2 (X8). The sedimented membranes wereused for sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis.

Sodium dodecyl sulphate/polyacrylamide-gel electro-phoresis

This was carried out on sodium dodecyl sulphate-solubilized 'ghost' membranes as described byFairbanks et al. (1971), with the previouslydescribed modifications (Brovelli et al., 1976). Thegels were stained with periodic acid/Schiff reagent(PAS) (Fairbanks et al., 1971). The electrophoreto-grams were recorded with a Saitron 803 densitometer(Florence, Italy) by using the yellow-green filter.

Analytical methods

Hexosamines and amino acids were determinedwith the Hitachi-Perkin-Elmer liquid chromato-graph by the method of Moore & Stein (1951).Total sialic acid content of 'ghost' membranes wasdetermined after hydrolysis of fresh 'ghosts' for 1 hat 80°C in 0.05M-H2SO4 and isolation of the sialicacid by chromatography on a column (1.5cmx2cm) of Dowex 2 (X8; acetate form) under theconditions of Brovelli et al. (1976). The sialic acidcontent of the glycopeptide released in the self-digestion experiments was determined by the sameprocedure, excluding the H2SO4 hydrolysis step.Adenosine triphosphatase was assayed by themethod of Kielley (1955). Phosphate was determinedby the molybdate/vanadate method of Zilversmith& Davis (1950). Protein content of 'ghost' membraneswas determined by the method ofLowry et al. (1951),with serum albumin as standard. GSH was assayedas described by Beutler et al. (1963). Inhibition ofM-N-group haemagglutination was tested as de-scribed by Lisowska & Duk (1975). Glycophorin wasprepared by the procedure of Marchesi & Andrews(1971).

Results

Self-digestion of intact erythrocytesSialic acid release from intact cells begins after

9h of incubation and reaches its highest value(50-70% of the total membrane sialic acid) after 25-30h of incubation. The product of this self-digestionprocess was isolated on a column (1.Scmx4cm) ofDowex 2 (X8) and purified by gel filtration on a

Table 1. Chemical composition ofthe sialic acid-containingmaterial isolated by gelfiltration on Bio-Gel P-30 or P-150

Gel filtration was carried out after incubation ofintact erythrocytes at pH7.4 and isolation of sialicacid-containing material on Dowex 2 (X8); sialo-glycopeptide was eluted from both the Bio-Gels inthe void volume. Basic amino acids are present in verysmall amounts and are not reported in the Table.Values are means± S.D. ofthree experiments.

Sialic acidGlucosamineGalactosamiineAspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineValineMethionineIsoleucineLeucine

Content(,mol/lOOpmol)

32.19± 1.664.79+0.2111.82+0.825.09+ 1.0710.60+1.0210.30+ 2.086.96± 1.03Traces

4.16±0.994.74± 0.084.21 ± 0.72Traces

2.82+ 0.862.33+0.62

column (1.5cmx85cm) of Bio-Gel P-30 or P-150under the conditions previously described (Brovelliet al., 1976). The sialoglycopeptide was eluted fromboth the columns in the void volume; this gel-filtration pattern supports the hypothesis that thesialoglycopeptide is obtained in an aggregated formand therefore behaves like the fragment released by'ghost' membranes (Brovelli et al., 1976). Itschemical composition is reported in Table 1; somecomponents are present in the same amount as inthe sialoglycopeptide released by 'ghost' membranes(sialic acid, aspartic acid, serine, proline, alanine,valine, isoleucine), but others are significantly different(glucosamine, galactosamine, threonine, glutamicacid, methionine, leucine). Boththesialoglycopeptidesreleased from 'ghosts' and from intact erythrocytesinhibit M-N-group haemagglutination. Electro-phoretograms of 'ghost' membranes preparedfrom incubated cells show that after 30h ofincubation a modification of the ratio of bandsPAS 1/PAS 2 appears (Fig. 1). It is noteworthy thatsialoglycopeptide release appears to be correlated withGSH content of the erythrocyte; at the point duringincubation when the cellular content of GSHdecreases, the self-digestion process begins. Theresults of six experiments indicate that, while GSHconcentration is maintained at about 60-80%(or above) of the concentration at zero time, no sialicacid release takes place, but when a 30-50% decreasein the GSH concentration occurs (9-12h of incu-bation) sialoglycopeptide release becomes evident(3-10% of the total membrane sialic acid). It wasalso shown that when 0.3 mM-adenine is present in the

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incubation medium a 20-40% inhibition of the self-digestion process occurs after 30h of incubation.These results seem to indicate that energy andredox metabolism affect the membrane self-digestionprocess.

Regulation ofthe membrane self-digestionprocessTo investigate the correlation between membrane

autolysis and the redox and energetic situation of the

PAS PAS 2 PAS 3

(a)

(c)

- 2 3 4 5 6

Distance migrated (cm)7 +

_ 100

50

100

50 -

IoiC

.100 ct0ca

50 0

i 100

Fig. 1. Sodium dodecylsulphate/polyacrylamide-gel electro-phoresis of sodium dodecyl sulphate-solubilized 'ghost'

membranes(a) Unincubated 'ghost' membranes; (b) membranesincubated for 4h at 37°C, pH7.4; (c) membranesincubated for 4h at 37°C, pH7.4, in the presence of0.6lM-ATP or 13mM-GSH; (d) membranes fromcells incubated for 30h at 37°C in 0.9% NaCI. Theelectrophoresis was carried out as described byFairbanks et al. (1971); the current used was

3-3.5mA/tube. Acrylamide concentration was 5.6%(w/v). Gels were stained with periodic acid/Schiffreagent (PAS).

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cell, the effect of different metabolites on this processwas studied by using 'ghost'-membrane preparations.The effects of the following compounds at physio-logical concentrations (Pennell, 1974) were tested(three to four experiments for each substance):ATP, ADP, AMP, NAD+, NADH, NADP+,NADPH, GSH and GSSG. Since in 'ghost'-mem-brane preparations a residual adenosine triphos-phatase activity is present, 4mM-ATP was added tothe incubation medium to obtain a physiologicalconcentration (0.6mM) of this nucleotide after 4hof incubation. In such conditions the sialoglyco-peptide release is completely inhibited. Sodiumdodecyl sulphate/polyacrylamide-gel electrophoresisof these membranes shows the same periodic acid/Schiff-stain pattern as that of unincubated mem-branes (Fig. 1). AMP and ADP have no effect. WhenGSH concentrations (3.25mM) similar to thosepresent in fresh erythrocytes were used, a 50%inhibition of the self-digestion process was achieved;for higher concentrations (6.5mM and 13mM), totalinhibition was observed; the glutathione concen-tration remains constant during the incubation.In these conditions, the periodic acid/Schiff-stainelectrophoretic pattern is superimposable on thatof unincubated 'ghost' membranes (Fig. 1). GSSG,NAD+, NADH, NADP+ and NADPH at physio-logical concentrations have no effect on membraneautolysis. Inhibition is restored if NADPH andglutathione reductase are added to 3mM-GSSG inthe incubation medium. Other thiol-group-containingcompounds (5 mM-dithiothreitol) do not influence theautolytic process.

Discussion

The mechanisms involved in erythrocyte agingare only partially known (Brewer, 1974), and, inparticular, no information is available about thefactors determining the removal of sialic acid fromold erythrocyte membrane (Balduini et al., 1974;Jancik & Schauer, 1974; Baxter & Beeley, 1975;Durocher et al., 1975; Gattegno et al., 1975). It hasbeen shown in our laboratory that human 'ghost'membranes are able to release, by an autolytic pro-cess, a glycopeptide in which 30-60% of totalmembrane sialic acid is present (Brovelli et al., 1976).Also intact human cells incubated at physiological pHdigest their own membrane glycoproteins and releasea sialoglycopeptide which is similar with respectto its chemical composition to that obtained after theincubation of 'ghost' membranes. Some substantialdifferences appear in the molar ratio of someconstituents; this can be ascribed to modificationsof the supramolecular arrangement occurring during'ghost'-membrane preparation, which could alsobe responsible for the different periodic acid/Schiff-stain electrophoretograms of residual membranes.

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472 A. BROVELLI AND OTHERS

However, immunological properties and aggrega-tion in solution indicate that both sialoglycopeptidesbehave like glycophorin (Janado, 1974; Lisowska& Duk, 1975). Moreover, the regulatory mechanismsof the autolytic process in 'ghost' membranes andintact cells show some similarities. The presence ofphysiological concentrations of GSH or ATP inthe incubation medium inhibits the sialoglycopeptiderelease from 'ghost' membranes. The similarity inperiodic acid/Schiff-stain electrophoretograms ob-tained from unincubated 'ghost' membranes andthose incubated with GSH and ATP confirms theprotective action of these two metabolites. The GSHeffect seems to be specific, since other thiol-group-containing reagents, such as dithiothreitol, are quiteineffective. Moreover, when GSSG, NADPH andglutathione reductase are present in the incubationmedium, the autolytic process is similarly inhibited;this must be a consequence of GSH production,since it has been shown that GSSG and reducednicotinamide nucleotides alone do not producesuch an effect. These results are in accordance withthe observations that sialoglycopeptide release issignificantly lower when intact cells are incubatedin the presence of adenine, and that the autolyticprocess only starts when a decrease in cellular GSHconcentration occurs. Our results suggest thatmembrane glycoprotein structure is related to cellularredox metabolism andATP synthesis. These processesare known to decrease in the old erythrocyte, sinceglucose 6-phosphate dehydrogenase activity andenergy charge are significantly decreased (Brewer,1974). Therefore it is suggested that, when theerythrocyte loses the ability to maintain its GSHand ATP concentrations, the membrane proteolyticmechanism becomes active, causing sialoglycopeptiderelease and surface modifications that enable haemo-catheretic organs to remove the old cells from thecirculation. A similar mechanism could also takeplace in the haematological disorders characterizedby a decrease in redox or energy metabolism.

This work was supported by a grant from the ConsiglioNazionale delle Ricerche, Italy.

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