T-cell specificity in murine autoimmune haemolytic anaemia induced by rat red blood cells.

Autoimmune haemolytic anaemia (AIHA) can be induced in mice by repeated injections with rat red blood cells (RBC). Here we describe the identification of rat and murine RBC antigens recognized by T-cells from mice with this disease. Splenic T-cells from mice with AIHA proliferated in response to multiple murine RBC membrane components, each of which is recognized by rat RBC induced autoantibodies. Thus, there were responses to murine autoantigen fractions that correspond in apparent molecular mass with the anion channel Band 3, with spectrin from the membrane skeleton and with the high and low molecular mass glycophorins, and the equivalent fractions from rat RBC also stimulated proliferation by T-cells. It was confirmed that purified Band 3 from murine and rat RBC also elicited responses. In contrast with the results in AIHA, T-cells from healthy control mice failed to respond to the antigens from either species, with the exception of proliferation induced by murine spectrin in one experiment and weak responses elicited by rat Band 3. It is suggested that T-cells activated by multiple cross-reactions between rat and murine RBC proteins, and by epitope spreading, are necessary to drive autoantibody production in this model of AIHA.

Recombinant forms of Gerbich blood group antigens: expression and purification.

Recombinant forms of normal glycophorin C (GPC), carrying the high frequency Gerbich blood group antigens, and its natural deletion mutants of Yus and Ge type (all combined with oligohistidyl tag) were expressed in CHO and COS 7 cells. The stable expression of all recombinant forms of GPC in CHO cells was obtained, but the level of expression was low and detectable only by flow cytometry. The high level of transient expression of GPC recombinant forms in COS 7 cells allowed their purification on Ni-NTA-agarose. The purified recombinant GPC and mutants of Yus and Ge type behaved in SDS-PAGE similarly to normal GPC forms from RBC membranes. The recombinant GPC.Yus and GPC.Ge mutants appeared as diffuse bands, suggesting the similar heterogeneity of glycosylation that was observed in natural GPC.Yus and GPC.Ge glycoproteins. The flow cytometry analysis of the transfected CHO and COS 7 cells showed that binding of anti-GPC monoclonal antibodies to GPC variants was accordant with the known fine specificity of these antibodies. The obtained recombinant forms of GPC carrying common Gerbich antigens may be useful in serology, and also as model molecules for structure-function studies.

High-yield synthesis and purification of an alpha-helical transmembrane domain.

Polypeptides corresponding to hydrophobic transmembrane alpha-helices, such as residues 69-101 of glycophorin A, are notoriously difficult to prepare in quantities sufficient for biophysical experiments. Simple synthetic and purification approaches reported here have been developed by combining a few modifications to standard procedures, without resorting to elevated temperatures, expensive activation strategies, or complex hydrophobic solvent mixtures. The cost of screening projects, preparing labeled peptides, and examining sequence variations is thereby significantly reduced. The quality of the peptide synthesized by this small-scale 9-fluorenylmethoxycarbonyl (Fmoc) strategy is comparable to that of the peptide synthesized by an experienced resource facility using a large-scale tert-butyloxycarbonyl strategy. Using reverse-phase HPLC, the desired peptide was separated from the primary side product (a Leu or Ile deletion) and quantitatively recovered at greater than 98% purity. Baseline resolution was achieved using a water:acetonitrile gradient to elute the peptides from a cyanopropyl column at ambient temperature. Combining these approaches readily yields 10 to 20 mg of pure transmembrane peptide from a small-scale Fmoc synthesis. The approaches are readily transferable to transmembrane sequences not previously synthesized and do not require setting up a specialized facility. The time and start-up expense required to launch new studies are thereby reduced expanding the range and detail with which questions in membrane protein biophysics can be explored.

Sialic acid-dependent binding of baculovirus-expressed recombinant antigens from Plasmodium falciparum EBA-175 to Glycophorin A.

The Plasmodium falciparum Erythrocyte Binding Antigen-175, EBA-175, is a soluble merozoite stage parasite protein which binds to glycophorin A surface receptors on human erythrocytes. We have expressed two conserved cysteine-rich regions, region II and region VI, of this protein as soluble His-tagged polypeptides in insect cell culture, and have tested their function in erythrocyte and glycophorin A binding assays. Recombinant region II polypeptides comprised of the F2 sub-domain or the entire region II (F1 and F2 sub-domains together) bound to erythrocytes and to purified glycophorin A in a manner similar to the binding of native P. falciparum EBA-175 to human red cells. Removal of sialic acid residues from the red cell surface totally abolished recombinant region II binding, while trypsin treatment of the erythrocyte surface reduced but did not eliminate recombinant region II binding. Synthetic peptides from three discontinuous regions of the F2 sub-domain of region II inhibited human erythrocyte cell binding and glycophorin A receptor recognition. Immune sera raised against EBA-175 recombinant proteins recognized native P. falciparum-derived EBA-175, and sera from malaria-immune adults recognized recombinant antigens attesting to both the antigenicity and immunogenicity of proteins. These results suggest that the functionally-active recombinant region II domain of EBA-175 may be an attractive candidate for inclusion in multi-component asexual blood stage vaccines.

New procedures for glycophorin A purification with high yield and high purity.

Glycophorin A (GPA) is the major glycoprotein of the human erythrocyte membrane. It is known to form, in SDS gels as well as in a membrane environment, homodimers, and also heterodimers with the homologous molecule Glycophorin B (GPB). It is shown in this report that the propensity of GPA to dimerize with GPB precludes satisfactory preparation with high yield of pure GPA using classical techniques including SEC and RPLC. It was demonstrated using multiple angle light scattering that GPA is eluted from RPLC columns as dimers. A convenient procedure was devised which allowed us to get pure GPA with high yield. This procedure consists of selectively blocking GPA-GPB heterodimer formation by selective modification of Cysteine 50 of GPB before RPLC.

Identification of blood group A and B antigens in human glycophorin.

Glycophorin A (GPA), the major sialoglycoprotein of human erythrocyte membranes, was isolated separately from blood group A and B erythrocytes using phenol-water extraction. After purification, performed as gel filtration in the presence of SDS, two glycophorin samples GPA-A and GPA-B were run, in duplicate, in SDS-PAGE and electroblotted onto Immobilon P. After staining with 1) anti-glycophorin antibody and 2) with relevant anti-blood group (A or B) antibody it was shown that the band pattern of the samples in each duplicate was the same. GPA-A and GPA-B samples were also degraded using Carlson degradation (beta-elimination in mild alkaline/strong reducing conditions) and from reaction products the fractions of O-glycans and N-glycans were isolated; they were used in hemagglutination inhibition test. It was shown that both sugar fractions derived from GPA-A did inhibit agglutination of blood group A erythrocytes by anti-A antibody, whereas oligosaccharide fractions derived from GPA-B inhibited agglutination of blood group B erythrocytes by anti-B antibody. These results, obtained using immunochemical methods, confirm the presence of blood group A and B determinants in the carbohydrate moiety of human glycophorin, derived from the blood group A or B erythrocytes, respectively.

Isolation and characterization of glycophorin from nucleated (chicken) erythrocytes.

A sialoglycoprotein fraction was isolated from chicken erythrocytes by two methods based on the phenol extraction or chloroform/2-propanol extraction of differently prepared erythrocyte membranes. Both preparations gave in SDS-PAGE two major PAS-stained bands (GP2 and GP3), which migrated as 60- and 33-kDa species, respectively, compared to reference proteins, or as 44- and 23-kDa molecules, compared to human glycophorins. Some less abundant slower migrating PAS-stained components, antigenically related to GP2 and GP3, also were detected. No evidence for the presence of antigenically distinct glycoproteins of leukosialin type was obtained. Interconversion in SDS-PAGE, similar carbohydrate composition, and similar antigenic properties of GP2 and GP3 indicated that they are a dimer and monomer, respectively, of the same glycoprotein which shows properties that allow it to be classified as a glycophorin. Lectin binding studies and methylation analysis of beta-elimination products of chicken glycophorin preparation showed the presence of O-glycans and N-glycans. The major O-glycans include sialylated Galbeta1-3GalNAc units and more complex GlcNAc-containing chains. Among the N-glycans, there are complex-type biantennary structures with a bisecting GlcNAc residue, accompanied by chains with additional antennas linked to alpha-mannose residues. A characteristic feature of the chicken glycophorin is a relatively high proportion of N-glycans to O-glycans, compared to the glycophorin A from human erythrocytes.

Glycophorin A in two patients with congenital dyserythropoietic anemia type I and type II is partly unglycosylated.

Glycophorins A from erythrocyte membranes of two patients with congenital dyserythropoietic anemia type I and type II (CDA type I and II) were analyzed for carbohydrate molar composition employing a modification of the recently published method that allowed simultaneous determination of carbohydrates and protein in electrophoretic bands of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Zdebska & Koscielak, 1999, Anal Biochem., 275, 171-179). The modification involved a preliminary extraction of erythrocyte membranes with aqueous phenol, subsequent electrophoresis and analysis of the extracted glycophorins rather than electrophoresis and analysis of the glycophorin from intact erythrocyte membranes. The results showed a large deficit of N-acetylgalactosamine, galactose, and sialic acid residues in glycophorin A from patients with CDA type I and type II amounting to about 45% and 55%, respectively. The results strongly suggest that glycophorin A in these patients is partly unglycosylated with respect to O-linked glycans. In addition, glycophorin A from erythrocytes of a patient with CDA II but not CDA I exhibited a significant deficit of mannose and N-acetylglucosamine suggesting that its N-glycosylation site was also partly unglycosylated.

Binding of bovine parvovirus to erythrocyte membrane sialylglycoproteins.

Bovine parvovirus (BPV), an autonomous parvovirus, haemagglutinates human type O erythrocytes and infects certain bovine cells in culture. Little is known about the receptor to which it attaches, either on nucleated host cells or on erythrocytes. Haemagglutination assays and radiolabelled virus-binding tests measuring the effects of trypsin, chymotrypsin, neuraminidase, phospholipase C and sodium periodate on attachment of BPV to receptors indicated that BPV interacted with N-acetylneuraminic acid-containing (sialyl) glycoproteins. SDS-polyacrylamide gel separation of erythrocyte ghost proteins and virus overlay protein-binding revealed BPV binding to glycophorin A. Confirmation testing showed BPV binding to purified glycophorin A on dot blots and on gels containing membrane glycophorin A and purified glycophorin A. Further, in competition assays, purified glycophorin A completely inhibited the BPV haemagglutination reaction. The results of this study indicate that BPV binds to sialated membrane glycoproteins, one of which is the major erythrocyte membrane glycoprotein, glycophorin A.

beta-Elimination of O-glycans from glycoproteins transferred to immobilon P membranes: method and some applications.

Selective beta-elimination of O-glycans from glycoproteins transferred from electrophoretic gels onto Immobilon P membranes is described. The experiments were performed with erythrocyte membrane proteins, in which glycophorins are the major poly-O-glycosylated components, and with lysates of human colon cancer cells CX-1.1. Lectins and monoclonal antibodies against peptidic, glycopeptidic, and carbohydrate epitopes were used to examine the effect of degradation. Experiments with erythrocyte membrane proteins showed that after heating the blots in 0.055 M NaOH for 16 h at 40 degrees C the O-glycans of glycophorins were undetectable, while N-glycans and peptidic epitopes of proteins were detected with unchanged or even increased intensity compared to untreated blots. The method was used to show that most protein-linked sialyl-Lea epitopes present on CX-1.1 cancer cells are located on O-glycosidic chains. Moreover, beta-elimination on the blots allows examination of the dependence of peptidic epitopes on O-glycosylation. This was shown using monoclonal antibodies specific for blood group M- or N-related epitopes of glycophorin A (GPA). Most of these antibodies recognize glycopeptidic epitopes dependent on O-glycosylation and, therefore, they did not detect GPA on NaOH-treated blots. Some less frequent anti-M antibodies cross-reacting with the rare GPA variant of Mg type are specific for a peptidic epitope which is unrelated to the MN blood group-specific amino acid sequence in unglycosylated peptides, but is recognized in GPA-M only in the glycosylated antigen. These antibodies, which showed specificity for GPA-M on untreated blots, detected GPA-M, GPA-N, and glycophorin B on NaOH-treated blots.

Interaction between complement proteins C5b-7 and erythrocyte membrane sialic acid.

The initial phase of membrane attack by complement is the interaction between C5b6, C7, and the cell membrane that leads to the insertion of C5b-7. Here we investigate the role of sialic acid residues in the assembly of C5b-7 intermediates on erythrocyte cell membranes. We find that C5b6 binds to glycophorin, whereas C5 or C6 does not bind, and desialylation of the glycophorin abolishes C5b6 binding. Complement lysis is inhibited by either masking glycophorin sialic acid with F(ab) fragments of an mAb, or by removal of the sialylated region of glycophorin by mild trypsinization. Gangliosides inhibit C5b-7 deposition when added to the aqueous phase. Asialogangliosides and synthetic gangliosides lacking the carboxylic acid residue have no inhibitory activity. We conclude that C5b6 binds to sialylated molecules on the erythrocyte surface. We propose a new model of membrane attack in which C5b6 initially binds to membranes via ionic forces. C7 then binds to C5b6, disrupting the ionic interaction and leading to the exposure of hydrophobic domains. Sialic acid is known to inhibit complement activation. Thus, these findings reveal a paradoxical role for sialic acid in complement attack; the presence of sialic acid inhibits the generation of C5b6, but once the membrane attack pathway is initiated, sialic acid enhances complement lysis.

Red blood cell glycophorins as B and T-cell antigens in canine autoimmune haemolytic anaemia.

Pathogenic autoantibodies from two dogs with autoimmune haemolytic anaemia (AIHA) were shown to react with glycophorin from the canine red blood cell (RBC) membrane. Autoantibodies in both cases bound to purified glycophorin in enzyme-linked immunosorbent assays (ELISAs), and the major autoantigen immunoprecipitated by the antibodies corresponded in apparent molecular mass with glycophorin. Furthermore, neuraminidase treatment of the precipitated antigen, or of canine glycophorin, resulted in identical changes in apparent molecular mass in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Such removal of sialic acid from glycophorins was demonstrated to cause shifts in SDS-PAGE migration that are unique among RBC membrane proteins. In two further cases of AIHA, where autoantibodies did not immunoprecipitate the glycophorin pattern, ELISAs revealed that RBC-reactive IgG was present in serum and RBC elutes, but that these antibodies failed to bind to canine glycophorin. Thus, we consider that autoantibodies specific for glycophorin are present in some, but not all, dogs with AIHA. T-cells from a case of AIHA proliferated in vitro in response to autologous RBC, or to multiple RBC membrane components fractionated by SDS-PAGE. Three fractions, corresponding to major glycophorins, to the RBC anion channel band 3, and to spectrin from the membrane skeleton, were stimulatory. In contrast, T-cells from healthy dogs failed to respond to RBC, or to any blot fractions with the exception, in one animal, of the fraction bearing spectrin. It is suggested that activation of autoreactive T-cells with multiple specificities may be necessary to provide sufficient help for pathogenic autoantibody production.

Identification of the membrane attachment sites for protein 4.1 in the human erythrocyte.

The nature of the membrane attachment site(s) for protein 4.1 in the human erythrocyte membrane has yet to be fully elucidated. In this paper we show that the major attachment site is glycophorin (GP) C/D, and that purified protein 4.1 can bind to two distinct sites on glycophorin C/D. One of these interactions is direct, involving residues 82-98 on glycophorin C (61-77 on glycophorin D), while the other interaction is mediated by p55. We have localized the binding site for p55 on glycophorin C to residues 112-128 (glycophorin D91-107). We also provide evidence that band 3 is an additional, minor, protein 4.1 binding site. The binding sites for band 3, glycophorin C/D, and p55 are all located within the 30-kDa domain of protein 4.1. We estimate that the relative utilization of the three sites in normal membranes comprises 40% to p55, 40% to GPC/D, and 20% to band 3. The same region of protein 4.1 binds GPC/D and band 3, while the p55 binding site is distinct. The interactions involving protein 4.1 with p55 and p55 with GPC/D are of high affinity (nM), while those involving GPC/D and band 3 are 100-fold lower (microM). These results suggest that the most significant interactions between protein 4.1 and the membrane are those involving p55.

Isolation and influenza virus receptor activity of glycophorins B, C and D from human erythrocyte membranes.

(1) Glycophorins (GPs) AM, AN, B, C and D were each isolated into a high state of purity from human erythrocyte membranes by a combination of lithium diiodosalicylate (LIS)-phenol extraction, gel-filtration with Bio-Gel A1.5m and HPLC with LiChrospher 1000 TMAE. (2) GPs-B, -C and -D reacted with influenza A and B viruses as well as GPs-AM and -AN and the order of reactivities against two viruses of the glycophorins was as follows: GP-B > GP-C > GP-AM = GP-AN >> GP-D for the former virus and GP-C > GP-B > GP-AM = GP-AN >> GP-D for the latter virus.

Differentiation of discolouration in a body by an erythrocyte membrane component, glycophorin A.

In a differential study to distinguish antemortem bruise from postmortem infiltration of haemoglobin on the skin, a component of erythrocyte membrane, glycophorin A was extracted from experimental bruise and haemoglobin infiltration lesions over set periods of time. This extraction was accomplished by utilising anti-glycophorin A serum, after which the difference between the two lesions was evaluated. The glycophorin A was recovered from the bruise lesions satisfactorily up to the ninth to twelfth days and showed good resistance to putrefaction. In contrast, no glycophorin A was detected in haemoglobin infiltration lesions taken at any time. Glycophorin A was also detectable in human vital bruises which were taken in autopsies of four hours to ten days postmortem. These results suggest that a differential diagnosis of antemortem bruise and postmortem haemoglobin infiltration is possible in advanced stages of death.

Immunochemical studies on the combining site of the A + N blood type specific Moluccella laevis lectin.

The specificity of the anti A+N lectin of Moluccella laevis (MLL) was examined by hemagglutination experiments with enzyme-modified human erythrocytes and by inhibition of hemagglutination. In addition, binding to various glycoproteins and inhibition by different sugars and glycoproteins were examined by enzyme immunoassay with antibodies to the lectin. Treatment of AMM erythrocytes with proteolytic enzymes increased their agglutinability by MLL 4-16-fold; similar treatment of ONN cells decreased their agglutinability 8-16-fold. This is in line with the known location and enzyme sensitivity of A and N specificity determinants. Treatment of the erythrocytes with sialidase increased their agglutinability and abolished the distinction between N and M cells. Hapten inhibition of hemagglutination of AMM and ONN erythrocytes by the lectin, and its binding to glycoproteins measured by enzyme immunoassay, confirmed the high specificity of MLL for N-acetyl-D-galactosamine (200-500 times more than for D-galactose) and suggested the presence of hydrophobic interactions around HO-2 of the D-galactose unit. The methyl alpha-glycosides of D-galactose and of N-acetyl-D-galactosamine were better inhibitors than the corresponding beta-glycosides; this preference was abolished, and sometimes reversed, when the p-nitrophenyl glycosides of the same monosaccharides were tested, stressing again the importance of hydrophobic interactions in the binding of carbohydrates to MLL. The lectin reacted well with ONN substance and with glycophorin A of the N phenotype (GPAN), but did not react with OMM substance or GPAM. The strongest inhibitor was asialo ovine submaxillary mucin, which contains many unsubstituted alpha-D-GalpNAc-(1-->3)-Ser/Thr residues; calculated per N-acetyl-D-galactosamine residue, it was 1500 stronger than free N-acetyl-D-galactosamine. In accordance with this result, it was found that the lectin strongly agglutinates Tn cells. The specificity of MLL can, thus, be defined as anti-Tn, crossreactive with blood types A and N, and with sialosyl-Tn. The N-specificity can best be explained by assuming that GPAN contains a small number of unsubstituted or partially sialylated alpha-D-GalpNAc-(1-->3)-Ser/Thr residues, which are present in smaller proportions, if at all, in GPAM.

An erythroid species-specific antigen of swine detected by a monoclonal antibody.

A monoclonal antibody (1AC11) has been produced which recognized the glycophorin of swine red blood cells. 1AC11 was specific for swine membrane erythrocytes. No other swine cells (leukocytes, macrophages, kidney and testis cells) nor red blood cells from all the tested mammalian species (goat, human, sheep, cattle, horse, rabbit, cat and guinea pig) were recognized. There was no blood group activity detected. Immunocytochemical analysis of blood vessel in the swine pituitary tissue showed that besides membrane erythrocytes, cytoplasmic molecules were recognized in some cells. Immunoblot analysis of both membrane and aqueous phase of chloroform/methanol fractions from swine erythrocytes showed that the monoclonal antibody 1AC11 reacts with the major sialoglycoprotein of apparent molecular weight 45,000 daltons.

O-linked oligosaccharides of glycophorins A and B in erythrocytes of two individuals with the Tn polyagglutinability syndrome.

Tn polyagglutinability syndrome is an acquired condition where erythrocytes express Tn neo-antigen and become susceptible to hemagglutination by the naturally occurring anti-Tn present in normal sera. Early studies had indicated that O-linked N-acetyl galactosamine was the sole serologic Tn determinant, but more recently O-linked NeuNAc alpha 2, 6GalNAc also has been implicated as a Tn antigen (sialosyl-Tn). However, none of these studies were performed on purified glycoproteins. In this report we examine oligosaccharides of glycophorins A and B purified from Tn erythrocytes of two affected individuals to establish how N- and O-linked saccharides differ from normal. Analysis of carbohydrate composition and treatment with N-glycanase showed that the Asn-linked unit of glycophorin A was not affected. O-linked oligosaccharides were obtained by beta-elimination in the presence of tritiated sodium borohydride. The reduced radiolabeled products were fractionated by Bio-Gel P-2 chromatography, and their structures were investigated by comparison with standards, by monosaccharide quantification, and by neuraminidases of known specificities. The results show that Tn glycophorins from both donors contain intact and truncated forms of trisaccharide and tetrasaccharide NeuNAc alpha 2,3Gal beta 1,3GalNAc and NeuNAc alpha 2,3Gal beta 1,3- (NeuNAc alpha 2,6)GalNAc usually present in glycophorins A and B. The truncated forms include the protein O-linked monosaccharide, GalNAc and disaccharide, NeuNAc alpha 2,6GalNAc (major isomer). The presence of intact glycans in the total population of Tn erythrocytes was confirmed by their susceptibility to T activation after treatment with neuraminidase. The proportion of the four species was not identical in glycophorins of these two donors but, in both, the truncated units predominated and the amount of the disaccharide was approximately one half of that of the monosaccharide. The data are consistent with alterations in UDPGal:GalNAc beta 1,3galactosyl transferase that may have multiple molecular origins and with induction of a specific GalNAc protein alpha 2,6 sialosyl transferase in Tn hematopoietic precursor cells. The molecular basis for these alterations awaits further study.

Rat erythrocyte glycophorins can be isolated by the lithium diiodosalicylate method used for other glycophorins.

1. The lithium diiodosalicylate/phenol method, widely employed for the isolation of membrane sialoglycoproteins (glycophorins) from mammalian erythrocytes, was applied for the first time to the purification of homologous glycoproteins from rat erythrocyte membranes. 2. The resulting preparations showed to be composed of four components, fractionated on SDS-PAGE. All four were positive for periodic acid-Schiff's reagent stain, the two largest of them being major. 3. Isolated rat glycophorins accounted for 60% of the ghost sialic acid and 1.5% of their protein. The presence of O-acetyl groups was confirmed in one-third of the sialic acid residues. 4. The molecular masses of the four glycophorin components were determined by a method which takes into account the anomalous mobility of glycoproteins on SDS-electrophoresis. Estimated values thus obtained for the actual molecular masses were 74, 32, 25 and 17 kDa.

Reaction of ozone with glycophorin in solution and in lipid vesicles.

Glycophorin from human red blood cells was exposed to ozone in aqueous solution. Amino acid analysis of glycophorin exposed to a 10-fold molar excess of ozone showed that the only residue affected was methionine. Both methionine residues of the protein were oxidized to methionine sulfoxide. Exposure of the oxidized protein to cyanogen bromide caused no cleavage of the polypeptide chain. Glycophorin was incorporated into unilamellar lipid vesicles made from phosphatidylcholine. The protein containing vesicles were exposed to ozone in a 10-fold molar excess to the glycophorin. Gas chromatography of the methyl esters showed negligible change in the fatty acid composition. Amino acid analysis of the ozone-treated protein showed the oxidation of only one methionine residue per polypeptide chain to methionine sulfoxide. Ghosts of human erythrocytes were exposed to ozone. Cyanogen bromide treatment of the oxidized glycophorin yielded fragments showing that the only methionine residue oxidized by ozone was residue 8. These results indicate that in this membrane model (a) amino acid is more susceptible to ozone than is the lipid, and (b) amino acids external to the membrane are more susceptible than those in the polypeptide chain spanning the membrane.