The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wiens, G. D.
Right arrow Articles by Rittenberg, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wiens, G. D.
Right arrow Articles by Rittenberg, M. B.
The Journal of Immunology, 2001, 167: 2179-2186.
Copyright © 2001 by The American Association of Immunologists

Mutation of a Single Conserved Residue in VH Complementarity-Determining Region 2 Results in a Severe Ig Secretion Defect1

Gregory D. Wiens2, Annemarie Lekkerkerker3, Imke Veltman4 and Marvin B. Rittenberg

Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 {lambda}1 L chain. These results represent the first demonstration that single somatic mutations in VH residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ability of somatic mutation to improve Ag-binding characteristics of Abs during immune responses is well documented (1, 2, 3, 4, 5); however, harmful effects of somatic mutation are less well characterized (6). The number of B cells directed to wastage pathways as a result of harmful somatic mutation probably far exceeds the number of cells with beneficial mutations (7, 8). Understanding the negative impact of somatic mutation on Ig structure is important as B cell survival appears to be dependent on the expression of functional Ig receptors (9). Failure to express functional Ig receptors due to harmful somatic mutations may also contribute to the genesis of pathological conditions such as Hodgkin’s disease (10, 11).

Previously, we created a collection of anti-phosphocholine (PC)5- and anti-nitrophenylphosphocholine (NPPC)-specific Abs as a model system to understand the deleterious nature of somatic mutation. In a panel of 160 random mutants generated from 2 Abs, T15 and PCG1-1, 16 VH mutants had defects in Ig trafficking and secretion (8, 12). These mutants secreted <10% of wild-type (WT) levels of Ig but expressed levels of VH mRNA and intracellular H chain equivalent to or greater than those of WT cells. Of the 16 secretion-defective mutants, all contain 2–4 residue changes, some in highly conserved residues in either VH complementarity-determining region 2 (CDR2) or framework 2 (FRW2). The role of these conserved residues in proper Ig folding and assembly is not clear, nor is it known how the quality control system within the endoplasmic reticulum (ER) of B cells maintains transport of properly assembled Ig while targeting improperly assembled Ig for degradation.

Here we conduct a molecular analysis of the secretion defect by determining the contribution of individual substitutions to the low secretion phenotype. Generation and analysis of a subset of single-site PCG1-1 mutants indicate that in each case, a single substitution in the highly conserved VH CDR2 position 51 impaired secretion markedly and led to intracellular accumulation of partially assembled H chains. A small amount of fully assembled Ab (1–3% of WT) was secreted and bound Ag similar to WT Ab, indicating the formation of functional combining sites. A computer model of the PCG1-1 combining site indicates that mutation at Ile51 indirectly impairs H and L chain pairing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructs and site-directed mutagenesis

The subcloning of the PCG1-1 VH gene into pTZ18U has been described previously (8). Single mutations Ile51->Lys and Ile51->Arg were introduced into the PCG1-1 VH gene using the Muta-Gene Phagemid in vitro mutagenesis kit (Bio-Rad, Richmond, CA). Mutations were introduced using the primers Ile51->Arg 5'-CCATCACCCCATCTCATTCCCAG-3' and Ile51->Lys 5'-CCATCACCCCATTTCATTCCCAG-3'. Mutant and WT VH genes were subcloned into the pSV2gpt expression vector containing a murine {gamma}2b C region gene (13), and the entire VH coding region was sequenced to ensure that no other mutations had been introduced. The forward primer (5'-TTAACCGTCTATGGTGTA-3') and the reverse primers (5'-TTGGCTCTGGGAGTTGTC-3' and 5'-CAGAATCCCCCCAACATG-3') were used.

Cell culture and stable transfection

The PCG1-1 heavy chain loss variant (PCG1-1 H- expressing the V{kappa}1 gene) has been described previously (8). J558L is a V{lambda}1-producing H chain loss variant of the IgA-secreting mouse myeloma J558 (14). Cell culture was done as described (8). Transfections were performed using the Lipofectin reagent (Life Technologies, Gaithersburg, MD), and mycophenolic acid-resistant clones were selected as described (13). Stable transfectants with intracellular H and L chains similar to WT, as determined by ELISA, were stored for further analysis.

Ig secretion assay

Stable transfectants were plated in triplicate at 1 x 106 cells in 1 ml of tissue culture medium in a 12-well plate. Culture supernatants and cell lysates were collected after 4 h as described (12).

Metabolic labeling, immunoprecipitation, and SDS-PAGE

Transfected cells were labeled with 150 µCi [35S]Express Protein labeling mix (NEN Life Sciences, Boston, MA) for 20 min for assembly experiments or for 4 h as previously described for chaperone-binding experiments (15). For chaperone-binding experiments, 100 µg/ml of the thiol-cleavable, chemical cross-linker dithiobis(succinimidyl propionate) (DSP; Pierce, Rockford, IL) was added to the lysis buffer immediately before use (16). Immunoprecipitates were washed with buffers as described previously (15). Ig was immunoprecipitated from clarified cell lysates or supernatants by incubation with protein A-Sepharose CL-4B (Pharmacia, Piscataway, NJ). All labeling experiments were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and IP lab gel software (version 1.5; Analytics, Vienna, VA).

ELISA

The concentration of Ig proteins in supernatants and cell lysates was determined by sandwich ELISA as described previously (12). Briefly, to determine H or H + L in the supernatant or lysate, ELISA plates were coated with rabbit anti-mouse IgG2b (Zymed Laboratories, San Francisco, CA), and the amount of Ab in the lysate or supernatant was then determined by a secondary alkaline phosphatase-conjugated goat anti-mouse {kappa} or rabbit anti-IgG2b Ab (Southern Biotechnology Associates, Birmingham, AL). All antisera were used at a 1/1000 dilution. Standard curves were generated using affinity-purified Ab from WT transfectant PCG1-1 ({gamma}2b, {kappa}). Ag binding was determined by direct binding ELISA on plates coated with PC-histone (1 µg/ml) as described (13). Hapten NPPC was obtained from Sigma-Aldrich (St. Louis, MO).

Computer modeling of WT PCG1-1 combining site

ABGEN, a fully automated web-based molecular modeling program (17), was used to construct a model of the PCG1-1 fragment variable (Fv) region.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ile51 is highly conserved in mouse and human VH

Previously, we generated 16 multisite secretion-defective mutants from 2 parent Abs; these mutants secrete <10% WT Ig levels but synthesize intracellular H and L chains at levels similar to WT transfectants (8, 12). Mutants P28 and P35 were identified for further analysis as both contain a nonconservative substitution at position 51, replacing isoleucine with the positively charged residue arginine or lysine (Fig. 1Go). None of the other mutations in P28 and P35 is sufficient to cause secretion defects because replacement of Asp54 with alanine or glutamic acid occurs singly in secretion competent Abs P20 and P9 (Fig. 1GoA). In addition, replacement of Met50 with valine occurs in a secretion competent anti-PC hybridoma, W140 (data not shown). Therefore, we focused our analysis on the position 51 substitutions. Strikingly, a compilation of all mouse VH sequences present in the Kabat database indicated that VH CDR2 Ile51 was highly conserved, occurring in 91% of all tabulated murine sequences (Fig. 1GoB). In addition, Ile51 is highly conserved in 81% (1348 of 1659) of human VH sequences. An explanation for the high degree of conservation of position 51 has not been reported. No VH sequences with arginine at position 51 were present in the Kabat database of murine VH as of March 10, 1998, and only 0.1% (3 of 2966) of the sequences contain a lysine residue at this position. In comparison, there is substantial amino acid variability in the neighboring positions 50 (Fig. 1GoC) and 52 (not shown).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 1. Amino acid sequences of VH CDR2 of the PCG1-1 WT and mutant Igs. A, PCG1-1 mutants P28, P35, P20, and P9 were created by random mutagenesis, and the secretion capacity (Sec.) was previously determined using a 4-h secretion assay; +, WT secretion; -, secretion <10% compared with WT (8 ). Amino acid positions are numbered according to the Kabat system (27 ). The frequency of occurrence is shown of each amino acid at VH position 51 (B) and position 50 (C). The total number of sequences (n) with an identified amino acid at position 51 was 2966; for position 50, n = 2944. Amino acids are denoted by the single letter abbreviation. Data were obtained from the Kabat database of murine VH sequences as of March 10, 1998 (27 ).

 
Mutation of Ile51 to arginine or lysine inhibits secretion and increases chaperone association

We tested whether a single substitution at position 51 was sufficient to impair secretion by introducing either an Ile51->Arg or Ile51->Lys into the VH CDR2 of PCG1-1. At least 20 clones of each mutant from 3 separate transfections were screened by ELISA. Stable transfectants secreted 1–3% of WT amounts of Ig (Table IGo). The presence of the intended mutation and lack of any other mutations in the V region were confirmed by directly sequencing VH RT-PCR products (data not shown). The secretion defect was not due to impaired H chain synthesis in that intracellular levels of the mutant H chains were 170% of WT as determined by H chain-specific ELISA (Table IGo). Similarly, the secretion defect was not due to decreased L chain synthesis as the amounts of intracellular and secreted L chain were similar between the WT and the mutants (Table IGo). To confirm the ELISA results, stable transfectants were labeled for 4 h with [35S]Met/Cys. Ig were immunoprecipitated from equal amounts of supernatant using protein A-Sepharose, which binds the H chain. Labeled proteins corresponding to H chains and L chains were present in the supernatants of both WT and the secretion-competent mutant P20 (Fig. 2GoA). The H chain doublet is characteristic of asymmetrical O-glycosylation of murine IgG2b H chains (18). Mutants, P35, P28, and site-directed mutants Ile51->Arg and Ile51->Lys secreted <3% of WT amounts of Ig as determined by densitometry confirming the ELISA results. The presence of abundant intracellular H chain of the correct size indicates that the secretion defect is not due to lack of H chain production (Fig. 2GoB). Less L chain coimmunoprecipitated with P35, P28, and Ile51->Arg and Ile51->Lys H chains as compared with WT suggesting a defect in H-L pairing. The PCG1-1 H chain loss variant cell line, H-, served as a control for nonspecific immunoprecipitation and demonstrates that the PCG1-1 L chain, in the absence of H chain, is not immunoprecipitated by protein A-Sepharose. In summary, both the ELISA and metabolic labeling experiments demonstrate that single substitutions of Ile51 to arginine or lysine were sufficient to impair Ig secretion in stably transfected cells.


View this table:
[in this window]
[in a new window]
  		Table I. Single amino acid mutations at position 51 in the VH impair Ig assembly and secretion in PCG1-1 cells expressing V{kappa}1

 


View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 2. Impaired Ig secretion mutants release little Ig into culture supernatant but contain intracellular H chain. Stably transfected cell lines were subject to 4-h steady state metabolic labeling with [35S]Met/Cys. A, Igs were immunoprecipitated from supernatants using protein A-Sepharose CL-4B. The PCG1-1 cell line, H-, produces L chain but does not produce H chain and is a control for nonspecific immunoprecipitation. B, Igs were immunoprecipitated from DSP-cross-linked cell lysates using protein A-Sepharose CL-4B. DSP was used to maintain H chain association with GRP94 as reported previously (16 ). Labels on the right mark the migration of GRP94, BiP, H chain, and L chain. Equal volumes of either supernatants or lysates were immunoprecipitated and loaded on 12% SDS-PAGE reducing gels. Results are representative of two to three experiments. The identity of BiP and GRP94 was confirmed by Western blot (data not shown).

 
Previously, we demonstrated that CDR2 mutations in the T15 H chain led to increased association of ER-localized chaperones BiP and GRP94 (15). Thus, we investigated whether a similar increase in chaperone association occurred with the PCG1-1 secretion mutants. In the presence of the chemical cross-linker dithiobis(succinimidyl propionate), secretion-impaired H chains exhibited a 7- to 13-fold increase in BiP association and a 10- to 16-fold increase in GRP94 association as compared with WT or the secretion-competent mutant P20 (Fig. 2GoB). Furthermore, intracellular H chains were sensitive to endoglycosidase H digestion, indicating that N-linked oligosaccharides on the majority of H chains had not undergone the normal enzymatic alterations in post-ER vesicles (data not shown). Taken together, these data suggest that the H chains of the multisite and single-site PGC1–1 low secretion mutants are retained within the ER as reported for other nonsecreted H and L chains (15, 19, 20, 21, 22).

Impaired L chain assembly of mutant H chains

To define the kinetics of secretion and H-L association, WT and Ile51->Arg cells were incubated for 20 min with medium containing [35S]Met/Cys followed by a chase period of 0, 2, or 10 h (Fig. 3Go). Immunoprecipitates were subjected to reducing SDS-PAGE to determine the Ig intracellular half-life and secretion kinetics (Fig. 3GoA), or subjected to nonreducing SDS-PAGE to follow H-L assembly (Fig. 3GoC). It is to be understood that H chains are secreted only as H-L complexes, but for simplicity we use the term H chain secretion. The WT H chains were secreted rapidly in comparison with H chains produced by the Ile51->Arg transfectant (Fig. 3GoA). The efficiency of H chain secretion was calculated by normalizing the amount of H chain to the amount of H chain present at time 0 (Fig. 3GoB). These analyses demonstrate that the majority of the labeled WT H chain was present in the supernatant by 2 h postchase. In contrast, only 1% of the labeled Ile51->Arg H chain was secreted into the supernatant by 2 h, and this percentage increased to only 5% by 10 h postchase. The WT H chain exhibited a shorter intracellular half-life (t1/2 = 6 h) than the Ile51->Arg H chain (t1/2 = 11 h). In summary, these data confirmed that H chains containing an arginine substitution for Ile51 were not efficiently secreted but displayed a prolonged intracellular persistence, suggesting that they are only slowly targeted for intracellular disposal.



View larger version (65K):
[in this window]
[in a new window]
  		FIGURE 3. Ig assembly and secretion in PCG1-1 WT and Ile51->Arg stable transfectants. A and C, Cells were 35S-pulse labeled for 20 min and chased for 0, 2, and 10 h. H chain and associated proteins were immunoprecipitated from cell lysates (Lysate) or supernatants (Sup). Samples were divided, and proteins were separated using reducing conditions (A) or nonreducing conditions (C). The identity of the assembly intermediates was confirmed by two-dimensional electrophoresis (data not shown). B, Kinetics of WT and mutant Ig secretion. Intracellular and secreted H chain are expressed as a percentage of intracellular H chain labeled after a 20-min incubation with [35S]Met/Cys (time 0). D, The amount of H2 assembly intermediate expressed as a percentage of total H chain. The total H chain was determined from samples separated using reducing SDS-PAGE conditions. Data in B and D are an average ± SEM of three independent experiments.

 
Interestingly, immunoprecipitation of intracellular mutant H chain from 0-, 2-, and 10-h chase time points coprecipitated a significant amount of L chain. We examined the stoichiometry of H chain to L chain to determine whether H-L chain association was altered in the Ile51->Arg mutant. The H:L ratio of secreted WT Ig was 1.1:1, and the H:L ratio in the small amount of secreted Ile51->Arg Ig was 0.9:1. Both ratios are within experimental error of the theoretical 1:1 ratio. However, the intracellular ratio of H to L in the Ile51->Arg mutant was higher than in WT. At the 2-h chase time point the ratio of H:L in the WT was 0.6:1, whereas in the mutant the ratio was 4.3:1. Thus, more L chain associates with WT H chain in WT cells than with H chain in the Ile51->Arg mutant cells, although the total amount of L chain present in the mutant cells is equal to that in WT cells (Table IGo).

To further investigate the kinetics of H-L assembly, samples were separated using nonreducing SDS-PAGE (Fig. 3GoC). In WT cells, H and L chain assembly occurred rapidly and was essentially complete in the 2-h lysate sample. In contrast, the majority of the Ile51->Arg H chain was blocked at the H2 assembly step. The amount of H2 in each sample was calculated using a PhosphorImager and normalized to the total H chain (Fig. 3GoD). At time 0, the WT H2 intermediate accounted for 20% of the total WT lysate H chain. This was reduced to 7% at 2 h and to <5% by 10 h. In contrast, the H2 intermediate in the Ile51->Arg mutant accounted for 55% of the total lysate H chain at 0 h, and this percentage remained stable at 2 h (59%) and 10 h (55%). During this time period, the total amount of H chain decreased by 54%. Furthermore, no discernable HL assembly intermediate was detected in the 0-h mutant lysate, whereas a strong band was present in the 0-h lysate of the WT (Fig. 3GoC). These data and the high ratio of H chain to L chain in the mutant suggest that impaired H-L assembly is responsible for the secretion defect. L chain that is coimmunoprecipitated with mutant H chain (Fig. 3GoA) is covalently associated because L chains were not dissociated under nonreducing SDS-PAGE conditions (Fig. 3GoC). Remarkably, the covalent attachment of L chain to small amounts of H chain occurs rapidly in that bands corresponding to H2L and H2L2 were present at the 0-h time point in the Ile51->Arg mutant. The failure to detect noncovalent L chains is not an artifact of insufficient denaturing conditions because BiP was displaced from the complex under the same conditions. Furthermore, it is unlikely that the covalent attachment is an artifact of the immunoprecipitation procedure because the lysis buffer contains 50 mM iodoacetamide to alkylate free sulfhydryl groups. A small amount of fully assembled H2L2 was present in the supernatant of the Ile51->Arg mutant, indicating that the block in secretion is not complete and is consistent with the small amount of Ig detected by the ELISA and steady state labeling experiments. In summary, these data indicate that the majority of H chain containing the Ile51->Arg mutation rapidly forms a covalent H2 assembly intermediate but that further assembly is blocked or aberrant.

PCG1-1 WT and single mutants bind to PC-histone

The presence of a small amount of fully assembled Ig in the supernatant led us to investigate whether this secreted Ig contained functional hapten-binding sites. We were able to purify a small amount of mutant Ig from culture supernatants using protein A chromatography followed by binding to PC-histone. Both the Ile51->Arg and Ile51-> Lys single mutants showed binding curves similar to those of PCG1-1 WT transfectant Ab (Fig. 4Go). Binding specificity was demonstrated by inhibition with 0.01 M NPPC, a hapten that mimics the diazophenyl linkage between PC and carrier protein (23). These data indicate that a low level of functional Ab was released from the mutants and that the conformation of this Ab is indistinguishable from that of WT, at least with respect to Ag binding.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 4. PCG1-1 WT and single Ile51->Arg or Ile51->Lys mutant proteins, purified from culture supernatant using protein A-Sepharose CL-4B, bind PC-histone with similar avidity. Binding was determined by direct ELISA. The binding of 5-ng/ml of each Ab was completely inhibited by preincubation with 0.01 mM of the hapten NPPC (shaded symbols). All samples were tested in triplicate and the results are representative of three separate experiments.

 
Partial rescue of secretion by expression with the J558 L chain

The capacity of mutant PCG1-1 H chains to form a small amount of functional Ab suggests that altering the fit between VH and VL may alter secretion. To test this possibility, we examined the secretion capacity of the PCG1-1 WT and mutant H chains with other L chains. Mutant H chains coexpressed with either the T15L or the V{kappa}10 L chain were not secretion competent (data not shown). However, coexpression of mutant PCG1-1 H chains with the J558 L chain partially rescued secretion (Table IIGo). Secretion of the Ile51->Arg mutant was 28% of WT amounts, whereas the Ile51->Lys mutant was 53% of WT amounts. Furthermore, abundant intracellular H-L complexes were present in both the WT and mutant cells (Table IIGo). L chain amounts were similar between transfectants (Table IIGo and data not shown). These results were confirmed by 4-h metabolic labeling experiments. Fully assembled Ig (H2L2) was released into the supernatant (Fig. 5GoA) containing H and L chains (Fig. 5GoB). Immunoprecipitation of intracellular H chain coprecipitated similar amounts of L chain (Fig. 5GoC). In contrast to WT H chain, mutant H chain coprecipitated with elevated amounts of BiP (4- to 6-fold) and GRP94 (6- to 8-fold). These data indicate that the PCG1-1 WT and mutant H chains fully assemble with J558 L chains, although the mutant H chains do so slightly less efficiently.


View this table:
[in this window]
[in a new window]
  		Table II. Secretion of PCG1-1 VH mutant H chains Ile51->Arg and Lys by the J558L cell line expressing {lambda}1

 


View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 5. Stably transfected J558L cells expressing PCG1-1 WT H chain (WT), the PCG1-1 Ile51->Arg (I51->R) H chain or the PCG1-1 Ile51->Lys (I51->K) H chain were subject to 4-h steady state metabolic labeling with [35S]Met/Cys. Igs were immunoprecipitated from supernatants (A and B) and lysates (C) using protein A-Sepharose CL-4B. Samples were run under nonreducing (nonred.) (A) or reducing (red.) conditions (B and C). Labels mark the migration of GRP94, BiP, H chain, and L chain.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Normal peripheral B cell survival is predicated on maintenance of Ig expression (9). During affinity maturation, maintenance of functional receptors is complicated by the extensive somatic hypermutation of the Ig VH and VL domains. Here, we report that a single V region mutation at a highly conserved residue in H chain CDR2 impairs secretion. Mutant H chains are sensitive to endoglycosidase H and coimmunoprecipitate with BiP and GRP94, consistent with retention in the ER (19, 20, 21, 22, 24). Furthermore, intracellular mutant H chains fail to associate covalently with L chain efficiently. These findings highlight the potential of somatic mutation in VH to alter the capacity of an H chain to assemble with its original VL counterpart (8, 25).

Sequence conservation of Ile51

Sequence analysis of 5000 Ig sequences has identified 47 positions conserved within the VH and VL of Igs (26). Most conserved residues are located in the FRW of the Ig domain and form contacts with residues in the same or adjacent {beta} sheets (26). Highly conserved residues are also present in CDRs; position 51 is an isoleucine in 91% of all murine and 81% of all human VH sequences compiled from the Kabat database (27). In addition, it has been noted that Ile occurs at position 51 in VH from the South American alligator Caiman, suggesting a conservation during vertebrate evolution (28). Although a systematic mutational analysis of conserved residues has not been performed, single mutations in other invariant residues Trp36 or Cys92 in VH do not alter Ig assembly or secretion (29, 30). Conservation of Ile51 in combination with our data indicating a functional role in assembly suggest that this position may be important to forming secretion competent Ig. It is also possible that the conservation of Ile51 in the Kabat database reflects a protection from the hypermutation mechanism. Ile51 in the VH CDR2 of PCG1-1 is encoded by the nucleotide triplet ATA and only single nucleotide substitutions are required to change Ile51 (ATA) to Arg (AGA) or Lys (AAA). Both AT and TA have a statistically higher dinucleotide mutation frequency than indicated by random chance (31). In addition, the T in the ATA triplet is more likely to be mutated than would be expected by chance as determined by analysis of murine intronic DNA and nonproductively rearranged human VH genes (31). This would argue against the proposition that there is an intrinsic bias against Ile51 mutations in the M141 germline gene of BALB/c mice, although it is formally possible that other factors may serve to shield this region from somatic mutation. Thus, the high degree of amino acid conservation and our functional data more likely suggest that Ile51 is conserved for its role in assembly and secretion.

Mechanism of the secretion defect

Ig that differ by a single amino acid are the simplest system for dissecting the structural requirements necessary for Ig assembly and secretion. To investigate the molecular environment surrounding Ile51, we constructed a computer generated model of the PCG1-1 WT combining site using the program ABGEN. This program constructs a molecular model of the Fv using an automated homology-based scaffolding technique (17). In the model of the PCG1-1 WT Fv (Fig. 6GoA), Ile51 does not contact L chain and is buried between VH CDR2 and FRW3. This is in agreement with the buried position of Ile51 in other Ig crystal structures (32, 33). Thus, if the Ile51 to arginine or lysine substitutions alter the interface of the H chain that contacts L chain, the mutations must do so indirectly. We have recently reported the structure of an anti-PC single-chain variable fragment, M3C65 at 2.35 Å, complexed with the hapten NPPC (34). M3C65 uses the same germline VH gene as PCG1-1, and both Abs bind NPPC. In the computer-generated PCG1-1 model and in the M3C65 crystal structure, Ile51 is surrounded by five residues, Gly55, Thr57, Ile69, Ser70, and Lys71 (Fig. 6GoB). Modeling the replacement of Ile51 with arginine in the PCG1-1 and M3C65 combining sites suggests that the side chain of arginine is accommodated within the folded Fv and that the side chain may extend into the solvent in between H chain CDR2 and FRW3 (data not shown). This accommodation would be consistent with the complete assembly of a small amount of mutant Ab that binds Ag indistinguishably from WT. One possibility is that the mutations at Ile51 have unfavorable interactions with the surrounding residues to create an energetic barrier to folding of CDR2 C' {beta} strand with the D and E {beta} strands of FRW3. Examination of the mutant side chain placement suggests potentially unfavorable electrostatic interactions with Lys71. Residue 71 of the heavy chain has been proposed to fix the relative dispositions of CDR1 and CDR2 depending on whether it is a bulky side chain (lysine or arginine) or a smaller side chain (alanine or valine) (35). Position 71 is a lysine or arginine in 38% (1125 of 2961 sequences) and an alanine or a valine in 56% (1661 of 2961) of sequences obtained from the Kabat database (27). Thus, we constructed a double mutant containing both Ile51->Arg and Lys71->Ala. This double mutant remained secretion impaired (data not shown), suggesting that an unfavorable interaction between the side chains of Arg51 and Lys71 is not the molecular explanation for the secretion defect. Furthermore, an unfavorable charge interaction would not explain the paucity of other substitutions at position 51 such as glycine, alanine, cysteine, tyrosine, phenylalanine, histidine, aspartic acid, glutamic acid, glutamine, and proline (Fig. 1GoB). Future experiments are required to determine the nature of interactions between Ile51 and surrounding residues, and the effects of substitutions at position 51 on the proper folding of the VH domain.



View larger version (57K):
[in this window]
[in a new window]
  		FIGURE 6. A, Computer-generated model showing a side view of the PCG1-1 WT Fv. Ile51 is buried between CDR2 and FRW3 and does directly contact L chain. H chain colors: yellow = CDR2; green = CDR3; red = CDR1; turquoise = FRW3. L chain is on the left. B, Close-up side view of Ile51 and the surrounding residues Thr57, Gly55, and Lys71. Atoms are displayed as balls and sticks. Colors: red, oxygen; blue, nitrogen. The C{alpha} backbone of Ile69 and Ser70 (side chains not shown) also encloses Ile51. Van der Waal’s radii surrounding each atom are represented as dots. The model was generated using ABGEN, and graphics were rendered using RasMol.

 
Besides influencing folding, it is possible that substitution of Ile51 may alter H chain interactions with molecular chaperones such as BiP. In our experiments, BiP coimmunoprecipitated with mutant H chain, whereas very little BiP was associated with WT H chain. Continuous binding of BiP to H chains in the absence of L chain results in H chain intracellular retention (19, 36) and that nonreleasing BiP ATPase mutants prevent secretion of L chains that are otherwise secretion competent (37). Furthermore, dissociation of BiP from the H chain is temporally linked to L chain arrival (38). Experiments using a truncated, two-domain heavy chain (VH-CH1) indicate that the disulfide bond within the CH1 domain is not formed until BiP dissociates (39). This suggests that the CH1 domain is not fully folded in the ER until it assembles with an L chain. One possibility is that the PCG1-1 L chain is unable to release BiP from the CH1 domain of the mutant H chains (39). It is also possible that mutation of Ile51 to arginine or lysine may directly enhance BiP binding or prevent its release from the V domain, thus impeding H chain maturation. Although BiP-binding sites on VH have not been precisely localized, a computer program (40) has predicted peptides from several VH that stimulate BiP ATPase activity in vitro (41). Interestingly, Knarr et al. (41) identified a potential BiP-binding site encompassing Ile51 in the VH of the 3D6 Ab. A peptide corresponding to VH residues 47–52a of the 3D6 Ab had a score of +7 (scores of +6 to +10 have a 3:1 chance of binding BiP) and stimulated BiP ATPase activity in vitro. We have used the same algorithm (40) to evaluate whether the single mutations affected the predicted BiP score in the same set of residues (47–53) from the VH of PCG1-1. The predicted BiP-binding score was +7 for WT but was reduced to -3 by the Ile51->Lys substitution or to +6 by the Ile51->Arg mutation. Thus, these results provide no evidence for the creation of a better BiP site, but rather predict reduced or no change in BiP binding to this putative site. Although the predicted BiP score does not suggest increased BiP binding, this result should be interpreted with caution given that Hellman et al. (42) demonstrated that there was no correlation between the presence of predicted BiP binding sequences and actual BiP binding to murine {lambda}I variable and constant domains. Further analysis is required to determine whether Ile51 is part of a BiP-binding site used in vivo and whether the single mutations affect this interaction.

Mutant {gamma}- and µ-chains, with reduced BiP binding due to a lack of the CH1 domain, partially assemble with L chain to form insoluble aggregates in the ER resembling Russell bodies (43, 44). However, our data do not support insoluble aggregation as the mechanism of secretion impairment, because soluble mutant PCG1-1 H chains were isolated at the 10-h chase time point, and we have failed to detect structures resembling Russell bodies within cells expressing mutant H chain (data not shown). The failure to form Russell bodies may be due to the isotype or specificity of the mutants (45). It is also possible that the increased association of BiP and GRP94 with the mutant H chains may prevent aggregation and thus formation of Russell bodies. Our results do not exclude the possibility that a small amount of H chain forms insoluble aggregates or that aggregation might be detected at a later time point.

Although sequence analysis indicates that Ile51 is highly conserved and the presence of arginine or lysine mutations at this position rare, we have found that an arginine substitution for isoleucine does not necessarily disrupt H-L pairing in all Igs. We have introduced this substitution into the M3C65 H chain where it is paired with the J558 {lambda}1 L chain. In this setting, the mutation had no effect on Ig secretion. However, the rate of intracellular assembly was modestly reduced (data not shown). Expression of the PCG1-1 Ile51 ->Arg H chain mutation in J558L {lambda}1 cells resulted in a partial rescue of the secretion defect, thus indicating that at least some L chains can compensate for a substitution at this residue. These results suggest that different combining sites may be differentially affected by changes at position 51. This is in accord with our previous findings that identical CDR2 mutations can have different effects on Ag binding and secretion depending on whether they are placed within the T15 or D16 Abs which use the same VH sequences (12, 46). Taken together, these results highlight the possibility that diversity in Ig sequence not only increases the repertoire for Ag but also reduces the potential for a particular mutation to harm Ig expression.

In summary, we demonstrate that even a single VH mutation may have dramatic consequences on Ig assembly and secretion. The PCG1-1 single-mutant H chains showed impaired association with L chains, although a small amount of covalently associated, functional H2L2 is secreted. These data and our data from multisite mutants (8) clearly demonstrate that mutations in H chain CDR2 and FRW can disrupt the production of functional Ig. Light chain somatic mutations in conserved residues can disrupt L chain secretion and also lead to L chain pathologies (47). Therefore, it is likely that somatic hypermutation in conserved residues in H and L chains may frequently generate B cells with impaired Ig formation with potentially pathological consequences. Single mutants provide a useful system for further elucidation of the molecular mechanisms governing B cell homeostasis and ER-mediated, Ig quality control.


   Acknowledgments
 
We thank M. Brown and Drs. G. Burrows, T. O’Hare, M. Stenzel-Poore, E. Whitcomb, and B. Wiens for critical comments and review of the manuscript.


   Footnotes
 
1 This work was supported in part by a Research Award Grant from the Oregon Chapter of the American Cancer Society (to G.D.W.) and National Institutes of Health Grants AI-14985 and AI-26827 (to M.B.R.). Back

2 Address correspondence and reprint requests to Dr. Gregory D. Wiens, Department of Molecular Microbiology and Immunology, L220, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97201. E-mail address: wiensg{at}ohsu.edu Back

3 Current address: Department of Immunology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands. Back

4 Current address: Department of Human Genetics-417, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Back

5 Abbreviations used in this paper: PC, phosphocholine; CDR, complementarity-determining region; ER, endoplasmic reticulum; FRW, framework region; NPPC, p-nitrophenylphosphocholine; WT, wild-type; BiP, Ig binding protein; Fv, variable region fragment of Ig; DSP, dithiobis(succinimidyl propionate). Back

Received for publication January 24, 2001. Accepted for publication June 15, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Griffiths, G. M., C. Berek, M. Kaartinen, C. Milstein. 1984. Somatic mutation and the maturation of immune response to 2-phenyl oxazolone. Nature 312:271.[Medline]
  2. Berek, C., C. Milstein. 1987. Mutation drift and repertoire shift in the maturation of the immune response. Immunol. Rev. 96:23.[Medline]
  3. Sharon, J., M. L. Gefter, L. J. Wysocki, M. N. Margolies. 1989. Recurrent somatic mutations in mouse antibodies to p-azophenylarsonate increase affinity for hapten. J. Immunol. 142:596.[Abstract]
  4. Brown, M., M. Stenzel-Poore, S. Stevens, S. K. Kondoleon, J. Ng, H. P. Bachinger, M. B. Rittenberg. 1992. Immunologic memory to phosphocholine keyhole limpet hemocyanin: recurrent mutations in the {lambda}1 light chain increase affinity for antigen. J. Immunol. 148:339.[Abstract]
  5. Milstein, C., M. S. Neuberger. 1996. Maturation of the immune response. Adv. Prot. Chem. 49:451.[Medline]
  6. Wiens, G. D., V. A. Roberts, E. A. Whitcomb, T. O’Hare, M. P. Stenzel-Poore, M. B. Rittenberg. 1998. Harmful somatic mutations: lessons from the dark side. Immunol. Rev. 162:197.[Medline]
  7. Shlomchik, M. J., S. Litwin, M. Weigert. 1989. The influence of somatic mutation on clonal expansion. Prog. Immunol. 7:415.
  8. Wiens, G. D., K. A. Heldwein, M. P. Stenzel-Poore, M. B. Rittenberg. 1997. Somatic mutation in VH complementarity-determining region 2 and framework region 2: differential effects on antigen binding and Ig secretion. J. Immunol. 159:1293.[Abstract]
  9. Lam, K. P., R. Kuhn, K. Rajewsky. 1997. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 90:1073.[Medline]
  10. Kanzler, H., R. Kuppers, M. L. Hansmann, K. Rajewsky. 1996. Hodgkin and Reed-Sternberg cells in Hodgkin’s disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells. J. Exp. Med. 184:1495.[Abstract/Free Full Text]
  11. Kuppers, R., K. Rajewsky. 1998. The origin of Hodgkin and Reed/Sternberg cells in Hodgkin’s disease. Annu. Rev. Immunol. 16:471.[Medline]
  12. Chen, C., T. M. Martin, S. Stevens, M. B. Rittenberg. 1994. Defective secretion of an immunoglobulin caused by mutations in the heavy chain complementarity determining region 2. J. Exp. Med. 180:577.[Abstract/Free Full Text]
  13. Chen, C., V. A. Roberts, M. B. Rittenberg. 1992. Generation and analysis of random point mutations in an antibody CDR2 sequence: many mutated antibodies lose their ability to bind antigen. J. Exp. Med. 176:855.[Abstract/Free Full Text]
  14. Weigert, M. G., I. M. Cesari, S. J. Yonkovich, M. Cohn. 1970. Variability in the {lambda} light chain sequences of mouse antibody. Nature 228:1045.[Medline]
  15. Martin, T. M., G. D. Wiens, M. B. Rittenberg. 1998. Inefficient assembly and intracellular accumulation of antibodies with mutations in VH CDR2. J. Immunol. 160:5963.[Abstract/Free Full Text]
  16. Melnick, J., S. Aviel, Y. Argon. 1992. The endoplasmic reticulum stress protein GRP94, in addition to BiP, associates with unassembled immunoglobulin chains. J. Biol. Chem. 267:21303.[Abstract/Free Full Text]
  17. Mandal, C., B. D. Kingery, J. M. Anchin, S. Subramaniam, D. S. Linthicum. 1996. ABGEN: a knowledge-based automated approach for antibody structure modeling. Nat. Biotechnol. 14:323.[Medline]
  18. Kim, H., Y. Yamaguchi, K. Masuda, C. Matsunaga, K. Yamamoto, T. Irimura, N. Takahashi, K. Kato, Y. Arata. 1994. O-Glycosylation in hinge region of mouse immunoglobulin G2b. J. Biol. Chem. 269:12345.[Abstract/Free Full Text]
  19. Bole, D. G., L. M. Hendershot, J. F. Kearney. 1986. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J. Cell Biol. 102:1558.[Abstract/Free Full Text]
  20. Dul, J. L., Y. Argon. 1990. A single amino acid substitution in the variable region of the light chain specifically blocks immunoglobulin secretion. Proc. Natl. Acad. Sci. USA 87:8135.[Abstract/Free Full Text]
  21. Reddy, P., A. Sparvoli, C. Fagioli, G. Fassina, R. Sitia. 1996. Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains. EMBO J. 15:2077.[Medline]
  22. Reddy, P. S., R. B. Corley. 1998. Assembly, sorting, and exit of oligomeric proteins from the endoplasmic reticulum. Bioessays 20:546.[Medline]
  23. Bruderer, U., M. P. Stenzel-Poore, H. P. Bachinger, J. H. Fellman, M. B. Rittenberg. 1989. Antibody combining site heterogeneity within the response to phosphocholine-keyhole limpet hemocyanin. Mol. Immunol. 26:63.[Medline]
  24. Melnick, J., J. L. Dul, Y. Argon. 1994. Sequential interaction of the chaperones BiP and GRP94 with immunoglobulin chains in the endoplasmic reticulum. Nature 370:373.[Medline]
  25. Primi, D., A. M. Drapier, P. A. Cazenave. 1987. Highly preferential VH-VL pairing in normal B cells results in antigen-independent selection of the available repertoire. J. Immunol. 138:1607.[Abstract]
  26. Gelfand, I. M., A. E. Kister. 1995. Analysis of the relation between the sequence and secondary and three-dimensional structures of immunoglobulin molecules. Proc. Natl. Acad. Sci. USA 92:10884.[Abstract/Free Full Text]
  27. Johnson, G., T. T. Wu. 2000. Kabat database and its applications: 30 years after the first variability plot. Nucleic Acids Res. 28:214.[Abstract/Free Full Text]
  28. Ohno, S., N. Mori, T. Matsunaga. 1985. Antigen-binding specificities of antibodies are primarily determined by seven residues of VH. Proc. Natl. Acad. Sci. USA 82:2945.[Abstract/Free Full Text]
  29. Sharon, J.. 1988. The invariant tryptophan in an H chain V region is not essential to antibody binding. J. Immunol. 140:2666.[Abstract]
  30. Rudikoff, S., J. G. Pumphrey. 1986. Functional antibody lacking a variable-region disulfide bridge. Proc. Natl. Acad. Sci. USA 83:7875.[Abstract/Free Full Text]
  31. Shapiro, G. S., K. Aviszus, D. Ikle, L. J. Wysocki. 1999. Predicting regional mutability in antibody V genes based solely on di- and trinucleotide sequence composition. J. Immunol. 163:259.[Abstract/Free Full Text]
  32. Chacko, S., E. A. Padlan, S. Portolano, S. M. McLachlan, B. Rapoport. 1996. Structural studies of human autoantibodies: crystal structure of a thyroid peroxidase autoantibody Fab. J. Biol. Chem. 271:12191.[Abstract/Free Full Text]
  33. Padlan, E. A.. 1994. Anatomy of the antibody molecule. Mol. Immunol. 31:169.[Medline]
  34. Brown, M., M. A. Schumacher, G. D. Wiens, R. G. Brennan, M. B. Rittenberg. 2000. The structural basis of repertoire shift in an immune response to phosphocholine. J. Exp. Med. 191:2101.[Abstract/Free Full Text]
  35. Tramontano, A., C. Chothia, A. M. Lesk. 1990. Framework residue 71 is a major determinant of the position and conformation of the second hypervariable region in the VH domains of immunoglobulins. J. Mol. Biol. 215:175.[Medline]
  36. Haas, I. G., M. Wabl. 1983. Immunoglobulin heavy chain binding protein. Nature 306:387.[Medline]
  37. Hendershot, L., J. Wei, J. Gaut, J. Melnick, S. Aviel, Y. Argon. 1996. Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants. Proc. Natl. Acad. Sci. USA 93:5269.[Abstract/Free Full Text]
  38. Hendershot, L. M.. 1990. Immunoglobulin heavy chain and binding protein complexes are dissociated in vivo by light chain addition. J. Cell Biol. 111:829.[Abstract/Free Full Text]
  39. Lee, Y. K., J. W. Brewer, R. Hellman, L. M. Hendershot. 1999. BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly. Mol. Biol. Cell 10:2209.[Abstract/Free Full Text]
  40. Blond-Elguindi, S., S. E. Cwirla, W. J. Dower, R. J. Lipshutz, S. R. Sprang, J. F. Sambrook, M.-J. Gething. 1993. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell 75:717.[Medline]
  41. Knarr, G., M. J. Gething, S. Modrow, J. Buchner. 1995. BiP binding sequences in antibodies. J. Biol. Chem. 270:27589.[Abstract/Free Full Text]
  42. Hellman, R., M. Vanhove, A. Lejeune, F. J. Stevens, L. M. Hendershot. 1999. 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. J. Cell Biol. 144:21.[Abstract/Free Full Text]
  43. Kaloff, C. R., I. G. Haas. 1995. Coordination of immunoglobulin chain folding and immunoglobulin chain assembly is essential for the formation of functional IgG. Immunity 2:629.[Medline]
  44. Valetti, C., C. E. Grossi, C. Milstein, R. Sitia. 1991. Russell bodies: a general response of secretory cells to synthesis of a mutant immunoglobulin which can neither exit from, nor be degraded in, the endoplasmic reticulum. J. Cell Biol. 115:983.[Abstract/Free Full Text]
  45. Tarlinton, D., I. Forster, K. Rajewsky. 1992. An explanation for the defect in secretion of IgM Mott cells and their predominant occurrence in the Ly-1 B cell compartment. Eur. J. Immunol. 22:531.[Medline]
  46. Chen, C., V. A. Roberts, S. Stevens, M. Brown, M. P. Stenzel-Poore, M. B. Rittenberg. 1995. Enhancement and destruction of antibody function by somatic mutation: unequal occurrence is controlled by V gene combinatorial associations. EMBO J. 14:2784.[Medline]
  47. Stevens, F. J., Y. Argon. 1999. Pathogenic light chains and the B-cell repertoire. Immunol. Today 20:451.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
G. D. Wiens, M. Brown, and M. B. Rittenberg
Repertoire Shift in the Humoral Response to Phosphocholine-Keyhole Limpet Hemocyanin: VH Somatic Mutation in Germinal Center B Cells Impairs T15 Ig Function
J. Immunol., May 15, 2003; 170(10): 5095 - 5102.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. D. Wiens, T. O'Hare, and M. B. Rittenberg
Recovering Antibody Secretion Using a Hapten Ligand as a Chemical Chaperone
J. Biol. Chem., October 26, 2001; 276(44): 40933 - 40939.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wiens, G. D.
Right arrow Articles by Rittenberg, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wiens, G. D.
Right arrow Articles by Rittenberg, M. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS