Mechanistic insights into the aggregation pathway of the patient-derived immunoglobulin light chain variable domain protein FOR005.

Systemic antibody light chain (AL) amyloidosis is characterized by deposition of amyloid fibrils. Prior to fibril formation, soluble oligomeric AL protein has a direct cytotoxic effect on cardiomyocytes. We focus on the patient derived λ-III AL variable domain FOR005 which is mutated at five positions with respect to the closest germline protein. Using solution-state NMR spectroscopy, we follow the individual steps involved in protein misfolding from the native to the amyloid fibril state. Unfavorable mutations in the complementary determining regions introduce a strain in the native protein structure which yields partial unfolding. Driven by electrostatic interactions, the protein converts into a high molecular weight, oligomeric, molten globule. The high local concentration of aggregation prone regions in the oligomer finally catalyzes the conversion into fibrils. The topology is determined by balanced electrostatic interactions in the fibril core implying a 180° rotational switch of the beta-sheets around the conserved disulfide bond.

Sensitivity-Enhanced Multidimensional Solid-State NMR Spectroscopy by Optimal-Control-Based Transverse Mixing Sequences.

Recently, proton-detected magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy has become an attractive tool to study the structure and dynamics of insoluble proteins at atomic resolution. The sensitivity of the employed multidimensional experiments can be systematically improved when both transversal components of the magnetization are transferred simultaneously after an evolution period. The method of preservation of equivalent pathways has been explored in solution-state NMR; however, it does not find widespread application due to relaxation issues connected with increased molecular size. We present here for the first time heteronuclear transverse mixing sequences for correlation experiments at moderate and fast MAS frequencies. Optimal control allows to boost the signal-to-noise ratio (SNR) beyond the expected factor of 2 for each indirect dimension. In addition to the carbon-detected sensitivity-enhanced 2D NCA experiment, we present a novel proton-detected, doubly sensitivity-enhanced 3D hCANH pulse sequence for which we observe a 3-fold improvement in SNR compared to the conventional experimental implementation. The sensitivity gain turned out to be essential to unambiguously characterize a minor fibril polymorph of a human lambda-III immunoglobulin light chain protein that escaped detection so far.

Protease resistance of ex vivo amyloid fibrils implies the proteolytic selection of disease-associated fibril morphologies.

Several studies recently showed that ex vivo fibrils from patient or animal tissue were structurally different from in vitro formed fibrils from the same polypeptide chain. Analysis of serum amyloid A (SAA) and Aß-derived amyloid fibrils additionally revealed that ex vivo fibrils were more protease stable than in vitro fibrils. These observations gave rise to the proteolytic selection hypothesis that suggested that disease-associated amyloid fibrils were selected inside the body by their ability to resist endogenous clearance mechanisms. We here show, for more than twenty different fibril samples, that ex vivo fibrils are more protease stable than in vitro fibrils. These data support the idea of a proteolytic selection of pathogenic amyloid fibril morphologies and help to explain why only few amino acid sequences lead to amyloid diseases, although many, if not all, polypeptide chains can form amyloid fibrils in vitro.

Solid state NMR assignments of a human λ-III immunoglobulin light chain amyloid fibril.

The aggregation of antibody light chains is linked to systemic light chain (AL) amyloidosis, a disease where amyloid deposits frequently affect the heart and the kidney. We here investigate fibrils from the λ-III FOR005 light chain (LC), which is derived from an AL-patient with severe cardiac involvement. In FOR005, five residues are mutated with respect to its closest germline gene segment IGLV3-19 and IGLJ3. All mutations are located close to the complementarity determining regions (CDRs). The sequence segments responsible for the fibril formation are not yet known. We use fibrils extracted from the heart of this particular amyloidosis patient as seeds to prepare fibrils for solid-state NMR. We show that the seeds induce the formation of a specific fibril structure from the biochemically produced protein. We have assigned the fibril core region of the FOR005-derived fibrils and characterized the secondary structure propensity of the observed amino acids. As the primary structure of the aggregated patient protein is different for every AL patient, it is important to study, analyze and report a greater number of light chain sequences associated with AL amyloidosis.

Domain Interactions Determine the Amyloidogenicity of Antibody Light Chain Mutants.

In antibody light chain amyloidosis (AL), mutant light chains (LCs) or their variable domains (VLs) form fibrils, which accumulate in organs and lead to their failure. The molecular mechanism of this disease is still poorly understood. One of the key open issues is whether the mutant VLs and LCs differ in fibril formation. We addressed this question studying the effects of the VL mutations S20N and R61A within the isolated VL domain and in the full-length LC scaffold. Both VL variants readily form fibrils. Here, we find that in the LC context, the S20N variant is protected from fibril formation while for LC R61A fibril formation is even accelerated compared to VL R61A. Our analyses revealed that the partially unfolded state of the VL R61A domain destabilizes the CL domain by non-native interactions, in turn leading to a further unfolding of the VL domain. In contrast, the folded mutant VL S20N and VL wt form native interactions with CL. These are beneficial for LC stability and promote amyloid resistance. Thus the effects of specific mutations on the VL fold can have opposing effects on LC domain interactions, stability and amyloidogenicity.

Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability.

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt ß-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

A single residue switch reveals principles of antibody domain integrity.

Despite their importance for antibody architecture and design, the principles governing antibody domain stability are still not understood in sufficient detail. Here, to address this question, we chose a domain from the invariant part of IgG, the CH2 domain. We found that compared with other Ig domains, the isolated CH2 domain is a surprisingly unstable monomer, exhibiting a melting temperature of ∼44 °C. We further show that the presence of an additional C-terminal lysine in a CH2 variant substantially increases the melting temperature by ∼14 °C relative to CH2 WT. To explore the molecular mechanism of this effect, we employed biophysical approaches to probe structural features of CH2. The results revealed that Lys101 is key for the formation of three secondary structure elements: the very C-terminal ß-strand and two adjacent α-helices. We also noted that a dipole interaction between Lys101 and the nearby α-helix, is important for stabilizing the CH2 architecture by protecting the hydrophobic core. Interestingly, this interaction between the α-helix and C-terminal charged residues is highly conserved in antibody domains, suggesting that it represents a general mechanism for maintaining their integrity. We conclude that the observed interactions involving terminal residues have practical applications for defining domain boundaries in the development of antibody therapeutics and diagnostics.

Prediction of human absorption of a trioxane antimalarial drug (CDRI 99/411) using an in-house validated in situ single-pass intestinal perfusion model.

OBJECTIVES: The aim of the study was to predict human intestinal permeability and the fraction absorbed of an oral dose of a promising trioxane anti-malarial drug (CDRI 99/411) using the single-pass intestinal perfusion technique (SPIP) in rats. METHODS: Effective permeability coefficients (P eff) in anaesthetized rats were determined for marker compounds and the trioxane derivative 99/411. Drug solution in perfusion buffer was perfused through intestine with a flow rate of 0.2 ml/min and samples were taken from an outlet tubing at different time points up to 120 min. Drug concentrations in samples were determined using RP-HPLC. KEY FINDINGS: The effective permeability coefficient values of marker compounds obtained in rats were compared with published data for human intestinal permeability (P eff (human)) and human fraction absorbed (F(a) (human)) to establish an in-house model. Strong correlations were found between rat and human values for markers (P eff (human) = 1.039 P eff (rat) - 0.1815; R2 = 0.970 and F(a) (human) = 0.15621n (P eff (rat) + 0.7232; R2 = 0.927). Subsequently the human permeability and fraction dose absorbed in human were predicted for 99/411 using the obtained rat permeability value and established correlations. P eff in human predicted from the model was found to be 7.05 x 10(-4) cm/s and F(a) value in human was predicted around 1. CONCLUSIONS: Considering the high correlation of rat Peff values with those of human reported values, it can be concluded that the developed in-house model is reliable and can be used preliminarily, to predict human permeability and fraction dose absorbed of any test compound. From predicted results, 99/411 was found to have high permeability and possibly complete absorption in human.

PAMPA permeability, plasma protein binding, blood partition, pharmacokinetics and metabolism of formononetin, a methoxylated isoflavone.

Formononetin (FMN) is a methoxylated isoflavone which is the major constituent in red clover and in commercially available extracts of this plant. In this study, we investigated the parallel artificial membrane permeability assay (PAMPA) permeability, protein binding, blood uptake characteristics, pharmacokinetics and metabolism of FMN. The permeability study samples were analyzed by HPLC-PDA method; whereas the pharmacokinetic study, protein binding and whole blood partitioning samples were analyzed by LC-MS/MS method. The PAMPA permeability of FMN was found to be high at pH 4.0 and 7.0. Plasma protein binding of FMN was found to be 93.61±0.44% and 96.14±0.15% at the tested concentration of 50 and 150 ng/mL, respectively. FMN reached equilibrium fast between red blood cells (RBCs) and plasma, and the partition coefficients between RBCs and plasma (K(RBC/PL)) were independent of the initial rat blood concentrations of FMN. The bioavailability of unchanged/free FMN was found to be poor, i.e. approximately 3%. FMN was found to have a high clearance (5.13 L/h/kg) and a large apparent volume of distribution (14.16L/kg). Circulating conjugates (glucuronides/sulfates) of FMN and daidzein (DZN) were quantified using enzymatic hydrolysis of plasma samples. The levels of isoflavone glucuronides/sulfates were found to be much greater than that of the corresponding aglycones.