Role of proline, glycerol, and heparin as protein folding aids during refolding of rabbit muscle creatine kinase

Aggregation of 3 M guanidine hydrochloride denatured creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) occurs after dilution into the refolding solution. Proline, glycerol and heparin sodium act as folding aids which can effectively inhibit aggregation of creatine kinase during refolding. Proline at 1 M concentration, glycerol at 10% concentration and heparin at 25 mg/ml not only completely prevented creatine kinase aggregation but also enabled the creatine kinase to return to its native state as well as to recover most of its native activity. The reactivity after the aggregation was completely blocked by the presence of each folding aid reached 65-80% of the native activity. Results of turbidity, activity, intrinsic fluorescence and 1-anilinonaphthalene-8-sulfonate binding fluorescence measurements suggested that the effect of heparin differs from that of proline and glycerol in its artificial chaperone-like behavior. Heparin may bind with creatine kinase both in the native state and during the refolding course. The results showed that this heparin-creatine kinase complex favorably restored the creatine kinase reactivity.     

Combination of the human prolyl isomerase FKBP12 with unrelated chaperone domains leads to chimeric folding enzymes with high activity

Folding enzymes often use distinct domains for the binding of substrate proteins ("chaperone domains") and for the catalysis of slow folding reactions such as disulfide formation or prolyl isomerization. The human prolyl isomerase FKBP12 is a small single-domain protein without a chaperone domain. Its very low folding activity could previously be increased by inserting the chaperone domain from the homolog SlyD (sensitive-to-lysis protein D) of Escherichia coli. We now inserted three unrelated chaperone domains into human FKBP12: the apical domain of the chaperonin GroEL from E. coli, the chaperone domain of protein disulfide isomerase from yeast, or the chaperone domain of SurA from the periplasm of E. coli. All three conveyed FKBP12 with a high affinity for unfolded proteins and increased its folding activity. Substrate binding and release of the chimeric folding enzymes were found to be very fast. This allows rapid substrate transfer from the chaperone domain to the catalytic domain and ensures efficient rebinding of protein chains that were unable to complete folding. The advantage of having separate sites, first for generic protein binding and then for specific catalysis, explains why our construction of the artificial folding enzymes with foreign chaperone domains was successful.     

The crystal structure of the fab fragment of the monoclonal antibody MAK33. Implications for folding and interaction with the chaperone bip

The Fab fragment of the murine monoclonal antibody, MAK33, directed against human creatine kinase of the muscle-type, was crystallized and the three-dimensional structure was determined to 2.9 A. The antigen-binding surface of MAK33 shows a convex overall shape typical for immunoglobulins binding large antigens. The structure allows us to analyze the environment of cis-prolyl-peptide bonds whose isomerization is of key importance in the folding process. These residues seem to be involved with not only domain stability but also seem to play a role in the association of heavy and light chains, reinforcing the importance of beta-strand recognition in antibody assembly. The structure also allows the localization of segments of primary sequence postulated to represent binding sites for the ER-specific chaperone BiP within the context of the entire Fab fragment. These sequences are found primarily in beta-strands that are necessary for interactions between the individual domains.     

Unique single‐domain antigen binding fragments derived from naturally occurring camel heavy‐chain antibodies

The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two heavy chains and no light chains. The amino acid sequence of the variable domain of the naturally occurring heavy‐chain antibodies reveals the necessary adaptations to compensate for the absence of the light chain. In contrast to the conventional antibodies, a large proportion of the heavy‐chain antibodies acts as competitive enzyme inhibitors. Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these heavy‐chain antibodies. The presence of the heavy‐chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only. These minimal antigen‐binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable. We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications. The structure of the camelid single domain is homologous to the human VH, however, the antigen‐binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs. This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti‐idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site. Copyright © 1999 John Wiley & Sons, Ltd.     

Hematopoietic cell kinase enhances osteosarcoma development via the MEK/ERK pathway

Osteosarcoma (OS) is a sarcoma with high rates of pulmonary metastases and mortality. The mechanisms underlying tumour generation and development in OS are not well-understood. Haematopoietic cell kinase (HCK), a vital member of the Src family of kinase proteins, plays crucial roles in cancer progression and may act as an anticancer target; however, the mechanism by which HCK enhances OS development remains unexplored. Therefore, we investigated the role of HCK in OS development in vitro and in vivo. Downregulation of HCK attenuated OS cell proliferation, migration and invasion and increased OS cell apoptosis, whereas overexpression of HCK enhanced these processes. Mechanistically, HCK expression enhanced OS tumorigenesis via the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway; HCK upregulation increased the phosphorylation of MEK and ERK and promoted epithelial-mesenchymal transition, with a reduction in E-cadherin in vitro. Furthermore, HCK downregulation decreased the tumour volume and weight in mice transplanted with OS cells. In conclusion, HCK plays a crucial role in OS tumorigenesis, progression and metastasis via the MEK/ERK pathway, suggesting that HCK is a potential target for developing treatments for OS.     

Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of Escherichia coli: evidence that chaperone activity is the prime effect of thioredoxin

In this study we investigate the effect of thioredoxin (Trx1) protein fusions in the production, oxidation, and folding of single chain Fv (scFv) antibodies in the cytoplasm of Escherichia coli. We analyze the expression levels, solubility, disulfide-bond formation, and antigen-binding properties of Trx1-scFv fusions in E. coli wild-type cells and isogenic strains carrying mutations in the glutathione oxidoreductase (gor) and/or thioredoxin reductase (trxB) genes. We compare the Trx1-scFv fusions with other reported systems for production of scFv in the cytoplasm of E. coli, including protein fusions to the maltose-binding protein. In addition, we analyze the effect of co-expressing a signal-sequence-less derivative of the periplasmic chaperone and disulfide-bond isomerase DsbC (DeltassDsbC), which has been shown to act as a chaperone for scFvs in the cytoplasm. The results reported here demonstrate that Trx1 fusions produce the highest expression level and induce the correct folding of scFvs even in the absence of DeltassDsbC in the cytoplasm of E. coli trxB gor cells. The disulfide bridges of Trx1-scFv fusions were formed correctly in E. coli trxB gor cells, but not in trxB single mutants. Antigen-binding assays showed that Trx1 has only a minor influence in the affinity of the scFv, indicating that Trx1-scFv fusions can be used without removal of the Trx1 moiety. In addition, we proved that a Trx1"AGPA" variant, having its catalytic cysteine residues mutated to alanine, was fully capable of assisting the folding of the fused scFvs. Taken together, our data indicate that the Trx1 moiety acts largely as an intramolecular protein chaperone, not as a disulfide bond catalyst, inducing the correct folding of scFvs in the cytoplasm of E. coli trxB gor cells.     

Dimeric trigger factor stably binds folding-competent intermediates and cooperates with the DnaK-DnaJ-GrpE chaperone system to allow refolding

Trigger factor (TF) is the first chaperone encountered by the nascent chain in bacteria and forms a stoichiometric complex with the ribosome. However, the functional significance of the high cytosolic concentration of uncomplexed TF, the majority of which is dimeric, is unknown. To gain insight into TF function, we investigated the TF concentration dependence of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reactivation yield in the presence and absence of the DnaK-DnaJ-GrpE chaperone system in vitro. Cross-linking results indicate that the observed decrease in the reactivation yield of GAPDH at high concentrations of TF is due to the formation of a stable complex between TF dimer and GAPDH intermediates. In the absence of TF, or at low TF concentrations, the DnaK-DnaJ-GrpE chaperone system had negligible effect on the GAPDH refolding yield. However, GAPDH intermediates bound and held by dimeric TF could be specifically rescued by the DnaK-DnaJ-GrpE chaperone system in an ATP-dependent manner. This indicates the potential of TF, in its dimeric form, to act as a binding chaperone, maintaining non-native proteins in a refolding competent conformation and cooperating with downstream molecular chaperones to facilitate post-translational or post-stress protein folding.     

Interaction of human creatine phosphokinase isoenzymes with rabbit antibodies and their Fab-fragments

The interaction of human creatine phosphokinase isoenzymes with rabbit antibodies and their Fab has been studied. It has been shown that Fab of the antibodies against MM or BB isoenzymes preserve high specificity of intact antibodies and the ability to inhibit creatine kinase isoenzymes. Differences between antibodies and their Fab have been found to exist with respect to the kinetics of binding with homologous isoenzymes: the rate of the complex formation for Fab is significantly higher. The interaction of creatine kinase isoenzymes with intact antibodies and their Fab is not affected by the addition of creatine kinase substrates. The antibodies against MM and BB isoenzymes have been used to study the individual properties of each subunit of the M- and B-type in a hybrid dimer MB. It has been shown that such properties of these subunits as the Michaelis constants, pH dependence and inhibition by homologous antibodies are identical to those of non-hybrid MM and BB isoenzymes, respectively.     

Catalytic activity and chaperone function of human protein-disulfide isomerase are required for the efficient refolding of proinsulin.

Protein-disulfide isomerase (PDI) catalyzes the formation, rearrangement, and breakage of disulfide bonds and is capable of binding peptides and unfolded proteins in a chaperone-like manner. In this study we examined which of these functions are required to facilitate efficient refolding of denatured and reduced proinsulin. In our model system, PDI and also a PDI mutant having only one active site increased the rate of oxidative folding when present in catalytic amounts. PDI variants that are completely devoid of isomerase activity are not able to accelerate proinsulin folding, but can increase the yield of refolding, indicating that they act as a chaperone. Maximum refolding yields, however, are only achieved with wild-type PDI. Using genistein, an inhibitor for the peptide-binding site, the ability of PDI to prevent aggregation of folding proinsulin was significantly suppressed. The present results suggest that PDI is acting both as an isomerase and as a chaperone during folding and disulfide bond formation of proinsulin.     

Effects of osmolytes on human brain-type creatine kinase folding in dilute solutions and crowding systems

The effects of osmolytes on the unfolding and refolding process of recombinant human brain-type creatine kinase (rHBCK) were comparatively, quantitatively studied in dilute solutions and macromolecular crowding systems (simulated by 100g/L polyethylene glycol 2000), respectively. The results showed that the osmolytes, including glycerol, sucrose, dimethylsulfoxide, mannitol, inositol, and xylitol, could both protect the rHBCK from denaturation induced by 0.8M GdnHCl and aid in the refolding of denatured-rHBCK in macromolecular crowding systems. When we examined the effects of sucrose and xylitol on the parameters of residual activity, reaction kinetics and intrinsic fluorescence of rHBCK during unfolding, it was found that the protecting effects of osmolytes in a macromolecular crowding system were more significant compared with those in a dilute solution, which resulted in more residual activities, protected the conformational changes and greatly decreased the rates of both the fast and slow tracks. Regarding the effects of glycerol, sucrose and mannitol on the denatured-rHBCK refolding parameters of refolding yield, reaction kinetics and aggregation, the results indicated that the osmolytes could alleviate the aggregation of rHBCK during refolding in both dilute solutions and macromolecular crowding systems, and the refolding yields and reaction rates under macromolecular crowding environment could be increased by the addition of osmolytes, though higher yields were obtained in the dilute solution. For further insight, osmolyte docking simulations and rHBCK denaturation were conducted successfully and confirmed our experimental results. The predictions based on the docking simulations suggested that the deactivation of guanidine may be blocked by osmolytes because they share common binding sites on rHBCK, and the higher number of interactions with rHBCK by osmolytes than guanidine may be one of the causes of rHBCK refolding. In brief, the additive effects of the exclusive volume effect from the macromolecular crowding system and the osmophobic effects from the osmolytes resulted in better performance of the osmolytes in a macromolecular crowding system, which also led to a better understanding of protein folding in the intracellular environment.     

Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain

Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4?nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles.     

具有分子伴侣功能的抗体

蛋白质的折叠问题一直是生物学研究的前沿之一，蛋白质稳态平衡的破坏与衰老及很多神经退行性疾病的发病机理密切相关，而蛋白质的正确折叠与蛋白质稳态在很大程度上取决于分子伴侣参与构建的复杂网络。许多研究表明，抗体可以作为分子伴侣促进蛋白质的正确折叠，并阻止蛋白质的异常聚集，抗体所具有的严格底物特异性使其具备了治疗特定蛋白质折叠病、帮助包涵体复性等应用潜力。本文简要介绍了分子伴侣的研究进展，详细阐述了具有分子伴侣功能的抗体及单链抗体的研究进展，最后重点讨论了可抑制蛋白质聚集的抗体的研究近况。     

A spin-label electron spin resonance study of the binding of mitochondrial creatine kinase to cardiolipin

The binding of the mitochondrial creatine kinase to aqueous dispersions of beef heart cardiolipin has been studied via the perturbation of the mobility of spin-labelled cardiolipin, using electron spin resonance (ESR) spectroscopy. In the presence of creatine kinase (1:1 protein/lipid ratio, by mass), the ESR spectra of cardiolipin labelled in a single acyl chain [n-(4,4-dimethyl-oxazolidinyl-N- oxy)stearoylcardiolipin] indicate a restriction of motion both at the C-5 and C-14 positions (n = 5, 14) of the lipid chains. The restriction in mobility was reversed by addition of phosphate or adriamycin, which are thought to inhibit the binding of creatine kinase to the mitochondrial membrane or to displace it from its binding site on the membrane. The effect of the protein on the chain mobility is consistent with surface binding of the protein; no positive evidence was obtained for penetration of the protein into the hydrophobic region of the membrane.     

Screening of single-chain variable fragments against TSP50 from a phage display antibody library and their expression as soluble proteins

A novel gene, testes-specific protease 50 (TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein inEscherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.     

Effect of osmolytes as folding aids on creatine kinase refolding pathway.

The influence of osmolytes, including dimethysulfoxide, glycine, proline and sucrose, on the refolding and reactivation courses of guanidine-denatured creatine kinase was studied by fluorescence emission spectra, circular dichroism spectra, recovery of enzymatic activity and aggregation. The results showed that low concentrations of dimethysulfoxide (<20%), glycine (<0.5 M), proline (<1 M) and sucrose (<0.75 M) improved the refolding yields of creatine kinase, but high osmolyte concentrations decreased its recovery. Sucrose favored the secondary structural formation of creatine kinase. Proline and sucrose facilitated refolding of the protein to its original conformation, while dimethysulfoxide and proline accelerated the hydrophobic collapse of creatine kinase to a packed protein. During the aggregation of creatine kinase, dimethysulfoxide and sucrose inhibited aggregation of creatine kinase, as did proline, but glycine was unable to inhibit aggregation. These systematic observations further support the suggestion that osmolytes, including low concentrations of dimethysulfoxide, proline or sucrose, possibly play a chaperone role in the refolding of creatine kinase. The results also indicate that sucrose and free amino acids are not only energy substrates and organic components in vivo, but also help correct protein folding.     

Improvement of antigen binding ability of human antibodies by light chain shifting

Human HB4C5 hybridoma cells produce a lung cancer-specific IgM human monoclonal antibody (mAb). HB4C5 human mAb cross-reacts with Candida cytochrome c (Cyt c) and carboxypeptidase (Cpase). Concanavalin A (ConA)-resistant variants of HB4C5 cells loss the original light chain followed by expression of various new light chains at a high incidence (light chain shifting) (Tachibana et al., 1996). HTD8 cells, one of the ConA-resistant variant subclones of HB4C5 cells, undergo the active light chain shifting and produce various sublines, each of which stably secretes new mAb consisting of a new light chain and a HB4C5 heavy chain. The new mAb exhibits altered antigen binding ability from that of the original antibody. We could expect that HTD8 cells can be used as ‘a light chain stem cell line’ to improve antigen binding ability and specificity of established human mAbs. A BD9D12 IgG human mAb recognizes lung cancer cells and cross-reacts with cytokeratin 8. Introduction of the heavy chain gene of BD9D12 mAb into HTD8 cells resulted in establishment of various sublines which secreted various kinds of hybrid antibodies consisting of different light chains derived from HTD8 subclones which underwent light chain shifting and a common IgG heavy chain derived from BD9D12. These hybrid antibodies exhibited different or improved reactivities to Cyt, Cpase, cytokeratin 8 and various cancer cells from those of parental mAb, demonstrating that light chain shifting can be applied to improve the affinity and specificity of human mAb. This revised version was published online in June 2006 with corrections to the Cover Date.     

Monoclonal antibody studies of creatine kinase. Antibody-binding sites in the N-terminal region of creatine kinase and effects of antibody on enzyme refolding.

(1) The binding sites of two monoclonal antibodies, CK-2A7 and CK-5H5, have been located to a 60-amino-acid sequence in the N-terminal region of creatine kinase (CK) by the use of chemical cleavage with formic acid (which cleaves proteins at Asp-Pro bonds) and cyanogen bromide (which cleaves at Met residues). (2) A simple method for preparing chemically-cleaved fragments of proteins for electrophoresis and Western blotting is described. (3) Binding studies with CK preparations from different animal species show that single amino acid changes at residues 39 or 82 prevent binding of CK-2A7 and CK-5H5 respectively. We suggest that Lys-39 and Glu-82 form parts of the binding sites on CK for the two monoclonal antibodies. The two sites lie in variable regions at each end of a highly-conserved sequence (residues 46 to 79) and are inaccessible to antibody in the native enzyme. (4) One of the antibodies, CK-2A7, inhibits the refolding of CK to native enzyme after denaturation by urea.     

Recombinant shark natural antibodies to thyroglobulin

As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.     

Folding on the chaperone: yield enhancement through loose binding

A variety of small cageless chaperones have been discovered that can assist protein folding without the consumption of ATP. These include mini-chaperones (catalytically active fragments of larger chaperones), as well as small proteins such as alpha-casein and detergents acting as "artificial chaperones." These chaperones all possess exposed hydrophobic patches on their surface that act as recognition sites for misfolded proteins. They lack the complexity of chaperonins (that encapsulate proteins in their inner rings) and their study can offer insight into the minimal requirements for chaperone function. We use molecular dynamics simulations to investigate how a cageless chaperone, modeled as a sphere of tunable hydrophobicity, can assist folding of a substrate protein. We find that under steady-state (non-stress) conditions, cageless chaperones that bind to a single substrate protein increase folding yields by reducing the time the substrate spends in an aggregation-prone state in a dual manner: (a) by competing for aggregation-prone hydrophobic sites on the surface of a protein, hence reducing the time the protein spends unprotected in the bulk and (b) by accelerating folding rates of the protein. In both cases, the chaperone must bind to and hold the protein loosely enough to allow the protein to change its conformation and fold while bound. Loose binding may enable small cageless chaperones to help proteins fold and avoid aggregation under steady-state conditions, even at low concentrations, without the consumption of ATP.     

Surface plasmon resonance (SPR) based binding studies of refolded single chain antibody fragments

Recent advances in Recombinant antibody technology / Antibody Engineering has given impetus to the genetic manipulation of antibody fragments that has paved the way for better understanding of the structure and functions of immunoglobulins and also has escalated their use in immunotherapy. Bacterial expression system such as Escherichia coli has complemented this technique through the expression of recombinant antibodies. Present communication has attempted to optimize the expression and refolding protocol of single chain fragment variable (ScFv) and single chain antigen binding fragment (ScFab) using E.coli expression system. Efficiency of refolding protocol was validated by structural analysis by CD, native folding by fluorescence and functional analysis by its binding with full length HIV-1 gp120 via SPR. Results show the predominant β–sheet (CD) as secondary structural content and native folding via red shift (tryptophan fluorescence). The single chain fragments have shown good binding with HIV-1 gp120 thus validating the expression and refolding strategy and also reinstating E.coli as model expression system for recombinant antibody engineering. SPR based binding analysis coupled with E.coli based expression and purification will have implication for HIV therapeutics and will set a benchmark for future studies of similar kind.