BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP-heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.     

ERdj3, a stress-inducible endoplasmic reticulum DnaJ homologue, serves as a cofactor for BiP's interactions with unfolded substrates

We recently identified ERdj3 as a component of unassembled immunoglobulin (Ig) heavy chain:BiP complexes. ERdj3 also associates with a number of other protein substrates, including unfolded light chains, a nonsecreted Ig light chain mutant, and the VSV-G ts045 mutant at the nonpermissive temperature. We produced an ERdj3 mutant that was unable to stimulate BiP's ATPase activity in vitro or to bind BiP in vivo. This mutant retained the ability to interact with unfolded protein substrates, suggesting that ERdj3 binds directly to proteins instead of via interactions with BiP. BiP remained bound to unfolded light chains longer than ERdj3, which interacted with unfolded light chains initially, but quickly disassociated before protein folding was completed. This suggests that ERdj3 may bind first to substrates and serve to inhibit protein aggregation until BiP joins the complex, whereas BiP remains bound until folding is complete. Moreover, our findings support a model where interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex.     

The In Vivo Association of BiP with Newly Synthesized Proteins Is Dependent on the Rate and Stability of Folding and Not Simply on the Presence of Sequences That Can Bind to BiP

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine λI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as “kinetic traps.” Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.     

In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo.     

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.     

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin

Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.     

Immunoglobulin heavy chain and binding protein complexes are dissociated in vivo by light chain addition

Immunoglobulin heavy chain binding protein (BiP, GRP78) associates stably with the free, nonsecreted Ig heavy chains synthesized by Abelson virus transformed pre-B cell lines. In cells synthesizing both Ig heavy and light chains, the Ig subunits assemble rapidly and are secreted. Only incompletely assembled Ig molecules can be found bound to BiP in these cells. In addition to Ig heavy chains, a number of mutant and incompletely glycosylated transport-defective proteins are stably complexed with BiP. When normal proteins are examined for combination with BiP, only a small fraction of the intracellular pool of nascent, unfolded, or unassembled proteins can be found associated. It has been difficult to determine whether these BiP-associated molecules represent assembly intermediates which will be displaced from BiP and transported from the cell, or whether these are aberrant proteins that are ultimately degraded. In order for BiP to monitor and aid in normal protein transport, its association with these proteins must be reversible and the released proteins should be transport competent. In the studies described here, transient heterokaryons were formed between a myeloma line producing BiP-associated heavy chains and a myeloma line synthesizing the complementary light chain. Introduction of light chain synthesis resulted in assembly of prelabeled heavy chains with light chains, displacement of BiP from heavy chains, and secretion of Ig into the culture supernatant. These data demonstrate that BiP association can be reversible, with concordant release of transportable proteins. Thus, BiP can be considered a component of the exocytic secretory pathway, regulating the transport of both normal and abnormal proteins.     

Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein

Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with light chains. The heavy chain-BiP complex can be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the CH1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line (CH1-) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.     

Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase

Myosin light chain kinases （MLCK） phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase （S-MLCK） to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase （L-MLCK） has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules （2Ig） in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased ＂spike＂-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.     

BiP and PDI cooperate in the oxidative folding of antibodies in vitro

Immunoglobulin heavy chain binding protein (BiP), a member of the Hsp70 chaperone family, and the oxidoreductase protein-disulfide isomerase (PDI) play an important role in the folding and oxidation of proteins in the endoplasmic reticulum. However, it was not clear whether both cooperate in this process. We show here that BiP and PDI act synergistically in the in vitro folding of the denatured and reduced Fab fragment. Several ATP-dependent cycles of binding, release, and rebinding of the unfolded antibody chains by BiP are required for efficient reactivation. Our data suggest that in the absence of BiP unfolded antibody chains collapse rapidly upon refolding, rendering cysteine side chains inaccessible for PDI. BiP binds the unfolded polypeptide chains and keeps them in a conformation in which the cysteine residues are accessible for PDI. These findings support the idea of a network of folding helper proteins in the endoplasmic reticulum, which makes this organelle a dedicated protein-processing compartment.     

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins

BACKGROUND: Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS: We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS: The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.     

BiP-binding sequences in HIV gp160. Implications for the binding specificity of bip.

BiP, a resident endoplasmic reticulum member of the HSP70 family of molecular chaperones, associates transiently with a wide variety of newly synthesized exocytotic proteins. In addition to immunoglobulin heavy and light chains, the first natural substrates identified for BiP, a number of viral polypeptides including the human immunodeficiency virus type 1 envelope glycoprotein gp160 interact with BiP during their passage through the endoplasmic reticulum. We have used a computer algorithm developed to predict BiP-binding sites within protein primary sequences to identify sites within gp160 that might mediate its association with BiP. Analysis of the ability of 22 synthetic heptapeptides corresponding to predicted binding sites to stimulate the ATPase activity of BiP or to compete with an unfolded polypeptide for binding to BiP indicated that about half of them are indeed recognized by the chaperone. All of the confirmed binding sites are localized within conserved regions of gp160, suggesting a conserved role for BiP in the folding of gp160. Information on the characteristics of confirmed BiP-binding peptides gained in this and previous studies has been utilized to improve the predictive power of the BiP Score algorithm and to investigate the differences in peptide binding specificities of HSP70 family members.     

Enzymatic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments

CD4, the cell-surface receptor for the human immunodeficiency virus (HIV), is a member of the immunoglobulin (Ig) gene superfamily. It contains four extracellular sequences homologous to Ig VL domains. The first of these (V1) is sufficient for binding to HIV; however, the structural basis for this binding has yet to be elucidated. While several models for the structure of Ig-like domains in CD4 have been proposed on the basis of crystal structures of Ig VL domains, direct evidence that CD4 and VL domains fold similarly has not been obtained. To produce individual domains of CD4 for structural studies, we used molecular fusions of such domains with Ig heavy chain (CD4 immunoadhesins), which are very efficiently expressed and secreted in mammalian cells and can be easily isolated in single-step purification with protein A. Since these fusion molecules are antibody-like homodimeric proteins, we investigated the possibility that they might be cleaved enzymatically to produce Fd-like and Fc fragments. We found that cleavage with papain releases an Fd-like fragment containing the V1 and V2 CD4 domains; this fragment fully retains the ability to bind to the HIV-1 envelope glycoprotein gp120 and to block HIV infection in vitro. Moreover, folding of the CD4 domains in the Fd-like fragment and in the parent immunoadhesin is indistinguishable, as indicated by circular dichroism. Spectral analysis of the Fd-like fragment suggests that secondary structure content is identical with that predicted from the known structure of Ig VL domains; this directly supports the hypothesis that the V1 and V2 domains of CD4 fold similarly to Ig VL domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation of a single conserved residue in VH complementarity-determining region 2 results in a severe Ig secretion defect

During an immune response, somatic mutations are introduced into the VH and VL regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile51 is present in 91% of murine VH complementarity-determining region 2 sequences, and we demonstrate that single Ile51-->Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1-3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig binding protein (BiP) and GRP94 and fail to form HL and H2L assembly intermediates efficiently. The mutations do not irreversibly alter the VH domain as the small amount of mutant H chain, which assembles with L chain as H2L2, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile51 is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile51-->Arg or Ile51-->Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 VH single Ile51-->Arg or Ile51-->Lys mutants were partially rescued when expressed with the J558L lambda1 L chain. These results represent the first demonstration that single somatic mutations in V(H) residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.     

A V kappa-J kappa junctional change in an antidigoxin recombinant antibody destroys digoxin-binding activity

A set of high affinity antidigoxin antibodies were previously identified with high homologous V kappa 1A L chain sequences but were associated with two entirely different VH regions and two dramatically different specificities for digoxin analogs. Antibodies 40-20, 40-60, 40-90, and 40-100 displayed similar binding specificities but differed from that of antibody 26-10. In a previous study using somatic cell fusion for Ig chain recombination we demonstrated that a recombinant antibody consisting of the H chain of antibody 26-10 and the L chain of antibody 40-20 retained digoxin binding and the 26-10 Id, but displayed a binding specificity pattern dominated by the 26-10 H chain donor. In the present study we produced three additional chain recombinant antibodies that contain the 26-10 H chain recombined with each of the L chains of antibodies 40-60, 40-90, and 40-100. All four recombinants expressed the 26-10 Id indistinguishably from the 26-10 antibody. Two of the recombinants (using the 40-60 and 40-90 L chains) bind digoxin; however, the recombinant using the 40-100 L chain failed to bind digoxin. Complete sequence analyses of the 40-20, 40-60, 40-90, and 40-100 VH and VL regions were performed. Antibodies 40-90 and 40-100 have identical VH region sequences but differed only in their L chains at position 96 (proline/leucine). This single difference at the VK-JK junction abolished digoxin binding in the context of one H chain (26-10), but does not cause a significant change in binding in association with the "normal" parental chains 40-90 and 40-100. Thus, structurally closely related VL regions can recombine with different VH regions to form digoxin binding sites of different specificity; in one binding site the identity of a L chain junctional residue is critical whereas in the second binding site that residue is unimportant. Molecular modeling studies revealed major differences between calculated binding site structures for 26-10 when leucine is substituted for proline at position 96 in the 26-10 VL region.     

Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.     

Phage display and peptide mapping of an immunoglobulin light chain fibril-related conformational epitope

Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human kappa4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O'Nuallain, B., et al. (2006) Biochemistry 46, 1240-1247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vkappa and Vlambda N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody's cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1-4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both kappa and lambda light chain fibrils. We posit that the associated binding site involves a rare type VI beta-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis.     

Clathrin heavy chain, light chain interactions

Purified pig brain clathrin can be reversibly dissociated and separated into heavy chain trimers and light chains in the presence of non-denaturing concentrations of the chaotrope thiocyanate. The isolated heavy chain trimers reassemble into regular polygonal cage structures in the absence of light chains. The light chain fraction can be further resolved into its two components L alpha and L beta which give different one-dimensional peptide maps. Radiolabelled light chains bind with high affinity (KD < 10(-10) M) to heavy chain trimers, to heavy chain cages and to a 110,000 mol. wt. tryptic fragment of the heavy chain. Both light chains compete with each other and with light chains from other sources for the same binding sites on heavy chains and c.d. spectroscopy shows that the two pig brain light chains possess very similar structures. We conclude that light chains from different sources, despite some heterogeneity, have a highly conserved, high affinity binding site on the heavy chain but are not essential for the formation of regular cage structures.