Formation Of High Molecular Weight Aggregates Research Articles

Organophosphate Hydrolase (OPH) Designed as a Functional Monomer

Organophosphate hydrolase (OPH) mutants haveshown potential use as a medical countermeasure against a range of differentorganophosphorus compounds (OPs). OPH is typicallyexpressed in bacteria as a homodimer. Two separate monomers (~37kDa each) self‐assemble throughnon‐covalent bonding at the enzyme face close to the putative active site, andenzyme activity is dependent upon dimer formation. OPH homodimers do notsecrete expediently from mammalian cells and are often misfolded. This causespotential problems when trying to express the protein from a heterologous plasmidor viral delivery system. To enhance secretion of OPH from mammalian cells, wesought to increase protein solubility without catastrophic detriment toactivity and without addition of fusion proteins. To this end, we designed OPH tobe expressed as a functional monomer by joining two OPH subunits with a poly‐glycinelinker and expressing it as a single polypeptide in bacteria to evaluateactivity. We created the single polypeptide OPH with a tether 10 or 35 aminoacids in length between the two halves, and named them T10 and T35, respectively. Western blot analysis and paraoxon hydrolysis assays revealedthat T10 was being produced and there was a 71% loss in specific activity comparedto an untethered homodimer positive control. This was a surprise as we expectedT10 to have no enzymatic activity since the tether was too short to allowproper dimer formation. The T35 functional monomer (~76 kDa) was produced showing 29% loss of specific activity. Both T10 and T35 OPH showed high molecular weight aggregate formation greater than 250 kDa in dynamic light scattering and native polyacrylamide gels. Construction of a functional OPH monomer with activity against OPs is instrumental in designing secretable OPHs for adeno‐associatedviral expression. It may also prove useful in the development of chimeric OPHs for maintaining control over the composition of each OPH half, allowing blended activities against a combination of organophosphorus substrates. This is the first attempt known for producing OPH as a functional monomer.Support or Funding InformationThe views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division. This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and USAMRMC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ameliorative effect of myo-inositol on red blood cell alterations in polycystic ovary syndrome: in vitro study

Polycystic ovary syndrome (PCOS)is a gynecological endocrine disorder which is associated with systemic inflammatory status inducing red blood cells (RBC) membrane alterations related to insulin resistance and testosterone levels which could be greatly improved by myo-inositol (MYO) uptake. In this study we aim to evaluate the effect of MYO in reducing oxidative-related alterations through in vitro study on PCOS RBC. Blood samples from two groups of volunteers, control group (CG, n = 12) and PCOS patient group (PG, n = 12), were analyzed for band 3 tyrosine phosphorylation (Tyr-P), high molecular weight aggregate (HMWA), IgG in RBC membranes, and glutathione (GSH) in cytosol, following O/N incubation in the presence or absence of MYO. PCOS RBC underwent oxidative stress as indicated by higher band 3 Tyr-P and HMWA and increased membrane bound autologous IgG. Twenty four hours (but not shorter time) MYO incubation, significantly improved both Tyr-P level and HMWA formation and concomitant membrane IgG binding. However, no relevant modification of GSH content was detected. PCOS RBC membranes are characterized by increased oxidized level and enhanced sensitivity to oxidative injuries leading to potential premature RBC removal. MYO treatment is effective in reducing oxidative related abnormalities in PCOS patients probably restoring the inositol phospholipid pools of the membranes.

Aggregation and Chemical Modification of Monoclonal Antibodies under Upstream Processing Conditions

To investigate antibody stability and formation of modified species under upstream processing conditions. The stability of 11 purified monoclonal human IgG1 and IgG4 antibodies, including an IgG1-based bispecific CrossMab, was compared in downscale mixing stress models. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. Analytical techniques include size exclusion chromatography (SEC), turbidity measurements, cation exchange chromatography (cIEX), dynamic light scattering (DLS) and differential scanning calorimetry (DSC). Sensitivity in downscale stress models varies among antibodies and results in formation of high molecular weight (HMW) aggregates. Stability is increased in cell culture medium and in bioreactors. Media components stabilizing the proteins were identified. Extensive chemical modifications were detected both in stress models as well as during production of antibodies in cell culture fermentations. Protective compounds must be present in chemically defined fermentation media in order to stabilize antibodies against the formation of HMW aggregates. An increase in chemical modifications is detectable in bioreactor stress models and over the course of cell culture fermentations; this increase is dependent on the expression rate, pH, temperature and fermentation time. Consequently, product heterogeneity increases during upstream processing, and this compromises the product quality.

Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions, Part 2: Nucleation and Growth Kinetics with Competing Growth Mechanisms

Aggregation mechanisms as a function of pH were assessed for the IgG1 antibody described in Part 1 (Brummitt RK, Nesta DP, Chang L, Chase SF, Laue TM, Roberts CJ. Non-native aggregation of an IGG1 antibody in acidic conditions: 1. Unfolding, colloidal interactions, and high molecular weight aggregate formation. J Pharm Sci. In press). Aggregation kinetics along with static light scattering and size-exclusion chromatography indicated that the aggregate nucleus was a dimer for all conditions tested, and this was semiquantitatively consistent with scaling of the characteristic time scale for nucleation (τ(n)) versus protein concentration at pH 4.5 and pH 5.5. Changing pH significantly altered the mechanism of aggregate growth, as well as the size and solubility of aggregates that were formed. Aggregates at pH 3.5 grew primarily by monomer addition and remained small and soluble. Aggregates at pH 4.5 grew first by chain polymerization, followed by condensation polymerization, leading ultimately to large insoluble particles. At pH 5.5, monomer loss resulted primarily in insoluble aggregate formation, with only low levels of soluble aggregate intermediates detected at early times. The influence of pH on aggregate solubility and the reversibility of aggregate phase separation were confirmed via cloud point titrations. Qualitatively, the global aggregation behavior was consistent with reduction of charge-charge repulsions as a primary factor in promoting larger aggregates and aggregate phase separation.

Conformational Implications of an Inversed pH-Dependent Antibody Aggregation

Antibody formulation development relies on accelerated stability data at elevated temperatures to optimize formulation parameters. However, the pH- and temperature-dependence of aggregation is complicated for antibody formulations. In this study, a human monoclonal IgG2 antibody exhibited typical pH-dependent dimer formation under normal storage conditions (4 and/or 29°C). However, an inversed pH-dependence was discovered for high molecular weight aggregate formation at elevated temperatures (37°C). The different stability profiles exhibited at the various storage conditions resulted in nonlinearity of the Arrhenius kinetics. Thermal unfolding at or below 37°C was not evident by differential scanning calorimetry. Enriched populations of the structural isoforms of the IgG2 subclass were tested for their unique temperature and pH-dependence of aggregation. The Arrhenius kinetics of aggregation for each of the individual IgG2 isoforms was also nonlinear. However, the temperature-dependence of clipping suggested that clip-mediated aggregation was responsible for the increased higher order aggregates at low pH and elevated temperatures. Unique clip species resulting from the conformational differences between the IgG2 isoforms lead to increased aggregation. These results have implications on the mechanisms of antibody aggregation and on the validity of accelerated data to predict shelf-life accurately. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3031–3042, 2009

Disulfide Bond Mediates Aggregation, Toxicity, and Ubiquitylation of Familial Amyotrophic Lateral Sclerosis-linked Mutant SOD1

Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathological hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insoluble SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermolecular disulfide bonds. Here we show that a non-physiological intermolecular disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high molecular weight aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degradation. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys(6)- and Cys(111)-disulfide cross-linked form and targeted it for proteasomal degradation.

Mutation in a short-chain collagen gene, CTRP5, results in extracellular deposit formation in late-onset retinal degeneration: a genetic model for age-related macular degeneration.

A primary feature of age-related macular degeneration (AMD) is the presence of extracellular deposits between the retinal pigment epithelium (RPE) and underlying Bruch's membrane, leading to RPE dysfunction, photoreceptor death and severe visual loss. AMD accounts for about 50% of blind registrations in Western countries and is a common, genetically complex disorder. Very little is known regarding its molecular basis. Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder with striking clinical and pathological similarity to AMD. Here we show that L-ORD is genetically heterogeneous and that a proposed founder mutation in the CTRP5 (C1QTNF5) gene, which encodes a novel short-chain collagen, changes a highly conserved serine to arginine (Ser163Arg) in 7/14 L-ORD families and 0/1000 control individuals. The mutation occurs in the gC1q domain of CTRP5 and results in abnormal high molecular weight aggregate formation which may alter its higher-order structure and interactions. These results indicate a novel disease mechanism involving abnormal adhesion between RPE and Bruch's membrane.

Functional and Structural Studies of α-Crystallin from Galactosemic Rat Lenses

Chaperone-like activity and structural changes of lens α-crystallin from rats fed with galactose at various time intervals have been studied using high-performance liquid chromatograph (HPLC), circular dichroism (CD), and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence emission. It was found that chaperone-like activity of α-crystallin from galactose-fed rats toward dithiothreitol (DTT)-induced insulin B aggregation started to decrease after 3 weeks and decreased significantly after 5 weeks. Consistent results were observed in lens morphology, and lens opacity slightly developed after 3 weeks and became obvious after 5 weeks. HPLC analysis for chaperone function showed that the formation of high molecular weight aggregates (HMWA) of α-/γ-crystallins decreases with the increase of galactose-feeding time, revealing that chaperone-like activity is concomitant with the formation of HMWA. Circular dichroism results showed the reduction of β-sheet structure and loss of microenvironment of aromatic-type amino acids for opaque lenses, indicating α-crystallin's secondary and tertiary structure changed with the development of the lens opacity. ANS binding site estimated by Klotz equation showed it is 1.5 times higher at room temperature and is 2.4 times higher at 58°C for age-matched normal α-crystallin than for 5-week galactose-fed lens α-crystallin, indicating opaque lens α-crystallin loses the ability to assemble into an appropriately placed hydrophobic regions. The overall results accordingly indicated that galactose-induced cataractous α-crystallin has disordered structure, leading to the loss of its chaperone-like activity.

The Formation of Oxidatively Induced High-Molecular-Weight Aggregate of α-/γ-Crystallins

α-/γ-Crystallin interactions under oxidation with ascorbate–FeCl3–EDTA–H2O2 followed by dialysis have been studied. A high-molecular-weight aggregate (HMWA) composed of α- and γ-crystallin was observed for the mixture of the dialyzed α-crystallin and the oxidized γ-crystallin through gel-filtration chromatography. This illustrates an interaction between α-crystallin and partially denatured γ-crystallin induced by oxidation. No HMWA formation was observed under the condition without dialysis and/or with the addition of catalase to the oxidized γ-crystallin prior to the addition of α-crystallin. More HMWA was formed by oxidized γ-crystallin followed by the addition of α-crystallin than by simultaneous oxidation of both α- and γ-crystallins. Conformational changes of α-crystallin during oxidation analyzed by circular dichroism spectra showed that oxidized α-crystallin can gradually be restored to an ordered structure through dialysis. The overall results imply that structural changes of both α- and γ-crystallins and dialysis are required to form HMWA. The observation of this oxidatively induced chaperone/substrate complex suggests that an efficient chaperone-like protective action against oxidative insults may exist in vivo.

Theoretical and experimental basis for the inhibition of cataract.

Aggregation of the lens proteins to form high molecular weight clusters is a major contributing factor in age-onset nuclear cataract [Benedek, G. B. (1971) Theory of transparency of the eye. Appl. Optics, 10, 459-473]. This aggregation occurs continually throughout life and contributes to an exponential increase, as a function of age, in the intensity of the light backscattered out of the lens. The time constant deltaT for this exponential increase in human populations is a valuable index, helpful for conducting clinical trials. In-vitro studies have identified reagents capable of inhibiting high molecular weight aggregate formation, as well as the non-covalent interprotein interactions responsible for phase separation. These reagents are also found experimentally to be effective cataract inhibitors in animal model systems in vivo. We believe that the stage is now set for human clinical trials of putative cataract inhibitors. We present rough quantitative estimates of the trial parameters needed to assure an unambiguous determination of efficacy in a trial population. Such a trial simply requires a measurement of the time constant deltaT in the treated population relative to the untreated population. A successful outcome of the trial is indicated if deltaT increases by 20% over that found for the untreated population. Our estimates suggest efficacy could be determined in a two year trial involving about 300 subjects in the treated group.

Glycation Mediated Lens Crystallin Aggregation and Cross-linking by Various Sugars and Sugar Phosphates In Vitro

Glycation of lens crystallins results in protein conformational changes, oxidation, browning and aggregation. Though glucose is the major sugar, other sugars and sugar phosphates generated as intermediates of metabolic pathways are present in the lens, albeit at low concentrations. In this study we incubated bovine lens soluble fraction with various sugars and sugar phosphates (5 mM for 10 days). The reactivity was in the order trioses > tetroses > penroses > hexoses. High molecular weight (HMW) aggregates were also formed at a comparable rate. Increased levels of fluorescence were associated with the HMW aggregates with fast reacting sugars. The phosphorylated derivatives were only slightly more reactive than their respective sugars. Interestingly, fructose-1,6-diphosphate was more reactive and cross-linked more readily than fructose-6-phosphate. Gel electrophoresis under reducing and nonreducing conditions showed formation of disulfide linked protein aggregates with slow reacting sugars such as glucose and non-disulfide covalent linked protein aggregates with fast reacting sugars such as erythrose. In contrast, if 0·1 M DTT was present in erythrose incubations (a fast reacting sugar), the HMW aggregate formation was significantly reduced. In order to show the reactivity among the slow reacting hexoses, we incubated lens proteins with 1 M hexoses for 30 days and the results showed that galactose was more reactive and showed higher cross-linking than fructose and glucose. These results thus indicate that relatively low levels of some sugars and sugar phosphates in the lens could be compensated by enhanced lens protein cross-linking and the combined effect could be rather significant with respect to cataractogenesis.

Camel lens crystallins glycosylation and high molecular weight aggregate formation in the presence of ferrous ions and glucose

The incubation of camel lens cortex homogenate with 100 microM ferrous ions and 5.5 mM glucose under sterile conditions caused rapid protein aggregation, but little or no reaction was seen with either 100 microM ferrous ions or 5.5 mM glucose alone. The formation of glycosylated high molecular weight (HMW) protein aggregates was confirmed by light scattering studies, a decreased level of free -SH groups, incorporation of [14C]-glucose and elution of HMW protein aggregate just after the void volume of a Sephacryl S-1000 column. The bonding involved in the formation of these aggregates was found to be mainly disulfide in nature. Isoelectric focusing (IEF) in the presence and absence of reducing conditions indicated that gamma-crystallins may be involved in the formation of HMW protein aggregates. The modifications observed were found to mimic those seen in cataractous lenses.

Progressive changes in lens crystallin glycation and high-molecular-weight aggregate formation leading to cataract development in streptozotocin-diabetic rats

Because of their remarkable longevity, lens crystallins undergo a substantial amount of glycation (non-enzymatic glycosylation) during diabetic hyperglycemia. These post-translational modifications have the potential to disrupt the structural and functional properties of the lens crystallins and contribute to the formation of cataracts. Streptozotocin-induced diabetic rats were used to study the relationship between glycation of lens proteins and the formation of insoluble high-molecular-weight (HMW) aggregates believed to be responsible for cataract formation. After the onset of diabetes, cataracts developed in about 12- to 13 weeks. The animals were followed in this manner until cataracts developed and for an additional 63 days. Five control and five diabetic rats were killed every 3 weeks and lenses removed. Levels of glycated protein and glycated amino acids in lenses from each animal were examined by affinity chromatography. In addition, the changes in crystallin composition and development of HMW aggregates were monitored by molecular-sieve HPLC techniques. As diabetic hyperglycemia continued there was a linear increase in glycated protein in both the soluble and insoluble fractions. This increase was paralleled by an increase in the soluble HMW and insoluble HMW aggregates. Other changes included a decrease in reactive sulfhydryls which indicates an increase in disulfide bond formation. The gamma-crystallin levels also decreased in a linear fashion during the hyperglycemic pre-cataract and cataract stages. It appears that the glycation of lens crystallin, the disappearance of reactive sulfhydryls and the formation of HMW aggregates are interrelated.

Influence of fructose 2,6-bisphosphate on the aggregation properties of rat liver phosphofructokinase.

The influence that fructose 2,6-bisphosphate (Fru-2,6-BP) has on the aggregation properties of rat liver phosphofructokinase has been studied by observing the fluorescence polarization of the enzyme covalently bound to the fluorescent probe pyrenebutyric acid. Fru-2,6-BP dramatically slows the dissociation of the high molecular weight aggregate forms of the enzyme when the enzyme is diluted to 3.2 micrograms/ml (4 X 10(-8) M subunits). Furthermore, Fru-2,6-BP is a strong promoter of reassociation to tetramer and larger forms if the enzyme has been previously allowed to dissociate to the dimer in its absence. Unlike many other positive effectors of liver phosphofructokinase, Fru-2,6-BP is also able to overcome the tendency of MgATP to promote tetramer formation and instead stabilize a very high degree of high molecular weight aggregate formation even in the presence of MgATP. The apparent affinity of liver phosphofructokinase for Fru-2,6-BP was measured by its ability to promote reassociation and compared to that for Fru-1,6-BP. The apparent dissociation constant for Fru-2,6-BP under these conditions is 36 microM, about 40-fold lower than the value of 1.4 mM measured for Fru-1,6-BP. Both ligands demonstrate synergism with the substrate Fru-6-P, which can lower the dissociation constant for Fru-2,6-BP 9-fold to 4 microM and that for Fru-1,6-BP 5-fold to 0.28 mM. These data are interpreted to suggest that influencing the aggregation state of rat liver phosphofructokinase may be one way in which Fru-2,6-BP achieves its effects on the enzyme in vivo.