Rapid,automated characterization of disulfide bond scrambling and IgG2 isoform determination

ABSTRACT

Human antibodies of the IgG2 subclass exhibit complex inter-chain disulfide bonding patterns that result in three structures, namely A, A/B, and B. In therapeutic applications, the distribution of disulfide isoforms is a critical product quality attribute because each configuration affects higher order structure, stability, isoelectric point, and antigen binding. The current standard for quantification of IgG2 disulfide isoform distribution is based on chromatographic or electrophoretic techniques that require additional characterization using mass spectrometry (MS)-based methods to confirm disulfide linkages. Detailed characterization of the IgG2 disulfide linkages often involve MS/MS approaches that include electrospray ionization or electron-transfer dissociation, and method optimization is often cumbersome due to the large size and heterogeneity of the disulfide-bonded peptides. As reported here, we developed a rapid LC-MALDI-TOF/TOF workflow that can both identify the IgG2 disulfide linkages and provide a semi-quantitative assessment of the distribution of the disulfide isoforms. We established signature disulfide-bonded IgG2 hinge peptides that correspond to the A, A/B, and B disulfide isoforms and can be applied to the fast classification of IgG2 isoforms in heterogeneous mixtures.     

Methods to identify the substrates of thiol‐disulfide oxidoreductases

The formation of a disulfide bond is a critical step in the folding of numerous secretory and membrane proteins and catalyzed in vivo. A variety of mechanisms and protein structures have evolved to catalyze oxidative protein folding. Those enzymes that directly interact with a folding protein to accelerate its oxidative folding are mostly thiol‐disulfide oxidoreductases that belong to the thioredoxin superfamily. The enzymes of this class often use a CXXC active‐site motif embedded in their thioredoxin‐like fold to promote formation, isomerization, and reduction of a disulfide bond in their target proteins. Over the past decade or so, an increasing number of substrates of the thiol‐disulfide oxidoreductases that are present in the ER of mammalian cells have been discovered, revealing that the enzymes play unexpectedly diverse physiological functions. However, functions of some of these enzymes still remain unclear due to the lack of information on their substrates. Here, we review the methods used by researchers to identify the substrates of these enzymes and provide data that show the importance of using trichloroacetic acid in sample preparation for the substrate identification, hoping to aid future studies. We particularly focus on successful studies that have uncovered physiological substrates and functions of the enzymes in the periplasm of Gram‐negative bacteria and the endoplasmic reticulum of mammalian cells. Similar approaches should be applicable to enzymes in other cellular compartments or in other organisms.     

Hydrogen exchange in BPTI variants that do not share a common disulfide bond.

Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-51, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-55]Ala and [30-51; 14-38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI folding intermediates containing either the 5-55 or the 30-51 disulfide bond.     

Evaluation of a noncanonical Cys40‐Cys55 disulfide linkage for stabilization of single‐domain antibodies

Incorporation of noncanonical disulfide linkages into single‐domain antibodies (sdAbs) has been shown to enhance thermostability and other properties. Here, we evaluated the effects of introducing a novel disulfide linkage formed between Cys residues at IMGT positions 40 and 55 on the melting temperatures (T _ms), reversibility of thermal unfolding, solubility, and antigen‐binding affinities of three types of sdAbs (V_HH, V_H, and V_L domains). The Cys40‐Cys55 disulfide linkage was tolerated by 9/9 V_HHs, 12/12 V_Hs, and 2/11 V_Ls tested and its formation was confirmed by mass spectrometry. Using circular dichroism, we found that the Cys40‐Cys55 disulfide linkage increased sdAb T _m by an average of 10.0°C (range: 0–21.8°C). However, enhanced thermostability came at the cost of a partial loss of refolding ability upon thermal denaturation as well as, for some sdAbs, significantly decreased solubility and antigen‐binding affinity. Thus, Cys40/Cys55 can be added to the panel of known locations for introducing stabilizing noncanonical disulfide linkages into antibody variable domains, although its effects should be tested empirically for individual sdAbs.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. I. Importance of hydrophobic interactions in stabilization of β‐hairpin structure

We previously studied a 16‐amino acid‐residue fragment of the C‐terminal β‐hairpin of the B3 domain (residues 46–61), [IG(46–61)] of the immunoglobulin binding protein G from Streptoccocus, and found that hydrophobic interactions and the turn region play an important role in stabilizing the structure. Based on these results, we carried out systematic structural studies of peptides derived from the sequence of IG (46–61) by systematically shortening the peptide by one residue at a time from both the C‐ and the N‐terminus. To determine the structure and stability of two resulting 12‐ and 14‐amino acid‐residue peptides, IG(48–59) and IG(47–60), respectively, we carried out circular dichroism, NMR, and calorimetric studies of these peptides in pure water. Our results show that IG(48–59) possesses organized three‐dimensional structure stabilized by hydrophobic interactions (Tyr50–Phe57 and Trp48–Val59) at T = 283 and 305 K. At T = 313 K, the structure breaks down because of increased chain entropy, but the turn region is preserved in the same position observed for the structure of the whole protein. The breakdown of structure occurs near the melting temperature of this peptide (T_m = 310 K) measured by differential scanning calorimetry (DSC). The melting temperature of IG(47–60) determined by DSC is T_m = 330 K and its structure is similar to that of the native β‐hairpin at all (lower) temperatures examined (283–313 K). Both of these truncated sequences are conserved in all known amino acid sequences of the B domains of the immunoglobulin binding protein G from bacteria. Thus, this study contributes to an understanding of the mechanism of folding of this whole family of proteins, and provides information about the mechanism of formation and stabilization of a β‐hairpin structural element. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Protein disulfide isomerase does not control recombinant IgG4 productivity in mammalian cell lines

Post‐translational limitations in the endoplasmic reticulum during recombinant monoclonal antibody production are an important factor in lowering the capacity for synthesis and secretion of correctly folded proteins. Mammalian protein disulfide isomerase (PDI) has previously been shown to have a role in the formation of disulfide bonds in immunoglobulins. Several attempts have been made to improve the rate of recombinant protein production by overexpressing PDI but the results from these studies have been inconclusive. Here we examine the effect of (a) transiently silencing PDI mRNA and (b) increasing the intracellular levels of members of the PDI family (PDI, ERp72, and PDIp) on the mRNA levels, assembly and secretion of an IgG4 isotype. Although transiently silencing PDI in NS0/2N2 cells suggests that PDI is involved in disulfide bond formation of this subclass of antibody, our results show that PDI does not control the overall IgG4 productivity. Furthermore, overexpression of members of the PDI family in a Chinese hamster ovary (CHO) cell line does not improve productivity and hence we conclude that the catalysis of disulfide bond formation is not rate limiting for IgG4 production. Biotechnol. Bioeng. 2010. 105: 770–779. © 2009 Wiley Periodicals, Inc.     

The effects of disulfide bonds on the denatured state of barnase

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended beta-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.     

A model for the interaction of the G3‐subdomain of Geobacillus stearothermophilus IF2 with the 30S ribosomal subunit

Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet‐tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2‐30S interaction, is positioned between the GTP‐binding G2 and the fMet‐tRNA binding C‐terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β‐sheets with each four anti‐parallel strands, followed by a C‐terminal α‐helix. In line with its role as linker between G3 and subdomain C1, this helix has no well‐defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB‐fold module, similar to that of the corresponding subdomain of Thermus thermophilus IF2, and to that of other known RNA‐binding modules such as IF2‐C2, IF1 and subdomains II of elongation factors EF‐Tu and EF‐G. Structural comparisons have resulted in a model that describes the interaction between IF2‐G3 and the 30S ribosomal subunit.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. III. Dynamics of long‐range hydrophobic interactions

A 20‐residue peptide, IG(42–61), derived from the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using circular dichroism, nuclear magnetic resonance (NMR) spectroscopy at various temperatures and by differential scanning calorimetry (DSC). Unlike other related peptides studied so far, this peptide displays two heat capacity peaks in DSC measurements (at a scanning rate of 1.5 deg/min at a peptide concentration of 0.07 mM), which suggests a three‐state folding/unfolding process. The results from DSC and NMR measurements suggest the formation of a dynamic network of hydrophobic interactions stabilizing the structure, which resembles a β‐hairpin shape over a wide range of temperatures (283–313 K). Our results show that IG (42–61) possesses a well‐organized three‐dimensional structure stabilized by long‐range hydrophobic interactions (Tyr50 ··· Phe57 and Trp48 ··· Val59) at T = 283 K and (Trp48 ··· Val59) at 305 and 313 K. The mechanism of β‐hairpin folding and unfolding, as well as the influence of peptide length on its conformational properties, are also discussed. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Engineered disulfide bonds improve thermostability and activity of L‐isoleucine hydroxylase for efficient 4‐HIL production in Bacillus subtilis 168

4‐Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l ‐Isoleucine hydroxylase specifically converts l‐ Ile to 4‐hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4‐hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l ‐isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half‐life of variant T181C was 4.03 h at 50°C, 10.27‐fold the half‐life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56‐fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole‐cell biotransformation, Bacillus subtilis 168/pMA5‐ido^T181C recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4‐hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4‐hydroxyisoleucine. Rational design of disulfide bond improved l ‐isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases.     

β‐amyloid model core peptides: Effects of hydrophobes and disulfides

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of β‐amyloid (Aβ21–30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aβ, and variants of these either cyclized or with an N‐terminal Cys. While Aβ21–30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aβ16–34 led to formation of typical amyloid fibrils. NMR showed no long‐range nuclear overhauser effect (nOes) in Aβ21–30, Aβ16–34, or their variants, however. Serial ¹H‐¹⁵N‐heteronuclear single quantum coherence spectroscopy, ¹H‐¹H nuclear overhauser effect spectroscopy, and ¹H‐¹H total correlational spectroscopy spectra were used to follow aggregation of Aβ16–34 and Cys‐Aβ16–34 at a site‐specific level. The addition of an N‐terminal Cys residue (in Cys‐Aβ16–34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aβ16–34 and Cys‐Aβ16–34, according to which Cys‐Aβ16–34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.     

ALS‐linked protein disulfide isomerase variants cause motor dysfunction

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) are ER foldases identified as possible ALS biomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized four ALS‐linked mutations recently identified in two major PDI genes, PDIA1 and PDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of these PDI variants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutant PDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of these PDI mutants. Finally, targeting ERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifies ER proteostasis imbalance as a risk factor for ALS, driving initial stages of the disease.     

Inhibition of bovine sperm‐oocyte fusion by a monoclonal antibody recognising the TEC‐2 epitope on bovine oocytes

The TEC‐2 antigenic determinant is a carbohydrate epitope located on a glycoprotein carrier molecule. In the mouse, this epitope is expressed on the zona pellucida and plasma membrane of the oocyte and is associated with the ZP2 glycoprotein and involved in the secondary sperm receptor mechanism. On the bovine oocyte expression is confined to the plasma membrane. The aim of this study was to determine the role the TEC‐2 epitope plays during fertilization in the bovine species using the monoclonal antibody TEC‐02. Incubating oocytes with the TEC‐02 antibody prior to fertilization inhibited cleavage in a dose‐dependent manner—the cleavage rate decreased as the concentration of the antibody increased. Significantly more sperm were bound to oocytes exposed to TEC‐02 (12 sperm/oocyte) compared to oocytes that were not incubated with the antibody (4 sperm/oocyte). Oocytes treated with the TEC‐02 antibody had a 7.5 ± 3.2% fusion rate and no cortical granule exocytosis compared with oocytes not exposed to the antibody, with 86.5 ± 5.8% of sperm‐oocyte fusions and release of cortical granules. The block to sperm‐oocyte fertilization observed in the pretreated group was overcome using intracytoplasmic sperm injection as the method of fertilization that bypassed the fusion process. Although sperm were binding to the oolemma these results suggest that fusion was not occurring and this may be due to the antibody occupying TEC‐2 epitope sites involved in the fusion process. In conclusion, the TEC‐2 epitope seems to be involved in sperm‐oocyte interaction in the bovine species and appears to be involved specifically during the fusion events of fertilization. Mol. Reprod. Dev. 54:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

Further experimental studies of the disulfide folding transition of ribonuclease A

Two very different mechanisms of folding have been proposed from experimental studies of disulfide formation in reduced ribonuclease A. (1) A pathway in which the rate-limiting step separates fully folded protein from all other disulfide intermediates and occurs solely in three-disulfide intermediates. (2) A multiple pathway mechanism with different rate-limiting steps for each pathway. The various rate-limiting steps involve disulfide breakage, formation, and rearrangement in intermediates with one, two, three, and four protein disulfides. To distinguish between these two mechanisms, we have carried out further studies of both unfolding and refolding. Refolding of reduced ribonuclease A requires three-disulfide intermediates to accumulate; negligible refolding occurs when only the nearly random one- and two-disulfide intermediate species are populated. Therefore, no rate-limiting steps of the type postulated in mechanism (2) occur in intermediates with one and two protein disulfides. Unfolding and disulfide reduction is an all-or-none process; no disulfide intermediates accumulate to detectable levels or precede the rate-limiting step. Mechanism (2) requires that such intermediates precede the rate-limiting step and accumulate to substantial levels. The different proposals were shown not to result from the use of different solution conditions or disulfide reagents; the two sets of data are not inconsistent. Instead, the inappropriate mechanism (2) resulted from an incorrect kinetic analysis and misinterpretation of the kinetics of disulfide formation and breakage.     

NMR characterization of the conformational fluctuations of the human lymphocyte function‐associated antigen‐1 I‐domain

Lymphocyte function‐associated antigen‐1 (LFA‐1) is an integrin protein that transmits information across the plasma membrane through the so‐called inside‐out and outside‐in signaling mechanisms. To investigate these mechanisms, we carried out an NMR analysis of the dynamics of the LFA‐1 I‐domain, which has enabled us to characterize the motions of this domain on a broad range of timescales. We studied first the internal motions on the nanosecond timescale by spin relaxation measurements and model‐free analysis. We then extended this analysis to the millisecond timescale motions by measuring ¹⁵N‐¹H residual dipolar couplings of the backbone amide groups. We analyzed these results in the context of the three major conformational states of the I‐domain using their corresponding X‐ray crystallographic structures. Our results highlight the importance of the low‐frequency motions of the LFA‐1 I‐domain in the inactive apo‐state. We found in particular that α‐helix 7 is in a position in the apo‐closed state that cannot be fully described by any of the existing X‐ray structures, as it appears to be in dynamic exchange between different conformations. This type of motion seems to represent an inherent property of the LFA‐1 I‐domain and might be relevant for controlling the access to the allosteric binding pocket, as well as for the downward displacement of α‐helix 7 that is required for the activation of LFA‐1.     

Synthetic sRNA‐Based Engineering of Escherichia coli for Enhanced Production of Full‐Length Immunoglobulin G

Production of monoclonal antibodies (mAbs) receives considerable attention in the pharmaceutical industry. There has been an increasing interest in the expression of mAbs in Escherichia coli for analytical and therapeutic applications in recent years. Here, a modular synthetic biology approach is developed to rationally engineer E. coli by designing three functional modules to facilitate high‐titer production of immunoglobulin G (IgG). First, a bicistronic expression system is constructed and the expression of the key genes in the pyruvate metabolism is tuned by the technologies of synthetic sRNA translational repression and gene overexpression, thus enhancing the cellular material and energy metabolism of E. coli for IgG biosynthesis (module 1). Second, to prevent the IgG biodegradation by proteases, the expression of a number of key proteases is identified and inhibited via synthetic sRNAs (module 2). Third, molecular chaperones are co‐expressed to promote the secretion and folding of IgG (module 3). Synergistic integration of the three modules into the resulting recombinant E. coli results in a yield of the full‐length IgG ≈150 mg L^?1 in a 5L fed‐batch bioreactor. The modular synthetic biology approach could be of general use in the production of recombinant mAbs.     

Monoclonal antibodies to the bovine immunoglobulin G2a allotypes A1 and A2.

Production of murine monoclonal antibodies (Mabs) to bovine IgG2a-A1 and A2 allotypes resulted in three Mabs being selected as anti-IgG2a-A1 and A2 reagents. Two Mabs recognize the A1 allotype and 1 recognizes the A2 allotype. Initial epitope mapping with the Mabs indicates that one of the A1 epitopes resides in the hinge region and the other epitope resides more toward the C-terminus of the immunoglobulin. The A2 epitope recognized by the A2 Mab does not appear to reside in the hinge region of the immunoglobulin but apparently resides more C-terminal.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. II. Interplay of local backbone conformational dynamics and long‐range hydrophobic interactions in hairpin formation

Two peptides, corresponding to the turn region of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus, consisting of residues 51–56 [IG(51–56)] and 50–57 [IG(50–57)], respectively, were studied by circular dichroism and NMR spectroscopy at various temperatures and by differential scanning calorimetry. Our results show that the part of the sequence corresponding to the β‐turn in the native structure (DDATKT) of the B3 domain forms bent conformations similar to those observed in the native protein. The formation of a turn is observed for both peptides in a broad range of temperatures (T = 283–323 K), which confirms the conclusion drawn from our previous studies of longer sequences from the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G (16, 14, and 12 residues), that the DDATKT sequence forms a nucleation site for formation of the β‐hairpin structure of peptides corresponding to the C‐terminal part of all the B domains of the immunoglobulin binding protein G. We also show and discuss the role of long‐range hydrophobic interactions as well as local conformational properties of polypeptide chains in the mechanism of formation of the β‐hairpin structure. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Solution structures of polcalcin Phl p 7 in three ligation states: Apo‐, hemi‐Mg2+‐bound,and fully Ca2+‐bound

Polcalcins are small EF‐hand proteins believed to assist in regulating pollen‐tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain‐swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.0, containing either 5.0 mM EDTA, 5.0 mM Mg²⁺, or 100 μM Ca²⁺. Phl p 7 is monomeric in all three ligation states. In the apo‐form, both EF‐hand motifs reside in the closed conformation, with roughly antiparallel N‐ and C‐terminal helical segments. In 5.0 mM Mg²⁺, the divalent ion is bound by EF‐hand 2, perturbing interhelical angles and imposing more regular helical structure. The structure of Ca²⁺‐bound Phl p 7 resembles that previously reported for Bet v 4—likewise exposing apolar surface to the solvent. Occluded in the apo‐ and Mg²⁺‐bound forms, this surface presumably provides the docking site for Phl p 7 targets. Unlike Bet v 4, EF‐hand 2 in Phl p 7 includes five potential anionic ligands, due to replacement of the consensus serine residue at –x (residue 55 in Phl p 7) with aspartate. In the Phl p 7 crystal structure, D55 functions as a helix cap for helix D. In solution, however, D55 apparently serves as a ligand to the bound Ca²⁺. When Mg²⁺ resides in site 2, the D55 carboxylate withdraws to a distance consistent with a role as an outer‐sphere ligand. ¹⁵N relaxation data, collected at 600 MHz, indicate that backbone mobility is limited in all three ligation states. Proteins 2013. © 2012 Wiley Periodicals, Inc.