(Received for publication, September 21, 1994; and in revised form, January 3, 1995)
From the
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.
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 -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)(
)(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.
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), ()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) .
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;
, 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;
,
GPC-3.
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). , 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;
, 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.
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).
, 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;
, GPC-3.
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.
, 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.
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 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
= 0.125 µM) and band 3 (K
= 0.11 µM). The measurement
of the K
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
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.