©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
BiP Binding Sequences in Antibodies (*)

(Received for publication, July 17, 1995)

Gerhard Knarr (1) Mary-Jane Gething (2)(§) Susanne Modrow (3) Johannes Buchner (1)(¶)

From the  (1)Institut für Biophysik & Physikalische Biochemie, Universität Regensburg, 93040 Regensburg, Germany, the (2)Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia, and the (3)Institut für Medizinische Mikrobiologie und Hygiene, Universität Regensburg, 93042 Regensburg, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(H) and C(H) 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.


INTRODUCTION

BiP, (^1)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 C(H)1 domain of the heavy chain being required for association with BiP (Hendershot et al., 1987). Thus, whereas mutant heavy chains lacking C(H)2 or C(H)3 domains are complexed with BiP and remain in the ER, chains lacking C(H)1 do not appear to interact with BiP and are transported along the secretory pathway. Other studies indicate that sequences within the V(H) 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(H) and C(H) 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.


MATERIALS AND METHODS

Purification of BiP from Bovine Pancreas

BiP used in ATPase stimulation assays was isolated from bovine pancreas based on a procedure described previously (Wiech et al., 1993). All steps of the isolation were performed at 4 °C. Bovine pancreas was homogenized, MgATP was added, and the homogenate was centrifuged twice to remove cell fragments, nuclei, and mitochondria. Subsequently, the microsomes were pelleted by ultracentrifugation. The post-microsomal supernatant contained a considerable amount of BiP, which could be separated from Hsc70 and Hsp90 by DE52 anion exchange chromatography with a linear NaCl gradient (100-200 mM). BiP-containing fractions were pooled, concentrated, and dialyzed against 40 mM HEPES, 100 mM KCl, 20 mM KOH, 10 mM (NH(4))(2)SO4, 4 mM MgCl(2), 2 mM potassium acetate, 1 mM dithiothreitol, 5% (v/v) glycerol, 0.005% (v/v) Triton X-100, pH 7.8 (buffer L). The resuspended rough microsomes were supplemented with NaCl and Triton X-100 to a final concentration of 100 mM and 0.1% (v/v), respectively. After homogenization, the membranes were removed by centrifugation at 140,000 times g for 120 min. Subsequent DE52 anion exchange chromatography was performed as described previously (Wiech et al., 1993). The dialyzed pools of BiP were then applied to a C8 ATP-agarose column (Sigma). After washing the column with buffer L containing 10 mM EDTA, but no MgCl(2), BiP was eluted with buffer L supplemented with 0.5 M KCl and 4 mM MgATP. The eluate was dialyzed against 40 mM potassium phosphate, pH 7.0 (buffer B), (NH(4))(2)SO(4) was added to a final concentration of 2 M, and the solution was then applied to a butyl-Sepharose 4 Fast Flow column (Pharmacia Biotech Inc.). BiP was released from the column by a linear gradient from 2 to 0 M (NH(4))(2)SO(4) (in buffer B). The fractions of pure BiP were concentrated and stored in buffer L containing 25% (v/v) glycerol at -70 °C.

ATPase Measurements

ATPase assays were performed according to Kornberg et al.(1978). Approximately 4 µg of purified BiP were incubated at 37 °C with 500 µM (final concentration) unlabeled ATP and 10 µCi of [alpha-P]ATP in a total volume of 20 µl containing 40 mM HEPES, pH 7.0, and 2 mM MgCl(2). The stimulation of the ATPase activity by peptide was determined in the presence of concentrations of 10 µM to 1 mM of the respective peptide. Following different times of incubation, 3-µl aliquots of the reaction mixture were added to 2 µl of a mixture of ATP, ADP, AMP, and EDTA (each 12 mM) to terminate the reaction. Thin layer chromatography was performed in 0.5 M LiCl and 2 N formic acid. The plates were dried, and the spots corresponding to ATP and ADP were located with UV light and excised; the radioactivity was then determined by liquid scintillation. The radioactivity in each spot was expressed as a fraction of the total recovered in each lane.

Peptide Synthesis and Purification

Peptides were synthesized using a 9050 PepSynthesizer (Milligen) and Fmoc-protected amino acids (Atherton et al., 1978). The first residue was coupled to polystyrene beads via an acid-labile linker (Tenta-Gel, Rapp-Polymere). Double coupling cycles were used for residues following proline or glycine and when identical or similar residues were clustered, in order to avoid incomplete coupling reactions. t-Butyl (Ser, Thr, Tyr), t-butylester (Asp, Glu), trityl (Cys, His, Gln, Asn), t-butyloycarbonyl (Lys), and 4-methoxy-2,3,6-trimethylbenzene-sulfonyl (Arg) groups were used for side chain protection. Fmoc-protected amino acids were converted to hydroxy-benzotriazol-activated esters with 1.5 mmol of hydroxybenzotriazol and 1.2 mmol of diisopropycarbodiimide per mmol of amino acid, prior to the coupling procedure. The subsequent coupling reaction was performed in N,N-1-dimethylformamide, and coupling times of 10 min were used. Fmoc groups were removed by 20% piperidine, and the completeness of this reaction was monitored by fluorometric measurements (all amino acids were purchased from Bachem AG, solvents were from Merck AG, and chemicals were from Aldrich). After synthesis, side chain protecting groups were removed by treatment for 4 h in 50% trifluoroacetic acid in dichloromethane or in 100% trifluoroacetic acid overnight when Arg residues were present in the peptide sequence. 3% anisol and 3% phenol was routinely added as scavengers including 5% 2-mercaptoethanol in cases where Arg and Trp residues were combined in the peptide sequence. Subsequently, solvents were evaporated, the peptide suspended in 1-2 ml acetic acid and precipitated in an excess of ice-cold t-butylethylether, washed several times, and suspended in water. The peptide was lyophilized and purified by reversed phase high performance liquid chromatography using a C2/C18 copolymer column (PepS, Pharmacia) and a gradient of 0-70% acetonitrile in 0.1% trifluoroacetic acid. Finally, the peptide-containing fractions were lyophilized.


RESULTS

Prediction of Potential BiP Binding Sites in the Sequences of Monoclonal Antibodies 3D6 and MAK33

The sequences of the MAK33 and the 3D6 antibodies were screened for potential BiP binding sites using the BiP Score computer program (Blond-Elguindi et al., 1993a). The algorithm used in this program scores amino acid sequences with a moving window of seven residues, the apparent size of the (poly)peptide binding cleft on BiP (Flynn et al., 1991; Blond-Elguindi et al., 1993a). An individual score is assigned to each amino acid depending on its position within the heptapeptide, and the sum of the seven scores provides a measure of the probability that a given heptapeptide would bind to BiP. Heptapeptides with scores greater than 10 are very likely to bind to BiP, while peptides with scores between +6 and +10 have odds of 3 to 1 of displaying binding activity. Scores from 0 to +5 have little predictive value, but peptides with negative scores almost invariably lack the capacity to bind to BiP.

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).





Many Synthetic Peptides Corresponding to Predicted BiP Binding Sites in Ig Heavy Chains Stimulate the ATPase Activity of BiP

To test the predictive capacity of the BiP Score program, we checked the ability of synthetic peptides corresponding to predicted BiP binding sites in the Ig heavy chains to stimulate the ATPase activity of BiP (Flynn et al., 1989). 34 peptides were synthesized. 26 of these corresponded to sequences from the heavy chain of antibody 3D6 (named HD14-183), including 15 potential binding sites with scores ranging from +5 to +12, and 11 sites with scores ranging from +1 to -28, which are unlikely to be BiP binding sequences. A further 8 peptides corresponding to potential sites in the heavy chain of MAK33 (named HM27 to 430) had scores of +7 to +20. The peptide HD111R does not correspond to a natural 3D6 peptide but is a randomized version of peptide HD111 having the same amino acid composition but a different sequence and a significantly lower BiP Score (-6 versus +10). Most of the synthetic peptides were heptamers, but when the carboxyl-terminal residue of the chosen heptapeptide was proline or tryptophan, thus causing problems with synthesis, octapeptides that included the next residue in the sequence were synthesized (see peptides HD47/48, HD133/134, and HM387/388). In each case, these peptides contain two potential seven-residue binding sites, and the overall BiP score for the second heptapeptide is also provided in Table 1.

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(m) 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(m) 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(m) value measured for ATPase stimulation (data not shown).

Localization of BiP Binding Sequences in the Three-dimensional Structure of Antibody 3D6

Finally we investigated the position in the three-dimensional structure of 3D6 of the BiP binding sequences that we had identified as causing significant stimulation of the ATPase activity of BiP. Fig. 4shows the V(H) and C(H)1 sequences of antibody 3D6, with the 10 peptides that were predicted and shown to be positive with respect to BiP binding outlined by frames. The residues of 3D6 involved in the interface between the H-chain and the L-chain (shown by shaded boxes) were determined by comparing the surface exposure of side chains in the crystal structure of the dimer and the artifically separated monomers. With only two exceptions (HD14 and HD167), the BiP binding sequences contain multiple residues that participate in the contact sites. In total, of 48 contact residues 25 are located within BiP binding sequences. These 25 residues include the majority of the contact site residues that are hydrophobic in character. These observations suggest that binding of BiP to one or more of several potential binding sites exposed within the contact interface on folded but unassembled heavy chains would shield the hydrophobic surfaces and inhibit unproductive aggregation reactions.


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.




DISCUSSION

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 geq+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 geq10 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(m) 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(H) and C(H)1 domains of the 3D6 heavy chain and within the C(H)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(H)2 domain. Thus, potential BiP binding sites are not confined to the C(H)1 domain of the heavy chain, as was previously suggested on the basis of experiments that showed that mutant heavy chains lacking C(H)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(H)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(H)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(H)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 H(2)L(2) 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.


FOOTNOTES

*
This work was supported in part by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to J. B.). 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.

§
Supported by an Investigatorship from the Howard Hughes Medical Institute and by a grant from the National Health and Medical Research Council of Australia.

To whom correspondence should be addressed. Tel.: 49-941-943-3039; Fax: 49-941-943-2813.

(^1)
The abbreviations used are: BiP, immunoglobulin heavy chain binding protein; ER, endoplasmic reticulum; Fab, proteolytically derived antibody fragment consisting of the entire immunoglobulin light chain and the two amino-terminal domains of the heavy chain; Fd, part of the immunoglobulin heavy chain consisting of the two amino-terminal domains (V(H) and C(H)1); Fc, part of the immunoglobulin heavy chain consisting of the two carboxyl-terminal domains (C(H)2 and C(H)3); MAK33, murine antibody of the subclass kappaIgG1, directed against human creatine kinase; 3D6, human monoclonal antibody of the subclass kappaIgG1 from the cell line 3D6 directed against the gp41 protein of the human immunodeficiency virus-1; Fmoc, N-(9-fluorenyl)methyloxycarbonyl.


ACKNOWLEDGEMENTS

We thank Martina Hilbert for identifying residues involved in interchain contacts by analysis of the crystal structure of the 3D6 antibody, Ursula Jakob for performing initial experiments on the ATPase activity of BiP, Lydia Nanu for performing the reduced and carboxymethylated lactalbumin competition assay, and Astrid Brunner for assistance during peptide synthesis. We are indebted to Ingrid Haas for stimulating criticism and to Linda Hendershot for fruitful discussions.


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