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Modification of membrane protein organization during in vitro aging of human erythrocytes
Int. J. Biochem. Vol. 16, No. 11, pp. I1 15-l 120, 1984 Printed in Great Britain. All rights reserved
Copyright
0
0020-711X/84$3.00+0.00 1984 Pergamon Press Ltd
MODIFICATION OF MEMBRANE PROTEIN ORGANIZATION DURING 1lV VITRO AGING OF HUMAN ERYTHROCYTES A. BROVELLI, C. SEPPI and Department
of Biochemistry,
University
of Pavia,
(Received 8 November Abstract-l. In in vitro aged membrane; these clusters are nearly completely dissociated 2. SDS-polyacrylamide gel are made of peptide fragments fragments correspond to an 3. Their formation results proteins.
Italy [Tel. (0382)33326]
1983)
human erythrocytes, the presence of protein clusters can be found on the made up of peptides held together by disulfide bridges, since they can be by dithiothreitol treatment. electrophoresis after dithiothreitol dissociation indicates that the aggregates with a molecular weight ranging from 20 to - 110 kdalton; none of these intact protein component of the membrane. from oxidation and proteolysis of membrane, and perhaps cytoplasmic
tively blood was defibrinated on glass pellets. Erythrocytes were collected by centrifugation at 1OOOg after repeated washes in 0.9% (w/v) NaCl. White cell contamination was minimized by sedimenting red cells on an isotonic Percoll (Pharmacia) suspension. Packed erythrocytes were suspended in one volume of Krebs-Ringer phosphate buffer pH 7.4, and incubated for different times under previously reported conditions (Brovelli et al., 1977). Dialysis tubing was boiled in ethylendiaminotetracetic acid (EDTA) at pH 8-9 and than rinsed in distilled water before use. Ghost membranes were prepared according to Marchesi and Palade (1967), using 0.2 mM phenylmethylsulfonylfluoride and 1 mM EDTA as protease inhibitors.
INTRODUCTION
A mature circulating erythrocyte undergoes many metabolic modifications during its life-span (Brewer, 1974) as a consequence of a decrease of several enzyme activities; significant changes in the surface properties correspond to this modified functional situation, as suggested by electrokinetic (Seaman, 1975) and lectin agglutination studies (Baxter and Beeley, 1975) and by chemical (Balduini et al., 1974; Bladier et al. 1980) and immunological investigations (Kay, 1975, 1978, 1981; Lutz, 1981). These modifications seem to involve not only the structure of membrane glycoproteins but also the topographic organization of proteins; several investigations indicate that metabolic impairment can produce the aggregation of some membrane proteins in high molecular weight complexes (for a review see Palek and Liu, 1979). Recently, we have investigated the relationships between metabolic events and membrane structure by means of in vitro aging experiments; we mimicked the aging through the starvation of cells of glucose and ATP and GSH precursors (Brovelli ef al., 1977) and we observed two phenomena modifying the membrane structure: (1) a breakdown of glycoproteins with a loss of sialopeptides mainly from glycophorins, (2) clustering in high molecular weight complexes of some membrane proteins (Balduini et al., 1979a). These phenomena seem to be controlled by the redox and energy capacities of the cell and seem to be a consequence of events occurring at the cytoplasmic surface of the membrane (Brovelli et al., 1982). In order to shed light on the mechanisms producing the membrane processes observed during the in vitro aging, the properties and the composition of the protein aggregates were investigated. MATERIALS
C. BALDUINI 27100 Pavia,
AND METHODS
In vitro aging of intact celfs Human blood was drawn from the veins of donors by using 3.8% sodium citrate (w/v) as anticoagulant. Alterna-
Geljltration
on Sepharose 2B and 48 and Ultrogel AcA34
Isolation of high molecular weight aggregates was obtained by gel filtration of 4% SDS dissociated erythrocyte membranes on Sepharose 2B (Lux and John, 1977), Sepharose 4B and Ultrogel AcA 34 columns (1.5 x 50 cm) equilibrated with 0.01 G Tris-HCl buffer, pH 7.8, contai&ng 1% SDS (w/v) and 0.025Y (w/v) NaN,. Elution was carried out with ihe same buffe; iflow ‘rate Sml/hr) and monitored at 280 nm with an ISCO U-A4 absorbance monitor. (a) Filtration on Sepharose 28 of solubilized membranes. An excluded peak (V,, - 2B) and a non-homogenous retarded peak are recovered. This second peak is composed of a “shoulder” in which protein aggregates are present and by more retarded components corresponding to nonaggregated membrane proteins (Fig. 1). (b) Filtration on Sepharose-4B. The “shoulder” material after filtration on Sepharose-4B is resolved in an excluded peak (V,, - 4B) and in a retarded peak. (c) Filtration on Ultrogel-AcA 34. Sepharose 4B retarded peak is resolved in an excluded peak (V,-AcA34) and in non-aggregated membrane components. The highest molecular weight aggregates ( VO- 2B), after refiltration on Sepharose 2B, were characterized in their amino acid composition and after dissociation with dithiothreitol (DTT), by SDS-polyacrylamide gel electrophoresis. Protein composition of VO- 4B and VO- AcA34 were determined by SDS-polyacrylamide gel electrophoresis after DTT dissociation. SDS-polyacrylamide
gel electrophoresis
SDS-polyacrylamide gel electrophoresis was carried out under the Laemmli (1970) conditions. Samples were dissociated for 40 min at 37°C or for 5 min at 100°C in 10 mM Tris-HCl buffer, pH 7.4, containing 40mM DTT, 1 mM 1115
A. BROVELLI et
1116
al
Characterization qf the complex eluted in the V, Sepharose 2B gel filtration (V, 2B) (a) Amino weight
Number
of
fractmns
Fig. 1. Gel filtration on Sepharose 2B of SDS-dissociated ghost membranes prepared from in vifro aged red cells. Protein clusters present on the membrane are eluted in the V0 and in the shoulder.
EDTA, 1% (w/v) SDS, 7% (w/v) saccharose and 0.01% (w/v) pyronin as tracking dye. Electrophoretic separation was done on polyacrylamide gel slabs (acrylamide concentration was 3% in the stacking gel and 8% (w/v) in the running gel: 14 x 13.5 cm, 1.5 mm thickness) with running times of about 5 hr at 40mA, without cooling of the plate. Protein contents of each sample were between 80 and 1OOpg. Molecular weight calibration was done using as standard two mixtures (Pharmacia), respectively, composed of (a) thyroglobulin, ferritin, catalase, lactate dehydrogenase, albumin and (b) phosphorylase b, albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and cc-lactalbumin, dissolved in the same buffer used for samples.
Analytical methods Total sialic acid content of ghost membranes was determined by the Svennerholm (1958) method after hydrolysis of ghosts for 1hr at 80°C in 0.05 M H,SO,, as previously reported (Brovelli et al., 1977). Protein was determined by the method of Lowry et a!. (1951), using serum albumin as a standard. Aminoacids and hexosamines, after hydrolysis of the sample, respectively, in 6N HCl (24 hr) and 4N HCI (7 hr) at lOYC, were determined by ion-exchange chromatography using the LKB 4101 Aminoacid analyzer.
acid composition.
aggregates
Membrane bation
protein organization
during in vitro incu-
Human phosphate
erythrocytes incubated buffer, pH 7.4, release
in
Krebs-Ringer
in the incubation medium glycopeptide fragments, as previously described; this phenomenon starts after 69 hr of incubation, when a consistent decrease of ATP and GSH occurs in the cell (Balduini et al., 1979b). Then, after 14-16 hours of incubation, the supramolecular arrangement of some membrane constituents begins to be modified and a macromolecular aggregate (mol. wt 140. IO3 kdalton) can be detected and isolated by Sepharose 2B gel filtration (Fig. 1). The formation of protein clusters and the glycopeptide release for different incubation times are reported in Table 1. Whatever the incubation time, smaller aggregates of proteins are also present on the membrane: these clusters can be detected as a shoulder eluted from the column of Sepharose 2B just before the peak containing all the membrane proteins (Fig. 1).
The high molecular the void volume of
in
Sepharose 2B gel-filtration have been characterized for their amino acid content. The results, reported in Table 2A, show that these protein clusters are different from the high molecular weight aggregates described by Lux and John (1977) in erythrocytes from splenectomized subjects and by Carraway et al. (1975) after Ca 2+ loading of ghost membranes. Moreover their amino acid content shows that these aggregates are not produced by unspecific clustering of all membrane proteins (the amino acid composition of whole membrane is reported in Table 2B). The clusters can be nearly completely dissociated by 40 mM dithiothreitol. (b) SDS-pol.vacrylamide-gel electrophoresis. In Fig. 2 the normal membrane protein pattern and the corresponding molecular weight obtained in our experimental conditions are reported. In order to study the protein composition of the high molecular weight aggregates, the protein material eluted in the void volume of Sepharose 2B gel filtration was collected,
Table
I. Effect of in t,irro incubation on protein sialopeptide release
Incubation time (hr)
_
0 9-12 14-16 24-30
Protein clustered (‘I”) 0.8 0.8 2.5 16.4
+ * i i
clustering
and
Sialopeptide release (4:, sialic acid)
0.5 0.5 1.3 4.3
IO 20 3&60
Values are mea” + SD of 8 experiments
Table 2. Amino acid composltion
of high molecular weight protein clusters (V,, - 2B) and ghost membranes
Ammo acid RESULTS
eluted
qf
Cysteic acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Methionine sulfoxide Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Areinine Triptopha” Glucosamine Galactosamine Galactose’ Glucose’
(A)
(B)
Protanclusters
Membrane proteins’
(C;, - 2B)” (~mol,‘100~mol) 1.08 k 0.24 8.20+0.17 5.17 kO.34 10.79 C_2.62 13.60 _+ 1.85 5.55 + 2.01 15.01 + 4.86 8.23 f 0.91 0.10t0.17 4.92 IO.60 0.15i0.13 0.68 k 0.61 3.05 k 0.32 6.92 ? 0.3 I I .08 i 0.93 4.94 ? 3.89 4.46 k 0.46 2.28 k 0.51 3.78 k 0.64 ND Traces TlXes Present Present
8.2-9.4 5.7-5.9 6.3-8.3 12.1~12.9 4.3-5.8 6&7. I 7.3-8.2 0.9-I .4 5.8-7. I L&2.4 4.4-5.3 I I .3-12.5 I .8-2.5 3.fF4.5 4.8-5.2 2.3-2.4 4.5-4 8 0.k2.5
“Values are mean f SD of 3 preparations. “Data from Bakerman and Wasemlller and from Rosenberg Guidotti. ‘Qualitatively determined by GLC.
and
MoLwt
-
(kd)
2
240.17 I 3.90 223.33 + 4.72
__192.4347.9z3
$0
l3ond
Mol.
wt
(kdl
Mol.wt (kd)
Protein
clusters
dialyzed, liophylized and then refiltered on a Sepharose 2B column. The protein complex eluted in the void volume was submitted to SDS-polyacrylamidegel electrophoresis under the Laemmli (1970) conditions, in the presence of dithiothreitol. Dithiothreitol dissociated the complex in protein fragments with a molecular weight ranging from 110 to 22 kdalton; none of these peptides correspond to a typical membrane component, identified on the basis of the molecular weight (Fig. 3A). Characterization of the Sepharose 2B gel jiltration
aggregates
retarded
by
Protein aggregates smaller than the clusters eluted in the V0 of Sepharose 2B gel filtration are retarded by this column and eluted in the shoulder (see the elution profile in Fig. 1). This material was seived on a Sepharose 4B and Ultrogel AcA34 column and the protein clusters excluded by Sepharose 4B (mol. wt 2 5.10’ kdalton) and excluded by Ultrogel AcA34 (mol. wt 2 700 kdalton) were submitted to SDSpolyacrylamide-gel electrophoresis in the Laemmli (1970) conditions, in the presence of dithiothreitol. The aggregates excluded by Sepharose 4B contain spectrin chains, linked by disulphide bridges. In some preparations ankyrin is also present (Fig. 3B). The aggregates excluded by Ultrogel AcA34 are composed of protein fragments with a molecular weight ranging between 60 and 75 kdalton (Fig. 3C).
DISCUSSION
During in vitro aging, human erythrocytes undergo a consistent modification of membrane protein and glycoprotein structure. In particular, in previous papers, the fragmentation of glycophorins has been characterized in detail (Brovelli et al., 1977; Pallavicini et al., 1981). This breakdown seems to be the expression of a more general mechanism of proteolysis at the membrane level, as proved by the evidence that clusters made up by protein fragments appear on the membrane during in vitro incubation. Two types of aggregates were isolated from the membrane of in vitro aged cells. The biggest ones (mol. wt. 2 40. 103kdalton), eluted in the void volume of Sepharose 2B gel filtration, are made up by peptide fragments held together by disulfide bridges. The dissociation of this aggregate by dithiothreitol and the characterization of its peptide components by polyacrylamide gel electrophoresis evidentiates the presence of fragments with a molecular weight ranging between 22 and 110 kdalton; none of these bands corresponds to an intact protein component of the membrane. The amount of these aggregates increases with the incubation time (Table 1) and can be controlled by the presence of ATP or reduced glutathione (Brovelli et al., 1982); on the contrary the amount of smaller aggregates present on the erythrocyte membrane (V, - 4B, V0 - AcA34) is independent on the incubation time and does not seem therefore to be influenced by the presence of ATP or GSH. The clusters excluded by Sepharose 4B are mainly composed of spectrin (band 1 and band 2). Sometimes
1119
in aged red cells
ankyrin is recovered in small amount in these aggregates. 40mM DTT can nearly completely dissociate these clusters. Disulfide bridges therefore hold the spectrin chains together, even if the presence of GSH in physiological concentration does not modify their appearance on the membrane. These aggregates are different from those described by Morrow and Marchesi (1981) which are composed of up to 11 dimers of spectrin, but not linked via disulfide bridges. The mol.wt 2 700 kdalton aggregates of ( V0 - AcA34) are present in a very small amount and their characterization by polyacrylamide gel electrophoresis indicates the presence of peptides with a molecular weight ranging between 60 and 75 kdalton. Some authors (Golovtchenko-Matsumoto et al. 1982) suggest the presence in this region of band 3 degradation products. In our conditions this material is held together by disulfide bridges, being completely dissociated by 40mM DTT. The V,, - 4B and V,, - AcA34 aggregates do not appear to be produced by in vitro aging, but by the conditions in which membranes are prepared. Only the V,, - 2B aggregates seem to be produced by in vitro aging conditions and their properties suggest that the proteolysis could act on proteins previously oxidized in their sulphydryl groups. It seems, therefore, that the two events occurring on the membrane during in vitro aging (proteolysis and oxidation) are related, since only oxidized fragments are present in the aggregates. As for the origin of the proteins present as oxidized fragments in the V, - 2B aggregates after incubation of intact cells (Brovelli et al., 1982) it cannot be excluded that some cytoplasmic component is present in the clusters, even if they were isolated after membrane preparation. It was in fact reported that hemoglobin is able to link band 3, glycophorin (Ravenbuehler et al., 1982) and spectrin (Alloisio et al., 1982) so that it can be easily hypothesized that these binding capacities can also concern some degradation product of hemoglobin or of other cytoplasmic components. Therefore our evidence seems to suggest that the metabolic impairment occurring during aging or in pathological conditions could damage membrane components by oxidative and proteolytic processes, so determining a modification of the structural organization of the cell surface. SUMMARY
During in vitro aging of red cells, a clustering of proteins occurs at the level of the membrane. This process can be found by gel filtration on Sepharose 2B of SDS-dissociated ghost membranes prepared from the cells aged in vitro. Clusters are eluted in the void volume (mol. wt 2 40. lo3 kdalton) and just before the retarded proteins. All clusters are made up of peptides linked through disulfide bridges. Only the aggregates eluted in the void volume from Sepharose 2B are specific for in vitro aging. Their appearance is detectable on the membrane after 14 hr of incubation of red cells and can be inhibited by physiological concentrations of ATP and GSH. SDSpolyacrylamide-gel electrophoresis after dith-
A. BROVELLI et al.
1120
iothreitol dissociation indicates that these clusters are made up of peptides with a molecular weight ranging from 22 to 110 kdalton. The clusters could result from oxidative and proteolytic damages produced by in vitro aging on membrane, and perhaps cytoplasmic, proteins.
Acknowledgements-Work supported by Minister0 Pubblica Istruzione and by Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie ereditarie of the C.N.R., Italy.
REFERENCES Alloisio N., Michelon D., Bannier E., Revel A., Beuzard Y. and Delaunav J. (1982) Alterations of red cell membrane proteins and hemoglobin under natural and experimental oxidant stress. Biochim. bioDhys. Acta 691, 3OG308. Bakerman S. and Wasemiller G. (1967) Studies on structural units of human erythrocyte membrane. I. Separation, isolation, and partial characterization. Biochemistry 6, 1100-1113. Balduini C., Balduini C. L. and Ascari E. (1974) Membrane glycopeptides from old and young human erythrocytes. Biochem. J. 140, 557-560. Balduini C., Brovelli A. and Pallavicini G. (1979a) In vitro aging - - of young and old human erythrocytes. In Glycoconjugates: Proceedings of the Vth International SymDosium (Edited bv Schauer R. et al.), __ PD. 478479. Thieme, StuttgartBalduini C., Brovelli A., Balduini C. L. and Sinigaglia F. (1979b) Extra bone-marrow remodeling of membrane glycoproteins during erythrocyte life-span. Bull. molec. Biol. Med. 4, 47-58. Baxter A. and Beeley J. G. (1975) Changes in surface carbohydrate of human erythrocytes aged in uivo. Biothem. Sot. Trans. 3, 134136. Bladier D., Gattegno L., Fabia F., Perret G. and Cornillot P. (1980) Individual variations of the seven carbohydrate components of human erythrocyte membrane during aging in vivo. Carbohy. Res. 83, 371-376. Brewer G. J. (1974) Red cell metabolism and function. In The Red Blood Cell (Edited by Surgenor D. MacN.) 2nd edn. Vol. I. on. 474508. Academic Press, New York. Brovehi A., Suhail M., Pallavicini G., Sinigagha F. and Balduini C. (1977) Self-digestion of human erythrocyte membranes. Role of ATP and GSH. Biochem. J. 164, 469472. Brovelli A.. Pallavicini G.. Airoldi G. and Balduini C. (1982) Supramolecular assembly of membrane constituents durine in vitro seine of human ervthrocvtes. In Protides ofyhe Biologica: Fiids (Edited by Peters H.) Vol. 29, pp. 337-340 Pergamon Press, Oxford.
Carraway K. L., Triplett R. B. and Anderson D. R. (1975) Calcium-promoted aggregation of erythrocyte membrane proteins. Biochim. biophys. Acta 379, 571-581. Golovtchenko-Matsumoto A. M., Matsumoto I. and Osawa T. (1982) Degradation of band-3 glycoprotein in vitro by a protease isolated from human erythrocyte membranes. Eur. J. Biochem. 121, 463467. Kay M. M. B. (1975) Mechanism of removal of senescent cells by human macrophages in situ. Proc. natn. Acad. Sci. U.‘.A. 72, 3521-3525. Kay 1~1. M. B. (1978) Role of physiologic autoantibody in the removal of senescent human red cells. J. supramolec. Strut?. 9, 555-567. Kay M. M. B. (1981) Isolation of the phagocytosis-inducing Ig-G binding antigen on senescent somatic cells. Nature 289, 49 l-494. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 221, 680-685. Lutz H. U. (1981) Elimination alter Erythrozyten aus der Zirkulation: Freilegung eines zellalter-spezifischen Antigens auf altemden Erythrozyten. Schweiz. med. Wschr. 111, 150771517. Lux S. E. and John K. M. (1977) Isolation and partial characterization of a high molecular weight red cell membrane protein complex naturally removed by the spleen. Blood 50, 625-641. Marchesi V. T. and Palade J. E. (1967) The localization of Mg-Na-K-activated adenosin tryphosphatase on red cell ghost membranes. J. Cell Biol. 35, 385-404. Morrow J. S. and Marchesi V. T. (1981) Self-assembly of spectrin oligomers in vitro: a basis for a dynamic cytoskeleton. J. Cell Biol. 88, 463468. Palek J. and Liu S. C. (1979) Dependence of spectrin organization in red blood cell membranes on cell metabol;m: implication for control of red cell shape, deformabilitv. and surface area. Semin. Haematol. 16, 75-93. Pallavicinj G., Brovelli A., Toma S. and Balduini C. (198 I) Structural investigations on sialoglycopeptide fragments released from ‘in vitro’ aged human erythrocytes. Xx1.X’ Annual CoNoquium on Protides in Biological Fluids, Poster 7.10, Brussels. Ravenbuehler P. B., Cordes K. A. and Salhany J. M. (1982) Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane, Biochim. biophys. Acta 692, 361-370. Rosenberg S. A. and Guidotti G. (1968) The protein of human erythrocyte membranes. Preparation, solubilization and partial characterization. J. biol. Chem. 243, 1985-1992. Seaman G. V. F. (1975) Electrokinetic behaviour of red cells. In The Red Blood Ce// (Edited by Surgenor D. MacN.) 2nd edn, Vol. II, pp. 113551229, Academic Press, New York. Svennerholm L. (1958) Quantitative estimation of sialic acid III. An anion exchange resin method. Acta them. stand. 12, 547-554.
Copyright
0
0020-711X/84$3.00+0.00 1984 Pergamon Press Ltd
MODIFICATION OF MEMBRANE PROTEIN ORGANIZATION DURING 1lV VITRO AGING OF HUMAN ERYTHROCYTES A. BROVELLI, C. SEPPI and Department
of Biochemistry,
University
of Pavia,
(Received 8 November Abstract-l. In in vitro aged membrane; these clusters are nearly completely dissociated 2. SDS-polyacrylamide gel are made of peptide fragments fragments correspond to an 3. Their formation results proteins.
Italy [Tel. (0382)33326]
1983)
human erythrocytes, the presence of protein clusters can be found on the made up of peptides held together by disulfide bridges, since they can be by dithiothreitol treatment. electrophoresis after dithiothreitol dissociation indicates that the aggregates with a molecular weight ranging from 20 to - 110 kdalton; none of these intact protein component of the membrane. from oxidation and proteolysis of membrane, and perhaps cytoplasmic
tively blood was defibrinated on glass pellets. Erythrocytes were collected by centrifugation at 1OOOg after repeated washes in 0.9% (w/v) NaCl. White cell contamination was minimized by sedimenting red cells on an isotonic Percoll (Pharmacia) suspension. Packed erythrocytes were suspended in one volume of Krebs-Ringer phosphate buffer pH 7.4, and incubated for different times under previously reported conditions (Brovelli et al., 1977). Dialysis tubing was boiled in ethylendiaminotetracetic acid (EDTA) at pH 8-9 and than rinsed in distilled water before use. Ghost membranes were prepared according to Marchesi and Palade (1967), using 0.2 mM phenylmethylsulfonylfluoride and 1 mM EDTA as protease inhibitors.
INTRODUCTION
A mature circulating erythrocyte undergoes many metabolic modifications during its life-span (Brewer, 1974) as a consequence of a decrease of several enzyme activities; significant changes in the surface properties correspond to this modified functional situation, as suggested by electrokinetic (Seaman, 1975) and lectin agglutination studies (Baxter and Beeley, 1975) and by chemical (Balduini et al., 1974; Bladier et al. 1980) and immunological investigations (Kay, 1975, 1978, 1981; Lutz, 1981). These modifications seem to involve not only the structure of membrane glycoproteins but also the topographic organization of proteins; several investigations indicate that metabolic impairment can produce the aggregation of some membrane proteins in high molecular weight complexes (for a review see Palek and Liu, 1979). Recently, we have investigated the relationships between metabolic events and membrane structure by means of in vitro aging experiments; we mimicked the aging through the starvation of cells of glucose and ATP and GSH precursors (Brovelli ef al., 1977) and we observed two phenomena modifying the membrane structure: (1) a breakdown of glycoproteins with a loss of sialopeptides mainly from glycophorins, (2) clustering in high molecular weight complexes of some membrane proteins (Balduini et al., 1979a). These phenomena seem to be controlled by the redox and energy capacities of the cell and seem to be a consequence of events occurring at the cytoplasmic surface of the membrane (Brovelli et al., 1982). In order to shed light on the mechanisms producing the membrane processes observed during the in vitro aging, the properties and the composition of the protein aggregates were investigated. MATERIALS
C. BALDUINI 27100 Pavia,
AND METHODS
In vitro aging of intact celfs Human blood was drawn from the veins of donors by using 3.8% sodium citrate (w/v) as anticoagulant. Alterna-
Geljltration
on Sepharose 2B and 48 and Ultrogel AcA34
Isolation of high molecular weight aggregates was obtained by gel filtration of 4% SDS dissociated erythrocyte membranes on Sepharose 2B (Lux and John, 1977), Sepharose 4B and Ultrogel AcA 34 columns (1.5 x 50 cm) equilibrated with 0.01 G Tris-HCl buffer, pH 7.8, contai&ng 1% SDS (w/v) and 0.025Y (w/v) NaN,. Elution was carried out with ihe same buffe; iflow ‘rate Sml/hr) and monitored at 280 nm with an ISCO U-A4 absorbance monitor. (a) Filtration on Sepharose 28 of solubilized membranes. An excluded peak (V,, - 2B) and a non-homogenous retarded peak are recovered. This second peak is composed of a “shoulder” in which protein aggregates are present and by more retarded components corresponding to nonaggregated membrane proteins (Fig. 1). (b) Filtration on Sepharose-4B. The “shoulder” material after filtration on Sepharose-4B is resolved in an excluded peak (V,, - 4B) and in a retarded peak. (c) Filtration on Ultrogel-AcA 34. Sepharose 4B retarded peak is resolved in an excluded peak (V,-AcA34) and in non-aggregated membrane components. The highest molecular weight aggregates ( VO- 2B), after refiltration on Sepharose 2B, were characterized in their amino acid composition and after dissociation with dithiothreitol (DTT), by SDS-polyacrylamide gel electrophoresis. Protein composition of VO- 4B and VO- AcA34 were determined by SDS-polyacrylamide gel electrophoresis after DTT dissociation. SDS-polyacrylamide
gel electrophoresis
SDS-polyacrylamide gel electrophoresis was carried out under the Laemmli (1970) conditions. Samples were dissociated for 40 min at 37°C or for 5 min at 100°C in 10 mM Tris-HCl buffer, pH 7.4, containing 40mM DTT, 1 mM 1115
A. BROVELLI et
1116
al
Characterization qf the complex eluted in the V, Sepharose 2B gel filtration (V, 2B) (a) Amino weight
Number
of
fractmns
Fig. 1. Gel filtration on Sepharose 2B of SDS-dissociated ghost membranes prepared from in vifro aged red cells. Protein clusters present on the membrane are eluted in the V0 and in the shoulder.
EDTA, 1% (w/v) SDS, 7% (w/v) saccharose and 0.01% (w/v) pyronin as tracking dye. Electrophoretic separation was done on polyacrylamide gel slabs (acrylamide concentration was 3% in the stacking gel and 8% (w/v) in the running gel: 14 x 13.5 cm, 1.5 mm thickness) with running times of about 5 hr at 40mA, without cooling of the plate. Protein contents of each sample were between 80 and 1OOpg. Molecular weight calibration was done using as standard two mixtures (Pharmacia), respectively, composed of (a) thyroglobulin, ferritin, catalase, lactate dehydrogenase, albumin and (b) phosphorylase b, albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and cc-lactalbumin, dissolved in the same buffer used for samples.
Analytical methods Total sialic acid content of ghost membranes was determined by the Svennerholm (1958) method after hydrolysis of ghosts for 1hr at 80°C in 0.05 M H,SO,, as previously reported (Brovelli et al., 1977). Protein was determined by the method of Lowry et a!. (1951), using serum albumin as a standard. Aminoacids and hexosamines, after hydrolysis of the sample, respectively, in 6N HCl (24 hr) and 4N HCI (7 hr) at lOYC, were determined by ion-exchange chromatography using the LKB 4101 Aminoacid analyzer.
acid composition.
aggregates
Membrane bation
protein organization
during in vitro incu-
Human phosphate
erythrocytes incubated buffer, pH 7.4, release
in
Krebs-Ringer
in the incubation medium glycopeptide fragments, as previously described; this phenomenon starts after 69 hr of incubation, when a consistent decrease of ATP and GSH occurs in the cell (Balduini et al., 1979b). Then, after 14-16 hours of incubation, the supramolecular arrangement of some membrane constituents begins to be modified and a macromolecular aggregate (mol. wt 140. IO3 kdalton) can be detected and isolated by Sepharose 2B gel filtration (Fig. 1). The formation of protein clusters and the glycopeptide release for different incubation times are reported in Table 1. Whatever the incubation time, smaller aggregates of proteins are also present on the membrane: these clusters can be detected as a shoulder eluted from the column of Sepharose 2B just before the peak containing all the membrane proteins (Fig. 1).
The high molecular the void volume of
in
Sepharose 2B gel-filtration have been characterized for their amino acid content. The results, reported in Table 2A, show that these protein clusters are different from the high molecular weight aggregates described by Lux and John (1977) in erythrocytes from splenectomized subjects and by Carraway et al. (1975) after Ca 2+ loading of ghost membranes. Moreover their amino acid content shows that these aggregates are not produced by unspecific clustering of all membrane proteins (the amino acid composition of whole membrane is reported in Table 2B). The clusters can be nearly completely dissociated by 40 mM dithiothreitol. (b) SDS-pol.vacrylamide-gel electrophoresis. In Fig. 2 the normal membrane protein pattern and the corresponding molecular weight obtained in our experimental conditions are reported. In order to study the protein composition of the high molecular weight aggregates, the protein material eluted in the void volume of Sepharose 2B gel filtration was collected,
Table
I. Effect of in t,irro incubation on protein sialopeptide release
Incubation time (hr)
_
0 9-12 14-16 24-30
Protein clustered (‘I”) 0.8 0.8 2.5 16.4
+ * i i
clustering
and
Sialopeptide release (4:, sialic acid)
0.5 0.5 1.3 4.3
IO 20 3&60
Values are mea” + SD of 8 experiments
Table 2. Amino acid composltion
of high molecular weight protein clusters (V,, - 2B) and ghost membranes
Ammo acid RESULTS
eluted
qf
Cysteic acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Methionine sulfoxide Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Areinine Triptopha” Glucosamine Galactosamine Galactose’ Glucose’
(A)
(B)
Protanclusters
Membrane proteins’
(C;, - 2B)” (~mol,‘100~mol) 1.08 k 0.24 8.20+0.17 5.17 kO.34 10.79 C_2.62 13.60 _+ 1.85 5.55 + 2.01 15.01 + 4.86 8.23 f 0.91 0.10t0.17 4.92 IO.60 0.15i0.13 0.68 k 0.61 3.05 k 0.32 6.92 ? 0.3 I I .08 i 0.93 4.94 ? 3.89 4.46 k 0.46 2.28 k 0.51 3.78 k 0.64 ND Traces TlXes Present Present
8.2-9.4 5.7-5.9 6.3-8.3 12.1~12.9 4.3-5.8 6&7. I 7.3-8.2 0.9-I .4 5.8-7. I L&2.4 4.4-5.3 I I .3-12.5 I .8-2.5 3.fF4.5 4.8-5.2 2.3-2.4 4.5-4 8 0.k2.5
“Values are mean f SD of 3 preparations. “Data from Bakerman and Wasemlller and from Rosenberg Guidotti. ‘Qualitatively determined by GLC.
and
MoLwt
-
(kd)
2
240.17 I 3.90 223.33 + 4.72
__192.4347.9z3
$0
l3ond
Mol.
wt
(kdl
Mol.wt (kd)
Protein
clusters
dialyzed, liophylized and then refiltered on a Sepharose 2B column. The protein complex eluted in the void volume was submitted to SDS-polyacrylamidegel electrophoresis under the Laemmli (1970) conditions, in the presence of dithiothreitol. Dithiothreitol dissociated the complex in protein fragments with a molecular weight ranging from 110 to 22 kdalton; none of these peptides correspond to a typical membrane component, identified on the basis of the molecular weight (Fig. 3A). Characterization of the Sepharose 2B gel jiltration
aggregates
retarded
by
Protein aggregates smaller than the clusters eluted in the V0 of Sepharose 2B gel filtration are retarded by this column and eluted in the shoulder (see the elution profile in Fig. 1). This material was seived on a Sepharose 4B and Ultrogel AcA34 column and the protein clusters excluded by Sepharose 4B (mol. wt 2 5.10’ kdalton) and excluded by Ultrogel AcA34 (mol. wt 2 700 kdalton) were submitted to SDSpolyacrylamide-gel electrophoresis in the Laemmli (1970) conditions, in the presence of dithiothreitol. The aggregates excluded by Sepharose 4B contain spectrin chains, linked by disulphide bridges. In some preparations ankyrin is also present (Fig. 3B). The aggregates excluded by Ultrogel AcA34 are composed of protein fragments with a molecular weight ranging between 60 and 75 kdalton (Fig. 3C).
DISCUSSION
During in vitro aging, human erythrocytes undergo a consistent modification of membrane protein and glycoprotein structure. In particular, in previous papers, the fragmentation of glycophorins has been characterized in detail (Brovelli et al., 1977; Pallavicini et al., 1981). This breakdown seems to be the expression of a more general mechanism of proteolysis at the membrane level, as proved by the evidence that clusters made up by protein fragments appear on the membrane during in vitro incubation. Two types of aggregates were isolated from the membrane of in vitro aged cells. The biggest ones (mol. wt. 2 40. 103kdalton), eluted in the void volume of Sepharose 2B gel filtration, are made up by peptide fragments held together by disulfide bridges. The dissociation of this aggregate by dithiothreitol and the characterization of its peptide components by polyacrylamide gel electrophoresis evidentiates the presence of fragments with a molecular weight ranging between 22 and 110 kdalton; none of these bands corresponds to an intact protein component of the membrane. The amount of these aggregates increases with the incubation time (Table 1) and can be controlled by the presence of ATP or reduced glutathione (Brovelli et al., 1982); on the contrary the amount of smaller aggregates present on the erythrocyte membrane (V, - 4B, V0 - AcA34) is independent on the incubation time and does not seem therefore to be influenced by the presence of ATP or GSH. The clusters excluded by Sepharose 4B are mainly composed of spectrin (band 1 and band 2). Sometimes
1119
in aged red cells
ankyrin is recovered in small amount in these aggregates. 40mM DTT can nearly completely dissociate these clusters. Disulfide bridges therefore hold the spectrin chains together, even if the presence of GSH in physiological concentration does not modify their appearance on the membrane. These aggregates are different from those described by Morrow and Marchesi (1981) which are composed of up to 11 dimers of spectrin, but not linked via disulfide bridges. The mol.wt 2 700 kdalton aggregates of ( V0 - AcA34) are present in a very small amount and their characterization by polyacrylamide gel electrophoresis indicates the presence of peptides with a molecular weight ranging between 60 and 75 kdalton. Some authors (Golovtchenko-Matsumoto et al. 1982) suggest the presence in this region of band 3 degradation products. In our conditions this material is held together by disulfide bridges, being completely dissociated by 40mM DTT. The V,, - 4B and V,, - AcA34 aggregates do not appear to be produced by in vitro aging, but by the conditions in which membranes are prepared. Only the V,, - 2B aggregates seem to be produced by in vitro aging conditions and their properties suggest that the proteolysis could act on proteins previously oxidized in their sulphydryl groups. It seems, therefore, that the two events occurring on the membrane during in vitro aging (proteolysis and oxidation) are related, since only oxidized fragments are present in the aggregates. As for the origin of the proteins present as oxidized fragments in the V, - 2B aggregates after incubation of intact cells (Brovelli et al., 1982) it cannot be excluded that some cytoplasmic component is present in the clusters, even if they were isolated after membrane preparation. It was in fact reported that hemoglobin is able to link band 3, glycophorin (Ravenbuehler et al., 1982) and spectrin (Alloisio et al., 1982) so that it can be easily hypothesized that these binding capacities can also concern some degradation product of hemoglobin or of other cytoplasmic components. Therefore our evidence seems to suggest that the metabolic impairment occurring during aging or in pathological conditions could damage membrane components by oxidative and proteolytic processes, so determining a modification of the structural organization of the cell surface. SUMMARY
During in vitro aging of red cells, a clustering of proteins occurs at the level of the membrane. This process can be found by gel filtration on Sepharose 2B of SDS-dissociated ghost membranes prepared from the cells aged in vitro. Clusters are eluted in the void volume (mol. wt 2 40. lo3 kdalton) and just before the retarded proteins. All clusters are made up of peptides linked through disulfide bridges. Only the aggregates eluted in the void volume from Sepharose 2B are specific for in vitro aging. Their appearance is detectable on the membrane after 14 hr of incubation of red cells and can be inhibited by physiological concentrations of ATP and GSH. SDSpolyacrylamide-gel electrophoresis after dith-
A. BROVELLI et al.
1120
iothreitol dissociation indicates that these clusters are made up of peptides with a molecular weight ranging from 22 to 110 kdalton. The clusters could result from oxidative and proteolytic damages produced by in vitro aging on membrane, and perhaps cytoplasmic, proteins.
Acknowledgements-Work supported by Minister0 Pubblica Istruzione and by Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie ereditarie of the C.N.R., Italy.
REFERENCES Alloisio N., Michelon D., Bannier E., Revel A., Beuzard Y. and Delaunav J. (1982) Alterations of red cell membrane proteins and hemoglobin under natural and experimental oxidant stress. Biochim. bioDhys. Acta 691, 3OG308. Bakerman S. and Wasemiller G. (1967) Studies on structural units of human erythrocyte membrane. I. Separation, isolation, and partial characterization. Biochemistry 6, 1100-1113. Balduini C., Balduini C. L. and Ascari E. (1974) Membrane glycopeptides from old and young human erythrocytes. Biochem. J. 140, 557-560. Balduini C., Brovelli A. and Pallavicini G. (1979a) In vitro aging - - of young and old human erythrocytes. In Glycoconjugates: Proceedings of the Vth International SymDosium (Edited bv Schauer R. et al.), __ PD. 478479. Thieme, StuttgartBalduini C., Brovelli A., Balduini C. L. and Sinigaglia F. (1979b) Extra bone-marrow remodeling of membrane glycoproteins during erythrocyte life-span. Bull. molec. Biol. Med. 4, 47-58. Baxter A. and Beeley J. G. (1975) Changes in surface carbohydrate of human erythrocytes aged in uivo. Biothem. Sot. Trans. 3, 134136. Bladier D., Gattegno L., Fabia F., Perret G. and Cornillot P. (1980) Individual variations of the seven carbohydrate components of human erythrocyte membrane during aging in vivo. Carbohy. Res. 83, 371-376. Brewer G. J. (1974) Red cell metabolism and function. In The Red Blood Cell (Edited by Surgenor D. MacN.) 2nd edn. Vol. I. on. 474508. Academic Press, New York. Brovehi A., Suhail M., Pallavicini G., Sinigagha F. and Balduini C. (1977) Self-digestion of human erythrocyte membranes. Role of ATP and GSH. Biochem. J. 164, 469472. Brovelli A.. Pallavicini G.. Airoldi G. and Balduini C. (1982) Supramolecular assembly of membrane constituents durine in vitro seine of human ervthrocvtes. In Protides ofyhe Biologica: Fiids (Edited by Peters H.) Vol. 29, pp. 337-340 Pergamon Press, Oxford.
Carraway K. L., Triplett R. B. and Anderson D. R. (1975) Calcium-promoted aggregation of erythrocyte membrane proteins. Biochim. biophys. Acta 379, 571-581. Golovtchenko-Matsumoto A. M., Matsumoto I. and Osawa T. (1982) Degradation of band-3 glycoprotein in vitro by a protease isolated from human erythrocyte membranes. Eur. J. Biochem. 121, 463467. Kay M. M. B. (1975) Mechanism of removal of senescent cells by human macrophages in situ. Proc. natn. Acad. Sci. U.‘.A. 72, 3521-3525. Kay 1~1. M. B. (1978) Role of physiologic autoantibody in the removal of senescent human red cells. J. supramolec. Strut?. 9, 555-567. Kay M. M. B. (1981) Isolation of the phagocytosis-inducing Ig-G binding antigen on senescent somatic cells. Nature 289, 49 l-494. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 221, 680-685. Lutz H. U. (1981) Elimination alter Erythrozyten aus der Zirkulation: Freilegung eines zellalter-spezifischen Antigens auf altemden Erythrozyten. Schweiz. med. Wschr. 111, 150771517. Lux S. E. and John K. M. (1977) Isolation and partial characterization of a high molecular weight red cell membrane protein complex naturally removed by the spleen. Blood 50, 625-641. Marchesi V. T. and Palade J. E. (1967) The localization of Mg-Na-K-activated adenosin tryphosphatase on red cell ghost membranes. J. Cell Biol. 35, 385-404. Morrow J. S. and Marchesi V. T. (1981) Self-assembly of spectrin oligomers in vitro: a basis for a dynamic cytoskeleton. J. Cell Biol. 88, 463468. Palek J. and Liu S. C. (1979) Dependence of spectrin organization in red blood cell membranes on cell metabol;m: implication for control of red cell shape, deformabilitv. and surface area. Semin. Haematol. 16, 75-93. Pallavicinj G., Brovelli A., Toma S. and Balduini C. (198 I) Structural investigations on sialoglycopeptide fragments released from ‘in vitro’ aged human erythrocytes. Xx1.X’ Annual CoNoquium on Protides in Biological Fluids, Poster 7.10, Brussels. Ravenbuehler P. B., Cordes K. A. and Salhany J. M. (1982) Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane, Biochim. biophys. Acta 692, 361-370. Rosenberg S. A. and Guidotti G. (1968) The protein of human erythrocyte membranes. Preparation, solubilization and partial characterization. J. biol. Chem. 243, 1985-1992. Seaman G. V. F. (1975) Electrokinetic behaviour of red cells. In The Red Blood Ce// (Edited by Surgenor D. MacN.) 2nd edn, Vol. II, pp. 113551229, Academic Press, New York. Svennerholm L. (1958) Quantitative estimation of sialic acid III. An anion exchange resin method. Acta them. stand. 12, 547-554.