©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Membrane Attachment Sites for Protein 4.1 in the Human Erythrocyte (*)

(Received for publication, September 21, 1994; and in revised form, January 3, 1995)

Nicola J. Hemming (§) David J. Anstee Marcelo A. Staricoff (1) Michael J. A. Tanner (1) Narla Mohandas (2)

From the  (1)International Blood Group Reference Laboratory, Bristol BS10 5ND, United Kingdom, the Department of Biochemistry, University of Bristol, University Walk, Bristol B58 1TD, United Kingdom, and the (2)Lawrence Berkeley Laboratory, Berkeley, California 94720

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 D 91-107). 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 (µM). These results suggest that the most significant interactions between protein 4.1 and the membrane are those involving p55.


INTRODUCTION

The structural integrity and deformability of the human erythrocyte, essential for its survival, are maintained by the skeleton, a network of proteins underlying the lipid bilayer membrane. The skeleton consists predominantly of spectrin, actin, and protein 4.1, as well as the minor components adducin, protein 4.2, protein 4.9, tropomyosin, and myosin (reviewed in (1) and (2) ). The membrane is connected to the skeleton by at least two protein interactions. Ankyrin links band 3 to beta-spectrin, an interaction which has been well studied(3) . The interaction of protein 4.1, the other protein linker, with the membrane has not been so well studied, and the nature of the membrane attachment site(s) has yet to be fully elucidated. Possible membrane attachment sites include band 3(4, 5, 6) , glycophorin C/D (GPC/D)(^1)(7, 8, 9) , glycophorin A(10) , and the lipid bilayer(11, 12) .

While there has been much circumstantial evidence for a protein 4.1-GPC/D interaction for several years(7, 8, 9) , direct evidence has only recently become available. This evidence suggests that GPC/D is the major membrane attachment site for protein 4.1(13, 14) . It has also been suggested that a third component, p55, is required for such an interaction(15, 16) , and that there are two protein 4.1 binding sites on GPC(13, 17) . In this paper we show that protein 4.1 can bind to GPC through two sites, one is a direct interaction between the two proteins, the other is an indirect interaction mediated by p55. We also provide evidence that band 3 is a minor, low affinity, protein 4.1 binding site.


MATERIALS AND METHODS

Fresh normal erythrocytes were available from the National Blood Service (Bristol, UK). Fresh Leach phenotype erythrocytes (which lack GPC/D) were obtained from donor PL(18) . Rabbit anti-protein 4.1 antibodies against the N- and C-terminal regions of the protein were prepared using synthetic peptides Cys-Lys in the 30-kDa N-terminal domain and His-Glu in 22/24-kDa domain. Rabbit anti-p55 was prepared using a synthetic peptide corresponding to amino acid residues 438-453 (Gly-Val-Asp-Glu-Thr-Leu-Lys-Lys-Leu-Gln-Glu-Ala-Phe-Asp-Gln-Ala-Cys-). Antisera were raised against synthetic peptides in a manner analogous to that described previously(14) .

Antibodies to band 3 (BRIC 169), (^2)glycophorin A (BRIC 163(20) ), and glycophorin C/D (BGRL 100(21) ) were available in-house. GPC-peptides and corresponding antisera were as described previously(14) . A synthetic peptide corresponding to the entire cytoplasmic domain of GPA was synthesized using methods previously described(14) .

Immunoblotting

Erythrocyte membranes were electrophoretically separated under reducing conditions on an 8% (w/v) acrylamide homogenous Laemmli gel. Immunoblotting was as described by Mallinson et al.(22) , except that 5% (w/v) bovine milk powder, resuspended in phosphate-buffered saline at pH 7.4, was used as the blocking agent.

Protein Purification

Protein 4.1 was isolated using a modified method of Tyler et al.(23) , as described previously(14) . To prepare protein 4.1 free of p55, the KCl concentration used to elute protein 4.1 from the DEAE-Sephacryl column was reduced from 100 mM to 90 mM. Contamination of protein 4.1 with p55 was detected by ELISA of the fractions using anti-p55. p55 was purified according to the method of Husain-Chishti et al.(24) . Protein determination was performed using the procedure of Lowry et al.(25) .

Limited Digestion of Protein 4.1 and Isolation of 30-kDa-containing Fragments

Protein 4.1 was digested with chymotrypsin at an enzyme to substrate ratio of 1:100, according to Leto and Marchesi(28) . Fragments containing the 30-kDa domain of protein 4.1 were isolated by passing the digestion mixture through a CNBr-activated Sepharose column to which anti-30-kDa antibody had been coupled. Unbound material (fragments devoid of the 30-kDa domain) was collected, and bound fractions (containing the 30-kDa domain) eluted with 0.1 M glycine HCl, pH 2.7. Following neutralization (1 M Tris, pH 9.0, 5 M NaCl), the purity of the fractions was confirmed by SDS-polyacrylamide gel electrophoresis and subsequent staining of the gels with Coomassie Blue. These fragments were subsequently used in the ELISA assay described below.

Alkali-stripping of Human Erythrocyte Membranes

Leaky membranes were prepared by the method of Dodge et al.(26) . Membranes were incubated with 2 mM phenylmethylsulfonyl fluoride at 0 °C for 5 min, before stripping of peripheral proteins by washing with ice-cold 0.1 M NaOH. They were then washed three times with 5 mM sodium phosphate buffer, pH 8.0 at 4 °C.

Proteolytic Digestion of Membranes

Unstripped and alkali-stripped membranes were diluted to 1 mg of protein/ml and incubated with 5 µg/ml trypsin or chymotrypsin in 5 mM sodium phosphate, pH 8.0, for 1 h on ice. Digestion was stopped by the addition of phenylmethylsulfonyl fluoride to 1 mM (final concentration). Alkali-stripped membranes were prepared, as above, and all membranes were washed three times with 5 mM sodium phosphate buffer, pH 8.0 at 4 °C.

Analysis of Protease-treated Membranes by ELISA

The wells of a round bottomed microtiter plate (Sterilin) were coated with 75 µl of lectin (from Agaricus bisporus; Sigma) (7 µg/ml in 20 mM sodium phosphate buffer, pH 7.0) and incubated for 1 h at room temperature(27) . All other procedures were carried out on ice, and all washes were in PBS, pH 7.4, 0.055% Tween 20. The lectin-coated plate was washed, 75 µl of a 5% suspension of protease-treated membranes was added, and the plate was centrifuged at 930 times g for 2 min. The wells were incubated with antibodies to band 3 (BRIC 169), glycophorin A (BRIC 163), or GPC/D (anti-GPC1, anti-GPC2, anti-GPC3, BGRL 100) (75 µl/well) for 2 h. After washing, the secondary antibody (rabbit anti-mouse or swine anti-rabbit peroxidase conjugate; DAKO) was added and incubated for 2 h. After further washing, 75 µl of substrate was added (1 mg of o-phenylenediamine and 0.4 µl of H(2)O(2) in 1 ml of 0.05 M citrate-phosphate buffer, pH 5.0). The color reaction was terminated by the addition of 75 µl of 1 M HCl after 3 min. The plate was read on a Titertek Multiskan ELISA reader (Flow Laboratories) at 492 nm.

Binding Assays Involving Protein 4.1, Fragments of Protein 4.1, p55, Erythrocyte Membranes, and GPC Peptides

Binding studies were carried out using the ELISA system previously described(14) . Preliminary experiments showed that equilibrium was reached after 4 h and that no peptide or protein binding was lost during washing. Within the limits of this assay, all plots appear linear. For inhibition of protein 4.1 or p55 binding to alkali-stripped membranes, serial dilutions of synthetic peptides were made in 3% (w/v) bovine serum albumin and incubated with an equal volume of protein 4.1 or p55 (final concentration 150 µg/ml) overnight at 4 °C before addition to the microtiter plate. For p55 binding GPC-3-saturated protein 4.1: purified protein 4.1 (500 µg/ml) on microtiter wells, prepared as described above, was incubated with saturating levels of GPC-3 (75 µl/well) overnight at 4 °C, before the addition of p55. For protein 4.1 binding p55-saturated alkali-stripped membranes: p55 (500 µg/ml) (75 µl/well) was incubated with microtiter plate-bound alkali-stripped membranes overnight at 4 °C, prior to the addition of protein 4.1.


RESULTS

Protein 4.1-GPC/D Interaction

In a previous report we provided evidence for a direct interaction between protein 4.1 and the region of GPC corresponding to amino acid residues 82-98(14) . However, the recent report showing that Leach phenotype membranes and membranes from individuals with protein 4.1 deficiency lack an additional protein, p55(15) , raised the possibility that p55 might be a contaminant of protein 4.1 preparations from normal erythrocytes and thereby influence protein 4.1 binding assays of the type described in our earlier study. Examination of protein 4.1 preparations from normal erythrocytes with anti-p55, either by ELISA or by immunoblotting, showed a small amount of p55 (approximately 5%, data not shown) and so the experiments were repeated with protein 4.1 preparations from normal cells which were demonstrably free of p55 (see Fig. 1and ``Materials and Methods''). Peptide GPC-3 bound directly to these purified protein 4.1 preparations (Fig. 2A) and completely inhibited their binding to alkali-stripped normal membranes (Fig. 2B), confirming that, under the conditions of this assay, protein 4.1 binds directly to GPC-3.


Figure 1: SDS-polyacrylamide gel electrophoresis analysis of membranes and purified protein 4.1 and p55 and immunoblotting with anti-protein 4.1 and anti-p55. Samples were run on a 4-16% polyacrylamide gradient Laemmli gel. Samples in lanes a-c were stained with Coomassie Blue. Samples in lanes d-f were probed with anti-protein 4.1 and samples in lanes g-j with anti-p55 (as described under ``Materials and Methods''). Lanes a, e, and g, normal membranes; lanes b, f, and j, purified protein 4.1; lanes c, d, and i, purified p55; lane h, Leach phenotype membranes.




Figure 2: A, binding of synthetic peptides to protein 4.1. GPC peptides were incubated with protein 4.1 (150 µg/ml) overnight at 4 °C. Binding was measured as described under ``Materials and Methods.'' Results are plotted with error bars corresponding to mean ± S.D. (n = 4) X, GPC-1; , GPC-2; bullet, GPC-3. B, inhibition of protein 4.1 binding to alkali-stripped normal membranes. Protein 4.1 (150 µg/ml) was incubated with GPC peptide for 5 h at 4 °C before incubation with stripped normal membranes. Binding was measured as described under ``Materials and Methods.'' Peptide concentrations are depicted on a logarithmic scale. Results are plotted with error bars corresponding to mean ± S.D. (n = 4). X, GPC-1; , GPC-2; bullet, GPC-3.



Role of p55 in Protein 4.1-GPC/D Interaction

Evidence that p55 is absent from Leach phenotype membranes and membranes from individuals with protein 4.1 deficiency suggests that p55 binds to both GPC and protein 4.1 in normal membranes. Indeed, purified p55 (Fig. 1) binds to protein 4.1 in a concentration-dependent, saturable (48 µg of p55/mg of protein 4.1) manner, with high affinity (K(d) = 1.27 ± 0.17 nM) (Fig. 3) and also binds saturably (63 µg of p55/mg of membrane protein) and with high affinity (K(d) = 4.54 ± 0.13 nM) to alkali-stripped normal membranes (Fig. 4A). As expected, p55 does not bind to alkali-stripped Leach phenotype membranes (Fig. 4A).


Figure 3: p55 binding protein 4.1. p55 was incubated with protein 4.1 (150 µg/ml) overnight at 4 °C. Binding was measured as described under ``Materials and Methods.'' Results are plotted with error bars corresponding to mean ± S.D. (n = 5). Binding capacity = 48 ± 3 µg of p55/mg of protein 4.1; K = 1.27 ± 0.17 nM.




Figure 4: A, p55 binding to alkali-stripped normal and Leach phenotype membranes and to trypsin/chymotrypsin (5 µg/ml)-treated alkali-stripped normal membranes. Binding was measured as described under ``Materials and Methods.'' Results are plotted with error bars corresponding to mean ± S.D. (n = 4). bullet, normal; X, Leach; , trypsin normal; , chymotrypsin normal. Normal binding capacity = 63 ± 5 µg of p55/mg of membrane protein; K = 4.54 ± 0.13 nM. B, binding of synthetic peptides to p55. GPC peptides were incubated with p55 (150 µg/ml) overnight at 4 °C. Binding was measured as described under ``Materials and Methods.'' Results are plotted with error bars corresponding to mean ± S.D. (n = 5). X, GPC-1; , GPC-2; bullet, GPC-3.



The p55 binding site on alkali-stripped normal membranes is lost if the membranes are pretreated with trypsin or chymotrypsin (Fig. 4A), which cleave the C terminus of GPC(13) . When unstripped normal membranes are trypsin-treated, only 88% ± 4% (n = 6) GPC is cleaved while 100% (n = 6) GPC is cleaved when stripped normal membranes are digested with trypsin. The ability of p55 to block the trypsin cleavage site on GPC was demonstrated by incubating p55 with alkali-stripped normal membranes prior to trypsin digestion. Under these conditions, only 43% ± 6% (n = 3) GPC was cleaved.

In an attempt to localize the p55 binding site on GPC, we investigated the ability of GPC peptides to bind to purified p55. The results (Fig. 4B) clearly show a concentration-dependent binding of GPC-1 to p55. The binding was saturable at 16 µg of GPC-1/mg of p55. This compares with a theoretical value of 22 µg/mg p55, assuming one GPC-1 binding site per molecule of p55. Some binding of the other peptides was observed, but this was consistently much lower than that of GPC-1. Further, GPC-1 can totally inhibit p55 binding to alkali-stripped normal membranes, while GPC-2 and GPC-3 have no effect (data not shown).

Taken together, these results indicate that p55 binds to the extreme C-terminal region of GPC.

GPC/D Independent Membrane Attachment Sites for Protein 4.1

In our previous paper(14) , we reported low levels of protein 4.1 binding to alkali-stripped Leach phenotype membranes (approximately 20% of normal). Since these membranes lack GPC/D and p55, additional membrane binding sites for protein 4.1 must exist. Band 3 and GPA have been postulated as protein 4.1 binding sites(4, 5, 6, 10) . To determine whether these sites are utilized, Leach phenotype stripped membranes were subjected to trypsin cleavage, which destroys the N-terminal domain of band 3, while only having a mild effect on the cytoplasmic domain of GPA (15% reduction) (as determined by monoclonal antibodies to the cytoplasmic domains of band 3 (BRIC 169) and GPA (BRIC 163) data not shown). Trypsin treatment destroyed all protein 4.1 binding to alkali-stripped Leach phenotype membranes, suggesting that band 3 rather than GPA is a binding site for protein 4.1. This finding is supported by the failure of a synthetic peptide corresponding to the entire cytoplasmic domain of GPA to inhibit binding of protein 4.1 to alkali-stripped Leach phenotype membranes and absence of binding of this peptide to purified protein 4.1 (data not shown).

Localization of Membrane Binding Sites on Protein 4.1

To identify the protein 4.1 domain(s) involved in binding to the membrane, chymotryptic fragments of protein 4.1 were separated (as described under ``Materials and Methods''), and their ability to bind GPC, p55, and band 3 was examined. Fragments containing the 30-kDa domain were able to bind to p55 (Fig. 5A) as well as to alkali-stripped normal membranes (Fig. 5B), while those lacking the 30-kDa domain were unable to bind. Thus, the membrane binding sites for GPC, p55, and band 3 appear to be located on the same domain.


Figure 5: Binding of chymotrypsin-digested protein 4.1 to p55 (A) and alkali-stripped (B) normal membranes. Protein 4.1 was partially digested with chymotrypsin, and the fragments were purified as described under ``Materials and Methods.'' Results are plotted with error bars corresponding to mean ± S.D. (n = 4). bullet, 30-kDa-containing fragments; X, 22/24-kDa-containing fragments.



In order to determine whether the binding sites were in the same or different regions of the 30-kDa domain, protein 4.1 was saturated with GPC-3, and its ability to bind p55 and alkali-stripped Leach phenotype membranes (band 3 sites) was studied. While there was no inhibition of binding to p55 (data not shown), binding to alkali-stripped Leach phenotype membranes was completely inhibited (Fig. 6). These results indicate that there are two distinct membrane binding sites on protein 4.1, one binds GPC-3 and band 3, while the other binds p55.


Figure 6: Inhibition of protein 4.1 binding to alkali-stripped Leach membranes. Protein 4.1 (150 µg/ml) was incubated with GPC peptides for 5 h at 4 °C, before incubation with Leach phenotype stripped membranes. Binding was measured as described under ``Materials and Methods.'' Peptide concentrations are depicted on a logarithmic scale. Results are plotted with error bars corresponding to mean ± S.D. (n = 4). X, GPC-1; , GPC-2; bullet, GPC-3.



Relative Utilization of the Membrane Binding Sites

We have sought to determine the proportion of protein 4.1 that binds to each of the three sites (GPC-3, p55, band 3) under the conditions of our assay. Determination of protein 4.1 binding to untreated and trypsin-treated alkali-stripped normal membranes which have been incubated previously with saturating quantities of p55 provides a means of determining the relative contribution of protein 4.1 binding sites on p55 and GPC-3, respectively. Interpretation of these experiments is possible because trypsin treatment destroys GPC independent protein 4.1 binding sites (see above) and because p55 binds, exclusively, to a trypsin-sensitive site at the extreme C terminus of GPC (see above).

Protein 4.1 binding to trypsin-treated membranes (GPC-3 sites) represents 55% of the total binding; protein 4.1 binding in the absence of p55 (band 3 + GPC-3 sites), 72% (Fig. 7; Table 1). A direct measure of the protein 4.1 binding to p55 was obtained by determining the amount of GPC-3-saturated protein 4.1 bound to p55-saturated alkali-stripped membranes. The expected value of 27% was obtained (Fig. 7; Table 1).


Figure 7: Binding of protein 4.1 to alkali-stripped normal membranes. X, purified protein 4.1 was incubated overnight at 4 °C with alkali-stripped normal membranes, binding capacity 192 ± 3 µg/mg membrane protein; K = 0.114 ± 0.11 µM. bullet, binding of protein 4.1 to alkali-stripped normal membranes saturated with p55, binding capacity 258 ± 5 µg/mg membrane protein; K = 0.955 ± 0.19 nM. , binding of protein 4.1 to trypsin-treated alkali-stripped membranes, binding capacity 151 ± 6 µg/mg membrane protein; K = 0.125 ± 0.16 µM. , binding of protein 4.1 to trypsin-treated alkali-stripped normal membranes saturated with p55, binding capacity 143 ± 8 µg/mg membrane protein; K = 0.119 ± 0.14 µM.






DISCUSSION

The location of protein 4.1 binding sites in the red cell membrane has been a matter of some controversy with GPC/D(7, 8, 9) , band 3(4, 5, 6) , glycophorin A(10) , and the lipid bilayer itself (11, 12) all being implicated. Under the conditions of the experiments reported in this paper, we find no significant binding to either glycophorin A or the lipid bilayer (all GPC independent binding is trypsin-sensitive). However, we provide evidence that protein 4.1 binds to alkali-stripped normal erythrocyte membranes through at least three distinct sites. Two sites are located on GPC/D, one is a direct interaction involving residues 82-98 of GPC, and the other an indirect interaction, mediated by p55. The third binding site is most likely located on the N-terminal cytoplasmic domain of the anion transport protein band 3 (syn AE-1). Under the conditions of our assay, the proportion of protein 4.1 occupying each of these sites is approximately 55% directly to GPC, 28% through p55, and 17% through band 3 (Table 1). Estimation of the dissociation constants for these interactions suggest that binding through p55 is of higher affinity (K(d) for GPC-p55 of 4.54 nM and for p55-protein 4.1 of 2.5 nM) than interactions involving GPC-protein 4.1 (K(d) = 0.125 µM) and band 3 (K(d) = 0.11 µM). The measurement of the K(d) for the GPC-protein 4.1 interaction is lower in this study than that reported previously(14) . This discrepancy may be attributable to p55 contamination in the protein 4.1 preparations used in the earlier study. It is interesting to note that Pasternack et al.(4) obtained similar high K(d) values using protein 4.1 prepared by the method of Tyler et al.(23) .

The involvement of p55 in the GPC-protein 4.1 interaction has recently been studied by Marfatia et al.(16) . These authors concluded that a recombinant fusion protein containing p55 was able to bind with high affinity to the 30-kDa domain of protein 4.1 and to GPC. They also concluded that the 30-kDa domain of protein 4.1 was able to bind to GPC with equally high affinity. While our data support the view of Marfatia et al.(16) that protein 4.1 can bind to both p55 and GPC, we suggest that the GPC-protein 4.1 interaction is of a lower affinity to the p55-protein 4.1 interaction. This conclusion is broadly consistent with the recent finding of Gascard and Cohen (17) that GPC contains both high and low affinity binding sites for protein 4.1. However, the proportion of high affinity sites on GPC reported by Gascard and Cohen (17) (10% on average) is much lower than our findings. This is likely to have arisen because Gascard and Cohen (17) did not consider the possible role of p55, and, thus, the high affinity sites detected are probably due to p55 contamination in their protein 4.1 preparations and/or IOVs used for reassociation assays. Gascard and Cohen (17) also describe low affinity binding sites on Leach inside out vesicles, again in agreement with our findings.

The results presented here were obtained using an in vitro assay. Hence, it does not automatically follow that the results of these binding assays can be directly related to the situation in native membranes. In particular, the amounts of protein 4.1 and p55 which are bound at saturation (after correction for loss of peripheral protein by alkali extraction) are 103 µg/mg for protein 4.1 and 25 µg/mg for p55, almost 2-fold higher than the amounts of protein 4.1 and p55 (59 µg/mg and 16 µg/mg), respectively, in normal membranes. Nevertheless, there is compelling evidence that GPC is a major site for protein 4.1 binding in native membranes. Up to 75% of protein 4.1 can be extracted from membranes of Leach phenotype under conditions of low ionic strength in comparison with approximately 25% from normal membranes(13, 14) . This result argues that up to 50% of protein 4.1 binding in normal membranes involves direct binding to GPC or binding via p55 to GPC. Consideration of the relative affinity of these interactions and the likely stoichiometry argues the latter since p55 is absent from both Leach phenotype membranes and protein 4.1-deficient membranes(15) , suggesting that all the p55 in normal membranes is simultaneously bound to GPC and protein 4.1. Estimates of the number of molecules of p55 in normal membranes are of the order 80,000/cell(34) , while protein 4.1 is 200,000/cell. Thus, a maximum of 40% of protein 4.1 molecules are likely to be involved in this high affinity interaction (assuming 1:1 stoichiometry). The marked increase in the protein 4.1 extracted by low ionic strength from Leach phenotype membranes may, for the most part, reflect the absence of the high affinity protein 4.1 binding site on GPC mediated by p55.

Peptide GPC-3 binds directly to Leach phenotype membranes in amounts sufficient to bind more than 85% of protein 4.1 present. In contrast, GPC-3 does not bind at all to normal membranes(14) . If the distribution is similar in normal membranes, then these results suggest that only a small proportion of protein 4.1 (approximately 15%) is bound to GPC-independent sites in these cells (presumably via band 3). The work presented here shows that the GPC-3 site on protein 4.1 is distinct from the p55 binding site (see above). Therefore, these results suggest that in normal red cells the utilization of the three protein 4.1 binding sites is 45% GPC, 40% p55, and 15% band 3. These conclusions are in agreement with those of Pinder et al.(13) who concluded that band 3 and GPA were not major binding sites for protein 4.1 and that GPC possesses two protein 4.1 binding sites.

The nature of protein 4.1 binding sites in the normal erythrocyte membrane has been the subject of considerable controversy over recent years. The realization that an additional protein (p55) also participates in this interaction (15) provides a possible explanation for many of the discrepancies since most studies have involved protein 4.1 reassociation with protein 4.1-depleted membranes without monitoring the level of p55 contamination in the isolated protein 4.1 or the protein 4.1-depleted membranes. Therefore, in most studies, the majority of sites examined have been low affinity sites and, of these, about 50% are trypsin-sensitive GPC-independent sites on band 3 with the possibility that a small number are associated with GPA and the lipid bilayer (this paper, 4, 35, 36). The remaining low affinity trypsin-resistant sites are likely to be located on GPC at the GPC-3 site (the GPC-3 and GPC-2 regions of GPC survive extensive protease digestion (papain, trypsin, and chymotrypsin, 50 µg/ml, 24 h at 37 °C) as judged by their failure to affect binding of specific antibodies to GPC-2 and GPC-3, data not shown).

It has been suggested that the binding site on protein 4.1 for band 3 and possibly also for GPC involves the negatively charged motif LEEDY (residues 37-41) on protein 4.1 and the oppositely charged motifs IRRRY (residues 386-390) and LRRRY(343-347) on band 3 and YHRKG (residues 85-89) on GPC(5) . The results presented here would be consistent with this hypothesis since the LEEDY sequence is located on the 30-kDa N-terminal domain of protein 4.1 and the GPC-3 peptide which contains the YHRKG motif completely inhibits protein 4.1 interaction with alkali-stripped normal and Leach membranes. Since Leach membranes lack GPC, it is necessary to postulate that the inhibition is due to blocking of the site on protein 4.1 which interacts with band 3.

The binding sites involved in protein 4.1-p55 interaction are unknown, but it is interesting to note that p55 contains an SH3 domain (29) and that the 30-kDa domain of protein 4.1 contains a proline-rich motif (PPDP residues 81-84) which might serve as a binding site for such an SH3 domain(32) .

The results presented here suggest two models of GPC-protein 4.1 interaction in native membranes (Fig. 8). In the first model (Fig. 8A), two molecules of protein 4.1 can, in some cases, bind to a single GPC molecule. One molecule of protein 4.1 binds through the GPC-3 site and the other through p55. In the second model (Fig. 8B), some GPC molecules have a single protein 4.1 molecule bound through the GPC-3 site while others have a single protein 4.1 molecule bound through the GPC-3 site and through p55. The first model (Fig. 8A) is compatible with the reassociation assays involving alkali-stripped membranes where the amount of protein 4.1 bound to alkali-stripped normal membranes at saturation is 1.75-fold greater than that found in normal membranes. The ratio of protein 4.1-GPC in normal membranes is approximately 1:1(33) . Since 55% of the protein 4.1 binding is directly to GPC through the GPC-3 site under these conditions (Table 1), most GPC molecules will have one protein 4.1 molecule bound at this site and about half the GPC molecules will have a second protein 4.1 molecule bound through p55. In native membranes, this model is less attractive. All of the p55 is likely to be bound to GPC (80,000 molecules/cell) accounting for approximately 40% of the protein 4.1 and GPC molecules, and the remaining protein 4.1 could be accommodated on other GPC molecules and band 3 rather than on the same GPC molecules. The observation that binding sites for GPC-3 are not available on normal membranes (14) would be consistent with this latter hypothesis.


Figure 8: Models of protein 4.1-GPC/D-p55 interactions. A, two molecules of protein 4.1 can bind to a single GPC molecule. One molecule of protein 4.1 binds through the GPC-3 site, the other through p55. B, a single molecule of protein 4.1 binds simultaneously to GPC-3 and p55.



The observations reported in this paper do not address the functional significance of protein 4.1-GPC interactions. Complete deficiency of protein 4.1 (and therefore, p55) results in elliptocytic cells and in several cases in hemolytic anemia(8) . In the case of the Leach phenotype, absence of GPC/D (and therefore, p55) results in a proportion of elliptocytic red cells(18) . Since Leach phenotype red cells also have a reduced content of protein 4.1 (80% of normal), it has been suggested that the slight elliptocytosis is a consequence of the protein 4.1 deficiency rather than the absence of GPC-protein 4.1-p55 interactions(15) . Support for this hypothesis has come from experiments in which the abnormal deformability properties (in the ektacytometer) of Leach phenotype red cells were corrected by addition of the spectrin binding domain of protein 4.1(34) . The functional role of interactions involving GPC/D, p55, and protein 4.1 in the red cell remains an enigma since, unlike protein 4.1 deficiency, there is no evidence of any pathological consequences of GPC/p55 absence in Leach phenotype. The absence of GPC/D in Leach phenotype results from a structural gene mutation (19) and so the concomitant absence of p55 is likely to be a consequence of the GPC deficiency rather than an abnormality in the p55 gene itself. It seems possible that p55 and analogues of protein 4.1 in other cells and tissues may interact with integral membrane proteins other than GPC so that the relatively benign consequences of GPC deficiency in red cells do not result in pathological consequences in other cells and tissues. It is also possible that alternative regulatory mechanisms operate in Leach phenotype red cells which assuage the deleterious consequences of GPC/p55 deficiency. At the present time, little is known about the regulatory pathways (phosphorylation, palmitoylation) that may involve p55, protein 4.1, and GPC in normal red cells.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: International Blood Group Reference Laboratory, Southmead Rd., Bristol BS10 5ND, UK. Tel.: 0272-507777; Fax: 0272-591660.

(^1)
The abbreviations used are: GP, glycophorin; ELISA, enzyme-linked immunosorbent assay.

(^2)
Smythe, J., Spring, F. A., Gardner, B., Parsons, S. F., Judson, P. A., and Anstee, D. J.(1995) Blood, in press.


ACKNOWLEDGEMENTS

We acknowledge the generous cooperation of donor PL.


REFERENCES

  1. Gilligan, D. M., and Bennett, V. (1993) Semin. Hematol. 30, 74-83 [Medline] [Order article via Infotrieve]
  2. Liu, S.-C., and Derick, L. H. (1992) Semin. Hematol. 29, 231-243 [Medline] [Order article via Infotrieve]
  3. Peters, L. L., and Lux, S. E. (1993) Semin. Hematol. 30, 85-118 [Medline] [Order article via Infotrieve]
  4. Pasternack, G. R., Anderson, R. A., Leto, T. L., and Marchesi, V. T. (1985) J. Biol. Chem. 260, 3676-3683 [Abstract]
  5. Jons, T., and Drenkhahn, D. (1992) EMBO J. 11, 2863-2867 [Abstract]
  6. Lombardo, C. R., Willardson, B. M., and Low, P. S. (1992) J. Biol. Chem. 267, 9540-9546 [Abstract/Free Full Text]
  7. Mueller, T. J., and Morrison, M. (1981) Erythrocyte Membranes , Vol. 2, Alan R. Liss, New York
  8. Alloisio, N., Morle, L., Bachir, D., Guertarni, D., Colanna, P., and Delauney, J. (1985) Biochim. Biophys. Acta 816, 57-62 [Medline] [Order article via Infotrieve]
  9. Reid, M. E., Takakuwa, Y., Conboy, J., Tchernia, G., and Mohandas, N. (1990) Blood 75, 2229-2234 [Abstract]
  10. Lovrien, R. E., and Anderson, R. A. (1980) J. Cell Biol. 85, 534-538 [Abstract]
  11. Sato, S. B., and Ohnishi, S. (1983) Eur. J. Biochem. 130, 19-25 [Abstract]
  12. Cohen, A. M., Liu, S. C., Lawler, J., Derick, L., and Palek, J. (1988) Biochemistry 276, 614-619
  13. Pinder, J. C., Chung, A., Reid, M. E., and Gratzer, W. B. (1993) Blood 82, 3482-3488 [Abstract]
  14. Hemming, N. J., Anstee, D. J., Mawby, W. J., Reid, M. E., and Tanner, M. J. A. (1994) Biochem. J. 299, 191-196 [Medline] [Order article via Infotrieve]
  15. Alloisio, N., Dalla Vanezia, N., Rana, A., Andrabi, K., Texier, P., Gilsanz, F., Cartron, J.-P., Delauney, J., and Chishti, A. H. (1993) Blood 82, 1323-1327 [Abstract]
  16. Marfatia, S. M., Lue, R. A., Branton, D., and Chishti, A. H. (1994) J. Biol. Chem. 269, 8631-8634 [Abstract/Free Full Text]
  17. Gascard, P., and Cohen, C. M. (1994) Blood 83, 1102-1108 [Abstract/Free Full Text]
  18. Anstee, D. J., Parsons, S. F., Ridgwell, K., Tanner, M. J. A., Merry, A. H., Thomas, E. E., Judson, P. A., Johnson, P., Bates, S., and Fraser, I. D. (1984) Biochem. J. 218, 615-619 [Medline] [Order article via Infotrieve]
  19. Tanner, M. J., High, S., Martin, P. G., Anstee, D. J., Judson, P. A., and Jones, T. J. (1988) Biochem. J. 250, 407-414 [Medline] [Order article via Infotrieve]
  20. Okubo, Y., Daniels, G. L., Parsons, S. F., Anstee, D. J., Yamaguchi, H., Tomito, T., and Seno, T. (1988) Vox Sang. 54, 107-111 [Medline] [Order article via Infotrieve]
  21. Reid, M. E., Mawby, W. J., Scott, M. L., Mushens, R. E., King, M.-J., and Holmes, C. H. (1990) Transfus. Med. 1, Suppl. 1, 66
  22. Mallinson, G., Martin, P. G., Anstee, D. J., Tanner, M. J. A., Merry, A. H., Tills, D., and Sonneborn, H. H. (1986) Biochem. J. 234, 649-652 [Medline] [Order article via Infotrieve]
  23. Tyler, J. M., Hargreaves, W., and Branton, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 5192-5196 [Abstract]
  24. Husain-Chishti, A., Faquin, W., Wu, C. C., and Branton, D. (1989) J. Biol. Chem. 264, 8985-8991 [Abstract/Free Full Text]
  25. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  26. Dodge, J. T., Mitchell, C., and Hanahan, D. J. (1963) Arch. Biochem. Biophys. 100, 119-130
  27. Scott, M. L. (May 27, 1992) U. K. Patent 9211176.4
  28. Leto, T. L., and Marchesi, V. T. (1984) J. Biol. Chem. 259, 4603-4608 [Abstract/Free Full Text]
  29. Ruff, P., Speicher, D. W., and Chishti, A. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6595-6599 [Abstract]
  30. Shiffer, K. A., and Goodman, S. R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4404-4408 [Abstract]
  31. Danilov, Y. N., Fennell, R., Ling, E., and Cohen, C. M. (1990) J. Biol. Chem. 265, 2556-2562 [Abstract/Free Full Text]
  32. Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, A. W., and Schreiber, S. L. (1994) Cell 76, 933-945 [Medline] [Order article via Infotrieve]
  33. Smythe, J., Gardner, B., and Anstee, D. J. (1994) Blood 83, 1668-1672 [Abstract/Free Full Text]
  34. Discher, D., Knowles, D., McGee, S., Chasis, J. A., Parra, M., Conboy, J., and Mohandas, N. (1993) Blood 82, Suppl. 1, 309A

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.