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Isolation, Characterization, and Immunoprecipitation Studies of Immune Complexes From Membranes of f3-Thalassemic Erythrocytes By Jie Yuan, Rama Kannan, Eilat Shinar, Eliezer A. Rachmilewitz, and Philip S.Low p-Thalassemia, a hemoglobinopathythat results in the precipitation of denatured *globin chains on the membrane, is characterized by erythrocytes with significantly reduced lifespans. We have demonstrated previously that hemoglobin denaturation on the membrane can promote clustering of integral membrane proteins, and that this clustering in turn leads t o autologous antibody binding, complement fixation, and rapid removal of the cell by macrophages. To evaluate whether this pathway also occurs in pthalassemic cells, we have isolated and characterizedthe immune complexes from the membranes of these cells. We observe that autologous IgG-containingcomplexes obtained by either immunoprecipitation or simple centrifugation of nondenaturing detergent extracts of pthalassemic cell membranes contain globin, band 3,IgG, and complement as major components. Absorp- tion spectra of these complexes demonstrate that the globin is, indeed, mainly in the form of hemichromes. Immunoblotting studies further show that much of the band 3 protein in the aggregates is covalently cross-linked t o a dimeric or tetrameric form, consistentwith the preferenceof the autologous IgG for clustered band 3. Although the insoluble aggregates constitute only -1.6% of the total membrane protein, they still contain 27% of the total IgG and 35% of the total complement C3 on the thalassemic cell surface. Because cell surface IgG and complement component C3 are thought t o trigger removal of erythrocytes from circulation, the hemichrome-induced clustering of band 3 may contribute t o the p-thalassemiccell’s shortened lifespan. o 1992by The American Society of Hematology. A MATERIALS AND METHODS LTHOUGH P-thalassemic erythrocytes are known to have shorter lifespans than normal the molecThalassemic erythrocyteswere obtained from untransfused spleular basis of their accelerated clearance has never been nectomized and nonsplenectomized patients with @-thalassemia intermedia from Kurdish Jewish and Arabic extraction. The determined. However, in the process of investigating noridentification of their genetic mutations has been reported elsemal and sickle cell clearance, we have formulated a hypothwhere.16These samples as well as control erythrocytesfrom healthy esis that explains how the lifespan of a cell can be donors were transported on ice for 1 to 2 days before use. determined by the integrity of its hemoglobinT6 Because IODO-BEADS and protein A beads were obtained from Pierce thalassemic cells are characterized by the early precipitaChemical Co (Rockford, IL) and Na’=I/NaOH was purchased tion of denatured globin chains on the membrane as well as from Amersham Corp (Arlington Heights, IL). Octaethylene glycol in the c y t ~ p l a s m ,we ~ - ~felt the premature demise of the mono-n-dodecylether (C12E8) was purchased from Nikko Chemithalassemic cell might arise in part from the mechanism we cal, Ltd (Tokyo, Japan) and nitrocellulose membranes were from have proposed. Schieicher and Schuell (Keene, NH). Antihuman C3c goat IgG Briefly, the clearance mechanism hypothesizes that shortly (“cyto grade,” affinity purified, polyclonal) was obtained from The Binding Site (San Diego, CA) and affinity-purifiedantihuman IgG before an erythrocyte’s removal by macrophages, hemoglowas from Miles Biochemical (Elkhart, IN). Polyclonalantibodies to bin begins to denature within the cell’s cytoplasm.6Because human hemoglobin and erythrocyte membrane proteins were the resultant hemichromes exhibit a high affinity for the cytoplasmic domain of the membrane protein band 3,loJ1 raised in rabbits and affinity purified against their respective antigens. All other reagents were purchased from Sigma (St Louis, pulling the associated copies of the anion transporter MO), Bio-Rad (Richmond, CA), or Boehringer Mannheim (Indiatogether into localized clusters in the membrane,3-6J1J2 napolis, IN) and were the highest purity available. hemoglobin denaturation, in effect, induces microscopic Isolation of hemichrome-rich membrane protein aggregates from changes in the external topography of the ce11.12 These Pthalassemic erythrocytes. Erythrocyte membrane proteins were microscopic clusters, which commonly contain less than 1% prepared from @-thalassemicand normal cells according to the procedure of Dodge et all7 by lysis at 4°C in 5 mmol/L sodium of the band 3 population of the ce11,5S6 are then rapidly 1mmol/L EDTA, pH 8.0 in the presence of phenylmeopsonized with autologous IgG and ~ o m p l e m e n t . ~ . ~ , ~ Jphosphate, ~J~ thylsulfonyl fluoride (PMSF) (20 p,g/mL final). The membranes Finally, the deposition of IgG and complement at one or more of these clustered sites triggers the recognition and removal of the cell by macrophages.13-15 Although substantial evidence exists for the participation From the Department of Chemishy, Purdue Universiiy, West of the proposed clearance mechanism in sickle cells and Lafayette, IN; and the Department of Hematology, Hadassah Medical senescent normal ~ e l l s , 3 - ~ ,the ” ~ ~involvement of hemiCenter, Jerusalem, Israel. Submitted October 31, 1991; accepted January 31, 1992. chrome-induced integral protein aggregation in thalassemic Supported by National Institutes of Health Grant GM24417. cell removal has never been evaluated. In this study, we Address reprint requests to Philip S. Low, PhD, Department of address this question by investigating the occurrence and Chemishy, Purdue Universiv, West Lafayette, IN 47907. composition of hemichrome-rich membrane protein aggreThe publication costs of this article were defrayed in part by page gates in P-thalassemic cells. We report that such aggregates charge payment. This article must therefore be hereby marked do exist, and that they contain elevated amounts of band 3, “advertisement” in accordance with 18 U.S.C. section I734 solely to autologous IgG, and complement. We suggest that these indicate this fact. membrane protein clusters may contribute to the premaQ 1992 by The American Socieiy of Hematology. ture clearance of P-thalassemic cells from circulation. 0006-4971/92/7911-0013$3.00/0 Blood, Vol79, No 11 (June 1). 1992: pp 3007-3013 3007 YUAN ET AL 3008 were subsequentlydepleted of spectrin and actin and converted to inside-out vesicles (IOVs) by incubation at 37°C for 30 minutes in 40vol of 0.3 mmol/L EDTA, 0.2 mmol/L dithiothreitol, 20 kg/mL PMSF, pH 8.0.l8 The resulting vesicles were collected by centrifugation at 17,ooOg. The protein content of the various IOV fractions was then determined by the BCA protein assay bicinchoninic acid (Pierce), and the hemoglobin content of the same vesicles was assayed by measuring the absorbance at 541 nm (Si: = 8.63).19 Equal amounts of IOVs, corrected for hemoglobin content, were then stirred for 20 minutes on ice in 5 vol of 5 mmol/L sodium phosphate, pH 8.0, containing 1% CIZE~. The insoluble pellet, if present, was collected by centrifugation at 35,ooOg for 45 minutes in an SS-34 Sorvall rotor and washed three times in 5 mL of 5 mmol/L sodium phosphate, pH 8.0, to remove weakly bound protein. Quantitative analysis of autologous IgG on iWuh.wemic erythrocytes and their derived aggregates. p-Thalassemic cells were washed four times in phosphate-buffered saline (PBS), pH 7.4, to remove plasma and butQ coat, after which they were incubated at 50% hematocrit for 3 hours with 30 kg/mL of '4-labeled GAH-IgG (1.96 x 102 cpm/mg). Radioiodination of the antihuman IgG was performed with IODO-BEADS (Pierce) and NalZI.6 The unl bound antibody was removed by washing three times in PBS containing 1% bovine serum albumin, and spectrin-depleted IOVs were prepared as described above. The IOVs were then assayed for protein content and the quantity of tightly bound lBI-GAH-IgG was determined by counting gamma emission. Detergent insoluble aggregates were then prepared as described earlier and the lZI-GAH-IgG content in the derived aggregates was also determined by gamma counting. The quantity of lZI-GAH-IgG bound per ghost was calculated from the cpm/mg protein in the IOVs, assuming that 0.65 mg of vesicle protein was equivalent to 1 mg of ghost protein, as determined experimentally. The reason for not measuring the counts on the intact cells directly was that some loosely associated IgG (presumably nonspecifically bound) were found to dissociateduring ghost and IOV preparation. Because the nature of these easily eluted antibodies was unknown, we felt it would be wisest to assign them neither to the aggregate nor to nonaggregated regions of the membrane. Immunoprecioiation of immune complms. Erythrocyte ghosts (7.2 mg/mL) 0.4 mL, from p-thalassemic or normal cells were solubilized in 10 vol of PBS containing c&8 (1% final). The resulting detergent solution was incubated for 30 minutes at 0°C with 100 FL of agarose beads (Sigma) to pre-absorb any polypeptides that might bind nonspecifically to the beads. After removing the beads and other pelletable material by centrifugation, the supernatant was allowed to react overnight at 4°C with 100 FL of protein A-linked agarose beads. These beads were then collected by centrifugation ( 1 , w 3 minutes) and washed five times in PBS containing 0.1% C12E8. Immune complexes were eluted and prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by boiling for 5 minutes in 100 ~ L osample f buffer containing 4% SDS with or without 5% f3-mercaptoethanol. Analytical procedures. Aggregates were prepared for SDSPAGE by solubilizing in SDS electrophoresis buffer, sonicating to disrupt tightly associated proteins, and heating for 5 minutes in a boiling water bath. The polypeptide composition of the aggregates was then analyzed on a 6% to 12% polyacrylamide gradient gel according to the method of Laemmli?O For immunoblotting studies, proteins were transferred to nitrocellulose membranes" and blocked with 4% bovine serum albumin in blotting buffer, consisting of 20 mmol/L Tris, 500 mmol/L NaCI, pH 7.4. The resulting blots were washed with blotting buffer containing 0.05% Tween 20 and reacted with the desired antibody diluted in blotting buffer. After further washing and labeling with second antibody conju- gated to horseradish peroxidase, the immunoblotswere developed using 4-chloronaphthol as the substrate. RESULTS SDS-PAGE analysis of hemichrome-stabilized aggregates isolated from @thalassemic cells. To evaluate the possible existence of hemichrome-stabilized membrane protein aggregates in p-thalassemic cells, membranes were prepared and extracted according to the protocol described in Materials and Methods. As shown in Fig 1lanes a through d, the Coomassie blue staining pattern of total membrane proteins from thalassemic cells was not significantly different from the staining pattern of total proteins from normal ghosts. Except for a new band appearing at -68,000 daltons, which was previously noted by JSahane and Rachmilewitz,22 and an unusually large amount of retained globin, no major distinction was observed. Whether the new 68,000-dalton band is a breakdown product of a higher molecular weight component, an over-expressed minor isoform of band 4.1, or a protein absorbed from the serum or cytoplasm cannot be evaluated from the data. However, band 3 4.19 4.2/ ? 5 6 7. Hblr Fig 1. Gel electrophoresis of detergent insoluble macromolecular aggregates isolated from equal amounts of control and p-thalassemic erythrocytes. The total detergent insoluble material (aggregates) isolated from 12 mg of @thalassemic lOVs (0.29 mg) and normal (control) IOVs (not detectable) was suspended in 2 vol of SDS electrophoresis buffer containing 5 % 2-mercaptoethanol, sonicated for 5 seconds to disrupt tightly associated proteins, heated for 5 minutes in a boiling water bath, and loaded onto a 6% to 12% Laemmli gradient SDS-polyacrylamide gel. The lanes contain (a) p-thalassemic ghosts from a splenectomized patient (50 pg), (b) pthalassemic ghosts from a nonsplenectomized patient (50 pa), (c) normal erythrocyte ghosts (travel control) (50 pg), (d) normal erythrocytes ghosts from a local donor (50 pg), (e) lOVs from a p-thalassemic splenectomized patient (50 pg), (f) lOVs from a pthalassemic nonsplenectomized patient (50 pg), (9) lOVs from the normal travel control (50 pg), (h) lOVs from the normal local donor (50 pg), (i) total detergent-insoluble aggregate from the p-thalassemic splenectomized patient (50 pg), (j) total aggregate from the p-thalassemic nonsplenectomized patient (4 pg), (k) total aggregate from the travel control (not detectable), (I)total aggregate from the local control (not detectable). The unidentified -68,000-dalton protein is designated with a question mark. IMMUNE COMPLEXES FROM P-THALASSEMIC CELLS it should be pointed out that this band is not enriched in the protein aggregate derived from thalassemic cells. When membrane protein aggregates were isolated from equal concentrations (6.8 mg/mL) of thalassemic and normal (control) IOVs, vastly different yields were obtained. Whereas virtually no detergent insoluble protein could be collected from control IOVs, (Fig 1,lanes k and I), roughly 1.6% of the total membrane protein could be harvested from thalassemic 10% Further, under identical conditions, there was an approximately 10- to 30-fold greater yield of aggregates from erythrocytes of splenectomized thalassemia patients than from erythrocytes of nonsplenectomized patients (Fig 1, compare lanes i and j). Densitometric analysis of the Pthalassemic membrane aggregates. The contribution of individual polypeptides to the total protein content of the aggregates (Fig 1, lane i) derived from erythrocytes of splenectomized thalassemic individuals was determined by scanning densitometric analysis. For this purpose the lane containing intact ghost was first scanned to determine the position of each major membrane polypeptide in the gel. Then, because the aggregate polypeptides migrated rather diffusely, the corresponding region of the scan from the aggregates lane was integrated for each polypeptide and listed in Table 1. As predicted, globin monomer represents the major polypeptide in the aggregates, comprising 43% to 52% of the total protein. The other major polypeptides were in the band 3 region (5% to lo%), bands 1,2, and 2.1 region (2% to 5%), band 4.1 (1% to 3%), band 5 (3% to 5%), and band 7 (5%). Thus, as with sickle cells,11globin and band 3 were found to be prominent proteins of the detergent insoluble thalassemic cell aggregates. Immunoblotting analysis of the Pthalassemic membrane aggregates. Because of the diffuse nature of the gel staining, we decided to rely on immunologic methods for identificationof the proteins in the aggregates. Immunoblotting analysis with affinity-purified polyclonal antibodies to Table 1. Relative Content of the Major Erythrocyte Membrane Polypeptides Isolated in the Detergent-Insoluble Aggregates Obtained From p-Thalassemic Erythrocytes Protein 1,212.1 Band 3, monomer Band 3, oligomer 4.1 4.2 5 6 7 Globin Unidentified polypeptides Protein in Aggregate (X) 2.1 5.7 3.2 1.6 1.6 3.4 1.9 4.7 44 32 Data were obtained by scanning densitometry using a Shimadzu CS9000 dual wavelength scanner of the Coomassie blue-stained gel in Fig 1, lane i. Because the aggregate polypeptides migrated diffusely in the polyacrylamide gel, the band area for each major membrane polypeptide was integrated over the banding region occupied by the same polypeptide in the gel of control ghosts. The location of the band 3 oligomer was determined by Western blotting, as in Fig 6c. lane 1. 3009 band 3 clearly demonstrated the presence of the anion transporter (Fig 2, lane C). Nonreducible cross-linked forms of band 3 were also seen in the same pellet at 190,000 daltons (band 3 dimer) and at the top of the gel (lane C). Normal cell ghosts stained for band 3 only at 100,000 daltons (monomer) and -62,OOO daltons (common proteolytic fragment; lane A). The control cell pellet (lane B) contained no detectable band 3. To evaluate whether autologous IgG and complement were also present at these sites of membrane protein reorganization, 40 pg of thalassemic cell aggregates were examined as above except they were immunoblotted with horseradish peroxidase conjugated to either antihuman IgG (Fig 3) or antihuman C3c (Fig 4) under nonreducing conditions. After staining with 4-chloronaphthol, the IgG (Fig 3, lane C) and complement (Fig 4, lane C) were easily detected in the aggregates from thalassemic cells but not in pelletable material from normal erythrocyte IOVs (lanes B of Figs 3 and 4). Furthermore, IgG and complement were undetectable in 50 pg of whole thalassemic cell ghosts immunoblotted under identical conditions (not shown). This indicates that autologous IgG and complement are significantly concentrated in hemichrome-enriched aggregates of @-thalassemiccells. Quantitative analysis of cell surface autologous IgG and complement component C3 associated with &thalassemic cell aggregates. To obtain a more quantitative estimate of the degree of autologous IgG enrichment at sites of integral membrane protein clustering, @-thalassemiccells were labeled with *=I-antihuman IgG and the antibody content of the whole membranes and derived aggregates was determined. As shown in Table 2, an average of 27% of the total cell surface autologous IgG was found to co-isolate with the aggregates from splenectomized individuals. Because the aggregates constitute only 1.6% of the membrane protein, this corresponds to a relative enrichment of autologous IgG at the aggregated sites over the remainder of the thalassemic cell membrane of 23-fold (Table 2). This suggests that the sites of integral protein clustering in thalassemic cells are indeed a major locus of IgG binding. Similar analysis of the enrichment of complement component C3 at the same sites shows a 40-fold enhancement of complement in the detergent-insolublemembrane material (Table 3). Absorption spectrum of membrane-associated globin in Pthalassemic cells. To evaluate whether the membraneassociated globin in the thalassemic cells was native or denatured, an absorption spectrum of extensively washed membranes was obtained. For this purpose thalassemic and normal cell membranes were washed in cold lysis buffer until no hemoglobin could be detected in the supernatant, after which the membranes were immediately solubilized in 10 vol of PBS containing C12E8(2% final) and scanned in a UV-visible spectrophotometer. The spectrum of p-thalassemic cell membranes shown in Fig 5 was obtained using control membranes with no bound hemoglobin or hemichromes as the reference. When compared with standard spectra of hemoglobin and hemichromes under similar conditions,1°the spectrum indicates that the globin bound - - YUAN ET AL 3010 A FF- B ~iw@?J!p C L i F F i V X ePolymer eDimer A agarose beads and determined whether the other three proteins were also present. After extensive washing, the proteins bound to the beads were eluted in SDS,separated by SDS-PAGE, transferred to nitrocellulose, and visualized with antibodies against IgG, C ~ Cband , 3, and Hb,as shown in Fig 6. Importantly, the immunoprecipitated proteins stained positively for IgG (lane al), complement compo- A B C a Monomer ._ Fig 2. lmmunoblotting analysis of the presence of band 3 in the insoluble aggregates isolated from 2.0 mg of normal and 6-thalassemic IOVs. Nitrocellulose blots of samples separated by SDS-PAGE were incubated in affinity-purified polyclonal rabbit anti-band 3 for 3 hours followed by goat anti-rabbit IgG conjugated t o horseradish peroxidase (GAR-HRP)for 2 hours and then developed with 4-chloronaphtol. The lanes contain (A) normal red blood cell ghosts (40 pg), (6) detergent-insoluble material from normal cells (not detectable), (C) detergent-insoluble material from 6-thalassemic cell (40 pg). to thalassemic cell membranes is mainly in the form of hemichromes. This observation is consistent with our earlier finding that hemichromes, not hemoglobin, have a strong affinity for the cytoplasmic domain of band I"unopwc@itation Of complms from pthalassemic Cell membranes. The fact that IgG, complement, band 3, and globin are present in the same detergentinsoluble membrane pellet suggests, but does not prove, that the four components are physical1y associated in a To examine this possibili@, we associated material from the detergent extracts on protein 3.1071* Fig 3. lmmunoblotting analysis of the presence of IgG in the insoluble aggregates isolated from 2.0 m g of normal and 6-thalassemic cell IOVs. Detergent-insoluble pellets were dissolved in 2 vol of Laemmli electrophoresis buffer without 6-mercaptoethanol, heated for 5 minutes, separated on a 6% t o 12% polyacrylamide gel, and transferred t o nitrocellulose. The blots were then incubated in GAHIgG for 3 hours followed by rabbit antigoat IgG conjugated t o horseradish peroxidase for 2 hours. The lanes contain (A) pure IgG (3 pg), (6) the entire normal cell pellet (not detectable), (C) the 6-thalassemic cell aggregates (40 pg). 3011 IMMUNE COMPLEXES FROM p-THALASSEMIC CELLS A .=w-?&p B ?mvw C Table 2. Estimation of the Fraction of Autologous Cell Surface IgG Associated With @-ThalassemicCell Aggregates Obtained From Splenectomized Individuals m= .- Total Aggregate IgG Membrane GAH-IgGI Associated Concentratedin Protein in Enrichment Patient Cell* GAH-IgGICeIl Aggregate (%I Aggregate (%) Factort .i' is; ., 1 2 3 Average : $ -185 kd 561 832 678 690 157 191 203 184 28 23 30 27 1.8 1.3 1.6 1.6 21 23 26 23 *Represents the number of GAH-IgGIcell. If more than one GAH-IgG can opsonize a surface-bound IgG, then the total number of IgG will be fewer by this factor. tThe enrichment factor represents the fraction of IgG in the aggregate ),;(f normalized to the fraction of protein in the aggregate (ftroIein) divided by the fraction of IgG in the remainder of the membrane normalized to the fraction of protein in the remainder of the membrane (fzoIein), as described in the following equation: (ftG) Enrichment Factor = ;GIf $rotein :GIf ZoIein also observed, suggesting abnormal reactions may be occurring in these immune complexes. Taken together, it would appear that autologous IgG, band 3, complement, and globin are all present in the same complexes, and that similar complexes can be isolated by both simple centrifugation and immunoprecipitation. DISCUSSION Y Fig 4. lmmunoblotting analysis of the presence of complement in the insoluble aggregates isolated from 2.0 mg of normal and @-thalassemic cell IOVs. Total pelletable material collected from detergenttreated lOVs was dissolved in an equal volume of electrophoresis buffer without @-mercaptoethanol,heated for 5 minutes, separated on a 6% t o 12% polyacrylamide gel, and transferred t o a nitrocellulose membrane. The blots were then incubated in GAH-C3c for 3 hours followed by rabbit antigoat horseradish peroxidase for 2 hours. The lanes contain (A) serum (5 pL), (B) the entire normal cell pellet (not detectable), (C) the @-thalassemiccell aggregates (40 Fg). nent C3 (lane bl), band 3 (lane cl), and globin (lane dl). In contrast, collection of similar immune complexes from normal cell membranes yielded no stainable material (Fig 6, lanes 2). One significant finding in the band 3 blot (lane cl) was that the dimer and polymer were present in enriched amounts. This could infer that autologous IgG on thalassemic cells display a preference for clustered over dispersed band 3, as has been recently demonstrated for other types of erythrocytes.z In the hemoglobin blot (lane dl), many nonreducible cross-linked species of globin were Numerous alterations in the structure and function of thalassemic eqthrocytes have been described,' among them Shinar et alZ4have shown that 3% to 8% of the protein in isolated P-thalassemia cell membranes is tightly bound globin. It would now appear that at least some of this globin is associated with immune complexes containing autologous IgG, complement component C3, and band 3. If the mechanism documented in model studies3J0J1J3 and observed at various stages in sickle cells5J2 and the densest fraction of normal cells6is correct, we suggest the instability Table 3. Estimation of the Fraction of Cell Surface C3 Associated With @-ThalassemicCell Aggregates Aggregate Total Associated Membrane GAH-C3cl GAH-C3cl C3 Concentratedin Protein in Enrichment Sample Cell" (xlO3)Cell (xlO3) Aggregate (%) Aggregate (%I Factort 1 2 3 Average 11.6 10.9 9.52 10.7 4.42 3.67 3.06 3.72 38 34 32 35 1.1 1.2 2.3 1.5 55 42 23 40 *Represents the number of GAH-C3clcell. If more than one GAH-C3c can opsonize a surface-bound C3, then the total number of C3 will be fewer by this factor. tThe enrichment factor represents the fraction of C3 in the aggregate (fe3) normalized to the fraction of protein in the aggregate (f$roIein) divided by the fraction of C3 in the remainder of the membrane (f&) normalized to the fraction of protein in the remainder of the membrane (fZotein),as described in the following equation: Enrichment Factor = - fkJf$rotein f % :oleinIf YUAN ET AL 3012 I I 510 I I I .... ........ 590 Wavelength (nm) 550 630 I ] Fig 5. The absorption spectrum of solubilized 6-thalassemic cell membranes. Ghosts from normal and pthalassemic cells (7.2 mg/ mL), 0.4 mL, were washed in cold lysis buffer until no hemoglobin could be detected in the supernatant. At this point the membranes from control erythrocyteswere white, while the membranesfrom the 6-thalassemic cells were distinctly reddish-brown. The ghosts were solubilized in 10 vol of PBS containing C,*E8 (2% final) and their UV-visible absorption spectrum was obtained in an IBM UV-visible The instrument was 9420 spectrophotometer (Danbury, CT) (-). balanced for this spectrum using control white ghosts (free of hemoglobin or hemichromes) in the reference cell. For comparison, and 0.12 the standard spectra of 0.2 mg/mL hemichrome (---) mg/mL oxyhemoglobin (--)shown. of a-globin chains along with their ability to cluster band 3 strongly influence the cell's lifespan. Once the denatured globin chains collect a few copies of band 3 into an a g g r e g a t e ? ~ ~ J autologous @~~ antibodies and complement likely associate spontaneously and promote the cell's removal.l3 Lessin et alZ have directly observed the clustering of integral membrane proteins at sites of brilliant cresyl blue-induced hemichrome binding to the a-thalassemic cell 1 2 - membrane (hemoglobin H disease) by electron microscopy. We have isolated related clusters from P-thalassemic erythrocytes and found them to also contain complement and IgG. While membrane altered ion permeability,27,28 and exposure of cryptic p-galactosyl residues29 etc may all contribute to the cell's early demise, an additional participant in the removal process must be the opsonization of clustered integral membrane proteins. Although the hemichrome-rich membrane protein aggregates contain 23 times as much autologous IgG per mg membrane protein as other regions of the membrane (see Table 2), this enrichment factor is still well below the value measured for similar aggregates isolated from sickle cells5 and the densest (most rapidly phagocy~tosed,3~.~*) fraction of normal cells? Thus, corresponding autologous IgG enrichment factors for these cells were calculated to be 275-fold and 680-fold, respectively. Whereas integral membrane protein clusters likely represent the predominant opsonization site on sickle and normal cells, other regions of P-thalassemic cell membranes could be equally or more important in accumulating IgG. It is possible that other mechanisms yet to be explored are involved in causing damage to the p-thalassemic cell surface. It would be interesting to know what fraction of the immune complexes in p-thalassemic cell membranes are in fact devoid of clustered band 3. Finally, as with sickle and dense normal cells,5S6 the globin in the p-thalassemic cell aggregate was heavily cross-linked. In fact, in the absence of reducing agent, little protein in any of these three types of aggregates was found to enter an SDS polyacrylamide gel. Because electrophoretic analysis of the unfractionated p-thalassemic cell 1 2 c3 - 1 2 1 2 band 3- globinD a b C d Fig 6. lmmunoblottlng analysis of proteins collected by adsorption of detergent-insolublemembrane aggregates to protein A agarose beads. IEI (1%final). After Erythrocyte ghosts (7.2 mg/mL), 0.4 mL, from thalassemic and normal cells were solubilized in 10 vol of PBS containing C2 pre-absorptionwith unmodified agarose beads, IgG-containingcomplexes were collected on protein A-agarose beads and extensively washed (see Materials and Methods). The absorbed proteins were eluted from the protein A beads with SDS and then analyzed by SDS-PAGE and immunoblotting with the desired antibody. Antibodies used were specific for human IgG (a), complement component C3c (b), band 3 (c), and human hemoglobin (d). Lanes 1 contain proteins collected from solubilized pthalassemic cell membranes, while lanes 2 contain proteins collected by the same procedurefrom solubilized normal cell membranes. IMMUNE COMPLEXES FROM P-THALASSEMIC CELLS 3013 membranes displays a relatively normal polypeptide banding pattern even in the absence of reducing agent, it can be concluded that intense oxidative reactions are concentrated in the compact membrane clusters. Perhaps once a dense mat of hemichromes forms at a site on the membrane, enzymes responsible for protecting the cell against reactive oxygen species are excluded and oxidative reactions proceed unabated. REFERENCES 1. Vigi V, Volpato S, Galiurro D: The correlation between red cell survial and excess of a-globin synthesis in P-thalassemia. Br J Haematol56:25,1969 2. 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