Crystal structure of 6aJL2-R24G light chain variable domain: Does crystal packing explain amyloid fibril formation?

Light chain amyloidosis is one of the most common systemic amyloidosis, characterized by the deposition of immunoglobulin light variable domain as insoluble amyloid fibrils in vital organs, leading to the death of patients. Germline λ6a is closely related with this disease and has been reported that 25% of proteins encoded by this germline have a change at position 24 where an Arg is replaced by a Gly (R24G). This germline variant reduces protein stability and increases the propensity to form amyloid fibrils. In this work, the crystal structure of 6aJL2-R24G has been determined to 2.0 Å resolution by molecular replacement. Crystal belongs to space group I2₁2₁2₁ (PDB ID 5JPJ) and there are two molecules in the asymmetric unit. This 6aJL2-R24G structure as several related in PDB (PDB entries: 5C9K, 2W0K, 5IR3 and 1PW3) presents by crystal packing the formation of an octameric assembly in a helicoidal arrangement, which has been proposed as an important early stage in amyloid fibril aggregation. However, other structures of other protein variants in PDB (PDB entries: 3B5G, 3BDX, 2W0L, 1CD0 and 2CD0) do not make the octameric assembly, regardless their capacity to form fibers in vitro or in vivo. The analysis presented here shows that the ability to form the octameric assembly in a helicoidal arrangement in crystallized light chain immunoglobulin proteins is not required for amyloid fibril formation in vitro. In addition, the fundamental role of partially folded states in the amyloid fibril formation in vitro, is not described in any crystallographic structure published or analyzed here, being those structures, in any case examples of proteins in their native states. Those partially folded states have been recently described by cryo-EM studies, showing the necessity of structural changes in the variants before the amyloid fiber formation process starts.     

Formation of Amyloid Fibers by Monomeric Light Chain Variable Domains

Systemic light chain amyloidosis is a lethal disease characterized by excess immunoglobulin light chains and light chain fragments composed of variable domains, which aggregate into amyloid fibers. These fibers accumulate and damage organs. Some light chains induce formation of amyloid fibers, whereas others do not, making it unclear what distinguishes amyloid formers from non-formers. One mechanism by which sequence variation may reduce propensity to form amyloid fibers is by shifting the equilibrium toward an amyloid-resistant quaternary structure. Here we identify the monomeric form of the Mcg immunoglobulin light chain variable domain as the quaternary unit required for amyloid fiber assembly. Dimers of Mcg variable domains remain stable and soluble, yet become prone to assemble into amyloid fibers upon disassociation into monomers.     

The amyloid fibrils of the constant domain of immunoglobulin light chain

Light chain-associated (AL) amyloidosis is characterized by dominant fibril deposition of the variable domain (VL) of an immunoglobulin light chain, and thus its constant domain (CL) has been considered not to be amyloidogenic. We examined the in vitro fibril formation of the isolated CL in comparison with β₂-microglobulin (β2-m), an immunoglobulin domain-like amyloidogenic protein responsible for dialysis-related amyloidosis. Two methods useful for β2-m at neutral pH also induced amyloid fibrils of CL, which were monitored by thioflavin-T binding and electron microscopy (EM). These results suggest that CL plays an important role, more than previously assumed, in the development of AL-amyloidosis.     

Influence of a Single Point Mutation in the Constant Domain of the Bence-Jones Protein BIF on Its Aggregation Properties

Multiple myeloma nephropathy occurs due to the aggregate formation by monoclonal immunoglobulin light chains (Bence-Jones proteins) in kidneys of patients with multiple myeloma. The mechanism of amyloid deposit formation is still unclear. Earlier, the key role in the fibril formation has been assigned to the variable domains that acquired amyloidogenic properties as a result of somatic mutations. However, fibril formation by the Bence-Jones protein BIF was found to be the function of its constant domain. The substitution of Ser177 by Asn in the constant domain of the BIF protein is most likely an inherited than a somatic mutation. To study the role of this mutation in amyloidogenesis, the recombinant Bence-Jones protein BIF and its mutant with the N177S substitution typical for the known immunoglobulin C_κ allotypes Km¹, Km^1,2, and Km³ were isolated. The morphology of aggregates formed by the recombinant proteins under conditions similar to those occurring during the protein transport in bloodstream and its filtration into the renal glomerulus, in the distal tubules, and in the proximal renal tubules was analyzed by atomic force microscopy. The nature of the aggregates formed by BIF and its N177S mutant during incubation for 14 days at 37°C strongly differed and depended on both pH and the presence of a reducing agent. BIF formed fibrils at pH 7.2, 6.5, and 10.1, while the N177S mutant formed fibrils only at alkaline pH 10.1. The refolding of both proteins in the presence of 5 mM dithiothreitol resulted in the formation of branched structures.     

Age delays thyroglobulin progression towards dense lysosomes in the cream hamster thyroid

We have shown that large lysosomes appear in thyroids of aging male cream hamsters. To investigate the role of this lysosomal change in the age-dependent reduction in hormone secretion, thyroids of young (<4 months of age) and old (>22 months of age) male and female hamsters were labeled with ¹²⁵I at near isotopic equilibrium. Changes in thyroid morphology were analyzed by light- and electron-microscopic morphometry. Changes in thyroglobulin processing were analyzed by subcellular fractionation and identification of ¹²⁵I-compounds by sucrose gradients and reverse-phase high-pressure liquid chromatography (HPLC). Sexual dimorphism present in thyroids of young animals became more marked upon aging. The parallel increase in thyroid weight and thyroglobulin content was more conspicuous in old females than in old males. Two morphological observations were specific to old females: (1) large follicles with flat epithelium and evenly labeled colloid and (2) deposits of amyloid material (possibly immunoglobulin light chain-related) between follicles. Although lysosomes were enlarged in female and male aged thyroids, they did not accumulate iodine. However, after isopycnic centrifugation of crude lysosomal fractions in Percoll gradients, ¹²⁵I in old thyroids was not distributed mainly in the dense fraction L1 (lysosomes) as in young thyroids, but partly in particles of lower density (light L2 and buoyant fractions). ¹²⁵I in the lighter particles was mostly found in intact thyroglobulin and in large iodopeptides. This ¹²⁵I shift towards less dense particles was more marked in females than in males. These results indicate that age delays thyroglobulin progression towards dense lysosomes and suggest that the slower traffic of thyroglobulin in the endocytic pathway contributes to the reduction in thyroid hormone secretion in the aged cream hamster.     

Hypervariable Region of Human Immunoglobulin Heavy Chains

THE variable regions of human immunoglobulin light chains contain three areas of unusually high variability^1–4. Similar hypervariable regions have been postulated for human heavy chains^{5, 6}, but there are no amino-acid sequence data to support this idea. These hypervariable regions are particularly interesting because they may be the areas of the immunoglobulin molecule involved in antibody complementarity.We have made use of the recent observation that a variable region subclass of heavy chains is characterized by an unblocked amino-terminal residue⁷ and of the availability of automated sequencing techniques^{8, 9} to study this question in detail with additional heavy chain sequences.     

Backbone and side-chain 1H, 13C and 15N resonance assignments of LEN, a human immunoglobulin κIV light-chain variable domain

¹H, ¹³C and ¹⁵N resonance assignments are presented for a recombinant 114 amino acid human immunoglobulin (Ig) κIV light-chain variable domain (V_L) LEN, which displays a high degree of sequence identity with another human Ig κIV V_L, SMA. While SMA is highly amyloidogenic in vivo and in vitro and has been linked to the pathogenesis of light-chain amyloidosis, LEN is non-amyloidogenic in vivo and can be converted to the amyloid state only in vitro under destabilizing conditions. Measurements of longitudinal and transverse amide ¹⁵N relaxation rates confirm that, as expected, LEN is a dimer at physiological pH and typical concentrations used for NMR studies, and the analysis of secondary chemical shifts indicates that the protein has a high β-sheet content. These findings are consistent with previously published biophysical data and the high-resolution X-ray structure of LEN.     

AL Amyloid Imaging and Therapy with a Monoclonal Antibody to a Cryptic Epitope on Amyloid Fibrils

The monoclonal antibody 2A4 binds an epitope derived from a cleavage site of serum amyloid protein A (sAA) containing a -Glu-Asp- amino acid pairing. In addition to its reactivity with sAA amyloid deposits, the antibody was also found to bind amyloid fibrils composed of immunoglobulin light chains. The antibody binds to synthetic fibrils and human light chain (AL) amyloid extracts with high affinity even in the presence of soluble light chain proteins. Immunohistochemistry with biotinylated 2A4 demonstrated positive reaction with ALκ and ALλ human amyloid deposits in various organs. Surface plasmon resonance analyses using synthetic AL fibrils as a substrate revealed that 2A4 bound with a K_D of ∼10 nM. Binding was inhibited in the presence of the –Glu-Asp- containing immunogen peptide. Radiolabeled 2A4 specifically localized with human AL amyloid extracts implanted in mice (amyloidomas) as evidenced by single photon emission (SPECT) imaging. Furthermore, co-localization of the radiolabeled mAb with amyloid was shown in biodistribution and micro-autoradiography studies. Treatment with 2A4 expedited regression of ALκ amyloidomas in mice, likely mediated by the action of macrophages and neutrophils, relative to animals that received a control antibody. These data indicate that the 2A4 mAb might be of interest for potential imaging and immunotherapy in patients with AL amyloidosis.     

Selective Cytotoxicity of Anti-Kappa Serum for B Lymphocytes

MOUSE lymphocytes can be divided into two distinct populations according to the density of immunoglobulin determinants on their surface. Lymphocytes with a high density of immunoglobulin are marrow-derived, nonthymus-processed, B cells, whereas lymphocytes with little or no immunoglobulin are thymus-derived, T cells^1–3. Since more than 95% of mouse immunoglobulin light chains are of the kappa type⁴, treatment of lymphocyte suspensions with an appropriate dilution of rabbit anti-mouse kappa serum and complement should be cytotoxic for only B lymphocytes. This prediction was tested by using lymphocyte populations enriched for either T or B cells or containing the two cell types in a known proportion.     

Residual Structures in the Acid-Unfolded States of Vλ6 Proteins Affect Amyloid Fibrillation

Many proteins form amyloid-like fibrils in vitro under partially or highly unfolding conditions. Recently, we showed that the residual structure in highly unfolded state is closely related to amyloid fibril formation in hen lysozyme. Thus, to better understand the role of the residual structure on amyloid fibril formation, we focused on AL amyloidosis, which results from the extracellular deposition of monoclonal immunoglobulin light-chain variable domains (V_Ls) as insoluble fibrils. We examined the relationship between the residual structure and amyloid fibril formation on three λ6 recombinant V_L (rVλ6) proteins, wild type, Jto, and Wil. Although rVλ6 proteins are highly unfolded in pH 2, ¹⁵N NMR transverse relaxation experiments revealed nonrandom structures in regions, which include some hydrophobic residues and a single disulfide bond, indicating the existence of residual structure in rVλ6 proteins. However, the residual structure of Wil was markedly disrupted compared with those of the other proteins, despite there being no significant differences in amino acid sequences. Fibrillation experiments revealed that Wil had a longer lag time for fibril formation than the others. When the single disulfide bond was reduced and alkylated, the residual structure was largely disrupted and fibril formation was delayed in all three rVλ6 proteins. It was suggested that the residual structure in highly unfolded state has a crucial role in amyloid fibril formation in many proteins, even pathogenic ones.     

Kinetic Control in Protein Folding for Light Chain Amyloidosis and the Differential Effects of Somatic Mutations

Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild-type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis–trans prolyl isomerization. Our data reveal that the deletion of proline 95a (AL-103 delP95aIns), which removes the trans–cis di-proline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH 2; reactions become slower and more stochastic as the pH increases up to pH 7. Based on these results, we propose that, in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis proline 95a) plays a major role in the AL-103 amyloid fibril formation process.     

Calcium-dependent aggregation of human serum amyloid P component: Inhibition by the cyclic 4,6-pyruvate acetal of galactose

Serum amyloid P component is a normal plasma glycoprotein which is the precursor of amyloid P component, a minor but universal constituent of amyloid deposits. When isolated human P component is exposed to free ionised Ca²⁺ it aggregates and precipitates. This phenomenon is completely inhibited by the presence of 10^?4?10^?2 M methyl 4,6-O-(1-car?yethylidene)-β-D-galactopyranoside, a recently synthesised specific ligand for amyloid P component. This observation suggests that the autoaggregation of human amyloid P component involves the Ca²⁺ dependent specific ligand binding property of P component, but does not distinguish between receptor-site-mediated and allosteric mechanisms.     

Asn27 is Essential for the Neurotoxicity of Amyloid β(1–42) Peptide

In contrast to the general tendency of hydrophobicity-toxicity relationship of amyloid ?? peptide, we have previously found that the replacement of Asn²⁷ of amyloid ??(25?C35) peptide with Ala yielded a more hydrophobic but less toxic analog and that of Met³⁵ gave a less hydrophobic but more toxic one. To reveal the unique role of these two residues in the neurotoxicity of amyloid ??(1?C42) peptide, the major peptide constituent of amyloid plaques in human brain, we synthesized two analogs N27A and M35A in which Asn²⁷ and Met³⁵ of amyloid ??(1?C42) peptide was replaced with Ala, respectively. The former showed much weaker toxicity than the native peptide, while the latter showed almost an equivalent toxicity, indicating that the side chain amide group of Asn²⁷ has an essential role for the toxicity of amyloid ?? peptides.     

Plasma Based Markers of [11C] PiB-PET Brain Amyloid Burden

Changes in brain amyloid burden have been shown to relate to Alzheimer''s disease pathology, and are believed to precede the development of cognitive decline. There is thus a need for inexpensive and non-invasive screening methods that are able to accurately estimate brain amyloid burden as a marker of Alzheimer''s disease. One potential method would involve using demographic information and measurements on plasma samples to establish biomarkers of brain amyloid burden; in this study data from the Alzheimer''s Disease Neuroimaging Initiative was used to explore this possibility. Sixteen of the analytes on the Rules Based Medicine Human Discovery Multi-Analyte Profile 1.0 panel were found to associate with [¹¹C]-PiB PET measurements. Some of these markers of brain amyloid burden were also found to associate with other AD related phenotypes. Thirteen of these markers of brain amyloid burden – c-peptide, fibrinogen, alpha-1-antitrypsin, pancreatic polypeptide, complement C3, vitronectin, cortisol, AXL receptor kinase, interleukin-3, interleukin-13, matrix metalloproteinase-9 total, apolipoprotein E and immunoglobulin E – were used along with co-variates in multiple linear regression, and were shown by cross-validation to explain >30% of the variance of brain amyloid burden. When a threshold was used to classify subjects as PiB positive, the regression model was found to predict actual PiB positive individuals with a sensitivity of 0.918 and a specificity of 0.545. The number of APOE ϵ 4 alleles and plasma apolipoprotein E level were found to contribute most to this model, and the relationship between these variables and brain amyloid burden was explored.     

Ca2+‐dependent endoplasmic reticulum stress correlates with astrogliosis in oligomeric amyloid β‐treated astrocytes and in a model of Alzheimer's disease

Neurotoxic effects of amyloid β peptides are mediated through deregulation of intracellular Ca²⁺ homeostasis and signaling, but relatively little is known about amyloid β modulation of Ca²⁺ homeostasis and its pathological influence on glia. Here, we found that amyloid β oligomers caused a cytoplasmic Ca²⁺ increase in cultured astrocytes, which was reduced by inhibitors of PLC and ER Ca²⁺ release. Furthermore, amyloid β peptides triggered increased expression of glial fibrillary acidic protein (GFAP), as well as oxidative and ER stress, as indicated by eIF2α phosphorylation and overexpression of chaperone GRP78. These effects were decreased by ryanodine and 2APB, inhibitors of ryanodine receptors and InsP₃ receptors, respectively, in both primary cultured astrocytes and organotypic cultures of hippocampus and entorhinal cortex. Importantly, intracerebroventricular injection of amyloid β oligomers triggered overexpression of GFAP and GRP78 in astrocytes of the hippocampal dentate gyrus. These data were validated in a triple‐transgenic mouse model of Alzheimer's disease (AD). Overexpression of GFAP and GRP78 in the hippocampal astrocytes correlated with the amyloid β oligomer load in 12‐month‐old mice, suggesting that this parameter drives astrocytic ER stress and astrogliosis in vivo. Together, these results provide evidence that amyloid β oligomers disrupt ER Ca²⁺ homeostasis, which induces ER stress that leads to astrogliosis; this mechanism may be relevant to AD pathophysiology.     

Conservatism in the Evolution of Immunoglobulin

IMMUNOGLOBULINS of all vertebrate species studied to date show two basic structural features¹: (1) they are composed of two types of polypeptide subunits termed light chains and heavy chains and (2) they possess various amino-acid sequences. The differences in primary sequence observed in single immunoglobulin classes reside chiefly in the amino-terminal portions of the polypeptide chains which have been classed as variable or V regions^2,3. This heterogeneity has provided an obstacle to the determination of the primary sequence of antibodies. Most knowledge of immunoglobulin structure therefore derives from analysis of homogeneous immunoglobulins produced by human and murine plasmacytomas^2,3. Definite assignments of homology among vertebrate species are difficult to make because of the dearth of amino-acid sequence data for lower species. The relatively small quantities of immunoglobulin, the difficulty of purification and the innate heterogeneity prevent the attainment of complete sequence information from critical species such as the lamprey^4,5.     

Recombinant full-size human antibody to Ebola virus

A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids, pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 × 10⁷ ± 1.5 × 10⁷ M^?1. Like the parent single-chain antibody 4D1, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.     

Comparative assay of amyloid and prion contents in yeast cells

Amyloid contents were quantitatively assayed in crude yeast lysates treated with thioflavin T that specifically stained amyloid fibrils. We demonstrated that guanidine hydrochloride (GuHCl) treatment and overexpression of Hsp104p chaperone resulted in the elimination of the [PSI ⁺] factor and that the stable decline in amyloid contents followed from the reduced fluorescence intensity (IF) of thioflavin T. Overexpression of the SUP35 gene coding the protein prionizable to [PSI ⁺] results in the generation of [PSI ⁺] clones with increased thioflavin T IF. Transmission of [PSI ⁺] factor by cytoduction in crossings of recipients with low IF was also accompanied by stable IF enhancement in cytoductants, indicating enriched amyloid contents. Thus, in model experiments, modifying the quantity of [PSI ⁺] factor, a yeast prion amyloid, the change in thioflavin T IF corresponds to the expected shift in amyloid contents, the IF shift behaving as a cytoplasm hereditary determinant. It is concluded that thioflavin T IF allows for the quantitative estimation of amyloid contents in cells. The stable mitotic IF shift induced by agents affecting the prion composition permits the quantitative evaluation of prion contribution into amyloid pool. It is possible to assume that the monitoring of thiophlavin T IF shifts under the exposure of agents affecting prion pattern may be helpful to disclose previously unknown prions in yeast strains.     

Glycosaminoglycans promote fibril formation by amyloidogenic immunoglobulin light chains through a transient interaction

Amyloid formation occurs when a precursor protein misfolds and aggregates, forming a fibril nucleus that serves as a template for fibril growth. Glycosaminoglycans are highly charged polymers known to associate with tissue amyloid deposits that have been shown to accelerate amyloidogenesis in vitro. We studied two immunoglobulin light chain variable domains from light chain amyloidosis patients with 90% sequence identity, analyzing their fibril formation kinetics and binding properties with different glycosaminoglycan molecules. We find that the less amyloidogenic of the proteins shows a weak dependence on glycosaminoglycan size and charge, while the more amyloidogenic protein responds only minimally to changes in the glycosaminoglycan. These glycosaminoglycan effects on fibril formation do not depend on a stable interaction between the two species but still show characteristic traits of an interaction-dependent mechanism. We propose that transient, predominantly electrostatic interactions between glycosaminoglycans and the precursor proteins mediate the acceleration of fibril formation in vitro.     

A new perspective on Hsp104-mediated propagation and curing of the yeast prion [PSI+]

Most prions in yeast form amyloid fibrils that must be severed by the protein disaggregase Hsp104 to be propagated and transmitted efficiently to newly formed buds. Only one yeast prion, [PSI⁺], is cured by Hsp104 overexpression. We investigated the interaction between Hsp104 and Sup35, the priongenic protein in yeast that forms the [PSI⁺] prion.¹ We found that a 20-amino acid segment within the highly-charged, unstructured middle domain of Sup35 contributes to the physical interaction between the middle domain and Hsp104. When this segment was deleted from Sup35, the efficiency of [PSI⁺] severing was substantially reduced, resulting in larger Sup35 particles and weakening of the [PSI⁺] phenotype. Furthermore, [PSI⁺] in these cells was completely resistant to Hsp104 curing. The affinity of Hsp104 was considerably weaker than that of model Hsp104-binding proteins and peptides, implying that Sup35 prions are not ideal substrates for Hsp104-mediated remodeling. In light of this finding, we present a modified model of Hsp104-mediated [PSI⁺] propagation and curing that requires only partial remodeling of Sup35 assembled into amyloid fibrils.