BiP Binding Sequences in Antibodies

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 VH and CH 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.

Overexpression of the 78-kDa Glucose-regulated Protein/Immunoglobulin-binding Protein (GRP78/BiP) Inhibits Tissue Factor Procoagulant Activity

“…First, immunoprecipitation experiments have failed to identify stable complexes between TF and GRP78/BiP in T24/83 cells. 2 Second, the BiP Score program (13), which has been successfully used to predict BiP binding sites within antibodies and HIV gp160 (13)(14)(15), indicates a low probability of TF-GRP78/BiP association. Third, TF PCA in rabbit brain thromboplastin is not inhibited by recombinant GRP78/BiP.…”

“…GRP78/BiP transiently associates with correctly folded proteins but forms more stable complexes with misfolded or incompletely assembled proteins (1,4,12). This association involves the binding of GRP78/BiP to hydrophobic motifs exposed on unfolded or unassembled polypeptides (13)(14)(15). Proteins stably bound to GRP78/BiP are subsequently translocated from the ER into the cytosol for proteasome-dependent degradation (16,17).…”

The Crystal Structure of the Fab Fragment of the Monoclonal Antibody MAK33

“…BiP ATPase Activity Assays-The binding of MAK33 peptides by BiP was monitored by the measurement of BiP ATPase activity as described previously (11). BiP used in this assay was purified from bovine pancreas as described (22), with the addition of gel filtration chromatography as a final purification step.…”

“…Notably leucine, but also tryptophan, residues are specifically enriched in BiP-binding sequences compared with the overall protein sequence. As evidenced from the stimulation of the ATPase activity of BiP upon heptapeptide binding in vitro, potential BiP-binding sites in vivo have been identified (11,12).…”

“…Based on the screening of a phage display peptide library for BiP-binding sequences (10), an algorithm has been developed that allows for the prediction of potential protein sequences, which might constitute BiP-binding sites in proteins (11,12). Analysis of the primary sequence of MAK33 as well as that of a related antibody indicated that binding does seem to require exposed hydrophobic residues.…”