(Received for publication, July 17, 1995)
From the
During the process of folding and assembly of antibody molecules
in the endoplasmic reticulum, immunoglobulin heavy and light chains
associate transiently with BiP, a resident endoplasmic reticulum
protein that is a member of the Hsp70 family of molecular chaperones.
BiP is thought to recognize unfolded or unassembled polypeptides by
binding extended sequences of approximately seven amino acids that
include bulky hydrophobic residues not normally exposed on the surface
of native proteins. We used a computer algorithm developed to predict
BiP binding sites within protein primary sequences to identify sites
within immunoglobulin chains that might mediate their association with
BiP. Very few of the sequential heptapeptides in the heavy or light
chain sequences were potential BiP binding sites. Analysis of the
ability of synthetic heptapeptides corresponding to 24 potential sites
in heavy chains to stimulate the ATPase activity of BiP indicated that
at least half of them were authentic BiP binding sequences. These
sequences were not confined to a single domain of the heavy chain but
were distributed within both the V and C
domains. Interestingly, when the BiP binding sequences were
mapped onto the three-dimensional structure of the Fd antibody
fragment, the majority involve residues that participate in contact
sites between the heavy and light chains. Therefore, we suggest that in vivo BiP chaperones the folding and assembly of antibody
molecules by binding to hydrophobic surface regions on the isolated
immunoglobulin chains that subsequently participate in interchain
contacts.
BiP, ()the sole ER-located member of the Hsp70 family
of molecular chaperones, was originally described as the immunoglobulin
heavy chain binding protein, found in non-covalent association with
heavy chains in myeloma cells that do not synthesize immunoglobulin
light chains (Haas and Wabl, 1983). Interestingly, BiP was
independently identified as a protein (GRP78) expressed in response to
glucose starvation (Pouyssegur et al., 1977). BiP is now known
to associate transiently not only with immunoglobulin heavy and light
chains during their folding and assembly (Bole et al., 1986;
Hendershot et al., 1987; Nakaki et al., 1989; Dul and
Argon, 1990, Ma et al., 1990; Knittler and Haas, 1992) but
also with a wide variety of other newly synthesized wild-type
exocytotic proteins (for reviews, see Gething and Sambrook(1992);
Gething et al.(1994); Haas (1994)). BiP interacts more
permanently with malfolded or unassembled proteins whose transport from
the ER is blocked but does not bind to native proteins. These
observations indicate that BiP plays a role in the folding and assembly
of newly synthesized proteins in the ER lumen. Like other Hsp70
proteins, BiP is thought to function by recognizing hydrophobic
sequences exposed on unfolded or unassembled polypeptides and, by
inhibiting intra- or intermolecular aggregation, maintaining them in a
state competent for subsequent folding and oligomerization (Gething and
Sambrook, 1992; Craig et al., 1993; Becker and Craig, 1994;
Hightower et al., 1994).
Although the transient interaction
between BiP and immunoglobulin molecules has been well documented,
little is known about the location or nature of the sequences on the
heavy and light chains that are recognized by the chaperone. Indirect
evidence points to the C1 domain of the heavy chain being
required for association with BiP (Hendershot et al., 1987).
Thus, whereas mutant heavy chains lacking C
2 or
C
3 domains are complexed with BiP and remain in the ER,
chains lacking C
1 do not appear to interact with BiP and
are transported along the secretory pathway. Other studies indicate
that sequences within the V
domain influence the
association of heavy chains with BiP (Haas, 1991). To identify
potential BiP binding sequences in Ig heavy chains, we took advantage
of a computer scoring system designed to predict BiP binding sites in
synthetic peptides and naturally occurring polypeptides (Blond-Elguindi et al., 1993a). We scored sequences from two different but
related antibodies, MAK 33 and 3D6. Monoclonal antibody MAK 33 has been
used in a number of studies on antibody folding in vitro (Buchner and Rudolph, 1991; Buchner et al., 1991; Lilie et al., 1995a, 1995b) and on the influence on this process of
folding catalysts and chaperones (Wiech et al., 1992; Schmidt
and Buchner, 1992; Schmidt et al., 1994; Lilie et
al., 1993, 1994; Lilie and Buchner, 1995). Since the
three-dimensional structure of this antibody is not yet known, we also
scored the Fd portion of the monoclonal antibody 3D6, which
three-dimensional structure had been determined to a resolution of 2.7
Å (He et al., 1992).
We report that it is possible to
predict potential BiP binding sites in the two different immunoglobulin
sequences using the scoring procedure. Only a small number of potential
BiP binding sites were identified, with the majority of the antibody
sequences having a very low probability of binding to BiP. The
potential binding sites in the Ig heavy chains are distributed within
both the V and C
domains, and the majority
involve residues that participate in contact sites between the heavy
and light chains. We suggest that BiP chaperones the folding and
assembly of antibody molecules by binding to hydrophobic surface
regions on the isolated immunoglobulin chains that subsequently
participate in interchain contacts.
Fig. 1shows the results of the scoring procedure for heavy chain sequences of the two antibodies. The scores range from -28 to +12 for heptapeptides contained in the 3D6 Fd fragment and from -31 to +20 for those from the MAK33 heavy chain (Fd + Fc). Only a small fraction of the heptapeptides (<8% for 3D6 sequences, <5% for MAK33 sequences) had scores greater than +6, indicating that they were potential sites for binding to BiP. The majority of these had scores in the range between +6 and +10 (14 out of 223 peptides in the case of 3D6, 18 out of 448 peptides in the case of MAK33) and should therefore bind to BiP with a probability of 3 to 1. Only a very few (3 in the case of 3D6 and 4 in the case of MAK33) had scores greater than +10, indicating a very high probability of binding. Peptides with positive scores were frequently clustered in small groups of 2-5 peptides but were distributed throughout the 3D6 Fd fragment and the MAK33 heavy chain, indicating no special preference for a distinct antibody domain. Very similar results were obtained when the light chains of the two antibodies were scored (Fig. 2).
Figure 1: Prediction of BiP binding sequences in the primary structure of the heavy chains of antibodies 3D6 and MAK33. Overall scores for each of the overlapping heptapeptides in these sequences were calculated using the BiP Score program described by Blond-Elguindi et al. (1993a) within the Fd fragment of antibody 3D6 (A) and the Fd (B) and the Fc (C) fragments of antibody MAK33, respectively. Asterisks indicate the positively scoring sequences in the 3D6 Fd fragment that, when tested as synthetic peptides, stimulated the ATPase activity of BiP (see Table 1).
Figure 2: Prediction of BiP binding sequences in the primary structure of the light chains of antibodies 3D6 (A) and MAK33 (B). Overall scores for each of the overlapping heptapeptides in these sequences were calculated using the BiP Score program described by Blond-Elguindi et al. (1993a).
Table 1presents the sequences and scores of the synthetic peptides together with the results of assays of their ability to stimulate the ATPase activity of BiP when added at a concentration of 500 µM. Of the 23 peptides having BiP scores of at least +5, 12 stimulated BiP ATPase activity by factors ranging from 1.9 to 3.8. These stimulation factors correspond well to those obtained in the same experiment for two peptides, pp28 and pp37, selected as BiP binding peptides by bacteriophage display (Blond-Elguindi et al., 1993a) and to those reported previously for other peptides tested at this concentration (Flynn et al., 1989; Blond-Elguindi et al., 1993b; Fourie et al., 1994). An additional two peptides displayed consistent but lower stimulatory capacity (1.4- and 1.6-fold, respectively). As ``negative controls'' we tested a set of 10 heptapeptides that had negative BiP scores and one peptide with a score of +1. All of these peptides failed to stimulate the ATPase activity of BiP (Table 1). The fact that peptide HD111 (score +10) displayed one of the highest stimulation factors (3.0) while its randomized version peptide 111R (score, -6) failed to stimulate BiP ATPase activity confirms that it is not the composition of amino acids that determines the ability of a peptide to bind to BiP but rather their specific sequence in the peptide.
The concentration dependence of
ATPase stimulation was measured for four peptides corresponding to
sequences from the 3D6 heavy chain and for the previously characterized
pp28 peptide. The data shown in Fig. 3yielded a K value of 60 ± 10 µM for
peptide HD111, defined as the concentration of peptide required for
half-maximal stimulation of the ATPase activity of BiP. As shown in Table 2, peptides HD131, HD133, and HD177 caused half-maximal
stimulation at even lower concentrations (6-17 µM),
while the K
obtained for peptide pp28 was 16
± 3 µM, which is in good agreement with the value
of 10 ± 2 µM obtained previously (Blond-Elguindi et al., 1993a).
Figure 3: Stimulation of the ATPase activity of BiP by peptide HD111. The concentration dependence of the stimulation of ATP hydrolysis by peptide HD111 was determined as described under ``Materials and Methods.'' The concentration of peptide necessary for half-maximal stimulation was calculated to be 60 µM.
Finally, the capacity of peptide HD111 to
bind to BiP was tested using the ability of the peptide to compete the
binding of BiP to reduced and carboxymethylated lactalbumin as
described previously (Fourie et al., 1994). Competition of
reduced and carboxymethylated lactalbumin binding was approximately
30-40% at a peptide concentration of 50 µM, while
100 µM peptide caused approximately 70-80%
competition. Extrapolation of this data suggests that half-maximal
competition of binding would be achieved at 60-70
µM, a value in excellent agreement with the K value measured for ATPase stimulation (data not
shown).
Figure 4: Localization of BiP binding sequences in the primary structure of the Fd fragment from antibody 3D6. Underlined amino acids correspond to the complementarity-determining regions (CDR's), while shaded segments in the 3D6 sequence represent amino acids that are involved in interchain contacts. These residues were determined by comparing the surface exposure of side chains in the crystal structure of the dimer and the respective monomers. Peptides that stimulate the ATPase activity of BiP are framed.
Using a computer program that predicts recognition sites
within protein sequences for binding of the ER-located chaperone BiP
(Blond-Elguindi et al., 1993a), we identified a number of
potential BiP binding sites in the primary sequences of antibodies 3D6
and MAK33. Only a small proportion of all the sequential heptapeptides
within the immunoglobulin sequences are potential BiP binders (i.e. have BiP Scores +5). Measurement of the ability of
synthetic peptides corresponding to 23 potential sites in the
immunoglobulin heavy chains to stimulate the ATPase activity of
purified BiP demonstrated that 12 of them were indeed BiP binding
sequences. Our results confirm that the predictive power of the scoring
program is greater than 50% for positive peptides, rising to
80%
when peptides with BiP scores
10 are considered, and it is close to
100% for negative peptides. Analysis of the concentration dependence of
stimulation of BiP by a subset of the peptides yielded apparent K
values of 6-60 µM that lie
within the range previously defined for high affinity sites
(Blond-Elguindi et al., 1993a; Fourie et al., 1994).
Confirmed BiP binding sequences are located within both the V and C
1 domains of the 3D6 heavy chain and within the
C
3 domain of the MAK33 heavy chain. In addition, a number
of untested sequences having BiP scores >10 (and therefore having a
high probability of being BiP binding sites) are present in the MAK33
C
2 domain. Thus, potential BiP binding sites are not
confined to the C
1 domain of the heavy chain, as was
previously suggested on the basis of experiments that showed that
mutant heavy chains lacking C
1 do not interact stably with
BiP (Hendershot et al., 1987). Rather our data are consistent
with the recent results of Kaloff and Haas(1995), who showed that the
C
1 domain is not the only portion of the heavy chain that
interacts with BiP during the biosynthesis and initial folding of the
molecule. What apparently sets the C
1 domain apart from the
other three domains of the heavy chain is its propensity to continue to
expose at least one BiP binding site after the heavy chain dimer is
formed, and this is probably the result of the C
1 domain
being uniquely unable to homodimerize.
Fig. 5presents a
schematic model of the role of BiP during the folding and assembly of
immunoglobulin molecules. During the translocation of heavy and light
chains as extended polypeptides into the lumen of the endoplasmic
reticulum, BiP binds to one or more of multiple potential sites present
within all four domains of the heavy chains and within both domains of
the light chains. Some potential binding sites become occluded as the
domains begin to fold, but sites displayed on surfaces destined to
become subunit interfaces remain exposed until assembly occurs, first
of heavy or light chain homodimers and then of the
HL
heterotetramers.
Figure 5: A schematic model of the role of BiP during the folding and assembly of immunoglobulin molecules. This model concentrates on the role of BiP in binding to linear epitopes exposed in the nascent chain and (partially) folded but unassembled structures. The simultaneous binding of BiP and protein disulfide isomerase (PDI) to antibody chains is still hypothetical. Furthermore, the interaction of BiP with antibodies may occur via ATP-dependent cycles of binding, release, and rebinding. The undoubtedly important roles of other chaperones and co-chaperones, for example Grp94 (Melnick et al. 1994), have not been addressed.
Our data suggest that BiP binding sites in immunoglobulins may in fact be concentrated in sequences that participate in subunit interfaces. The localization within the three-dimensional structure of antibody 3D6 of confirmed potential binding sites in the Fd portion of the heavy chain showed that almost every site includes amino acids that are part of the contact surface between the heavy and light chains. Furthermore, the hydrophobic residues that are destined to become buried in the interface are disproportionately present within the potential BiP binding sites. By protecting hydrophobic patches on the surface of Ig folding intermediates from forming inappropriate interactions with other partially folded polypeptides, nonspecific aggregation and unproductive side reactions would be suppressed, and the generation of correctly assembled antibodies would be favored. It will be of great interest to determine if BiP binding sites in other multisubunit proteins are also preferentially localized in sequences that form subunit interfaces, thus indicating a general and important role for BiP during subunit assembly.