Recruitment of Light Chains by Homologous and Heterologous Fibrils Shows Distinctive Kinetic and Conformational Specificity

Light chain amyloidosis is a protein misfolding disease in which immunoglobulin light chains aggregate as insoluble fibrils that accumulate in extracellular deposits. Amyloid fibril formation <i>in vitro</i> has been described as a nucleation–polymerization, autocatalytic reaction in which nascent fibrils catalyze formation of new fibrils, recruiting soluble protein into the fibril. In this context, it is also established that preformed fibrils or “seeds” accelerate fibril formation. In some cases, seeds with a substantially different sequence are able to accelerate the reaction, albeit with a lower efficiency. In this work, we studied the recruitment and addition of monomers in the presence of seeds of five immunoglobulin light chain proteins, covering a broad range of protein stabilities and amyloidogenic properties. Our data reveal that in the presence of homologous or heterologous seeds, the fibril formation reactions become less stochastic than <i>de novo</i> reactions. The kinetics of the most amyloidogenic proteins (AL-T05 and AL-09) do not present significant changes in the presence of seeds. Amyloidogenic protein AL-103 presented fairly consistent acceleration with all seeds. In contrast, the less amyloidogenic proteins (AL-12 and κI) presented dramatic differential effects that are dependent on the kind of seed used. κI had a poor efficiency to elongate preformed fibrils. Together, these results indicate that fibril formation is kinetically determined by the conformation of the amyloidogenic precursor and modulated by the differential ability of each protein to either nucleate or elongate fibrils. We observe morphological and conformational properties of some seeds that do not favor elongation with some proteins, resulting in a delay in the reaction

DataSheet_1_Pathologic light chain amyloidosis oligomer detection in urinary extracellular vesicles as a diagnostic tool for response and progression of disease.zip

Light Chain (AL) Amyloidosis is a plasma cell dyscrasia producing amyloidogenic light chains (LC) that misfold and form amyloid deposits that cause damage in vital organs, primarily the heart and kidneys. Urinary extracellular vesicles (uEVs) are nanoparticles produced by renal epithelial cells throughout the nephron. We previously showed that uEVs from active renal AL amyloidosis patients contain LC oligomers that are large (>250kDa), resistant to heat and chemical denaturation, but of low abundance. Renal dysfunction in AL amyloidosis results in high urine protein, compounding technical challenges to use uEVs as analytical tools. In this study, we assess the use of uEVs as analytical diagnostic tools for response and disease progression in AL amyloidosis. Our results suggest that uEV protein concentration, urine volume, and particle concentrations are not directly correlated. Multiple strategies for overcoming non-specific antibody binding in uEV samples were validated in our study. We demonstrated that the sensitivity for pre-clinical testing is improved with a urine sample requirement algorithm that we developed. The findings of our study will provide a pathway toward development of critically needed tools for patient management. Sensitive detection of LC oligomers from a non-invasive urine sample rather than an invasive renal biopsy will reduce patient burden and healthcare costs. The ability to detect LC oligomers in patients with renal progression, despite positive hematologic response; will allow clinicians to confidently treat, but not overtreat, patients at risk of ongoing significant renal injury.</p

Immunoglobulin Light Chains Form an Extensive and Highly Ordered Fibril Involving the N- and C-Termini

Light-chain (AL)-associated amyloidosis is a systemic disorder involving the formation and deposition of immunoglobulin AL fibrils in various bodily organs. One severe instance of AL disease is exhibited by the patient-derived variable domain (V_L) of the light chain AL-09, a 108 amino acid residue protein containing seven mutations relative to the corresponding germline protein, κI O18/O8 V_L. Previous work has demonstrated that the thermodynamic stability of native AL-09 V_L is greatly lowered by two of these mutations, Y87H and N34I, whereas a third mutation, K42Q, further increases the kinetics of fibril formation. However, detailed knowledge regarding the residues that are responsible for stabilizing the misfolded fibril structure is lacking. In this study, using solid-state NMR spectroscopy, we show that the majority of the AL-09 V_L sequence is immobilized in the fibrils and that the N- and C-terminal portions of the sequence are particularly well-structured. Thus, AL-09 V_L forms an extensively ordered and β-strand-rich fibril structure. Furthermore, we demonstrate that the predominant β-sheet secondary structure and rigidity observed for in vitro prepared AL-09 V_L fibrils are qualitatively similar to those observed for AL fibrils extracted from postmortem human spleen tissue, suggesting that this conformation may be representative of a common feature of AL fibrils

Collection of representative immuno-gold labeling images of MM patient exosomes, fractions 2, 5 and 9.

<p>There is a diversity of sizes and labeling within the fractions of this MM patient.</p

Description of the patients involved in this study.

<p>AL =  light chain amyloidosis; MM =  multiple myeloma; MGUS = monoclonal gammopathy of undetermined significance; M-protein =  monoclonal protein; sFLC = serum free light chain concentration in milligrams per deciliter. Proteinuria is the concentration of protein in urine in grams per day. * This patient in currently in complete hematologic and near complete renal response after autologous stem cell transplantation.</p

Western blot showing free light chain immuno-reactive proteins for controls.

<p>Control-ex1 is a patient with membranous glomerulonephritis. Control-ex2 is a patient with IgGλ+λ MGUS. Control-ex3 is a patient with IgA nephropathy. No free light chains were detected for control-ex4 (monoclonal λ protein) or for a healthy normal control (control-ex5), so no blots are shown for these two control samples.</p

Collection of representative immuno-gold labeling images of exosomes from normal control, fraction 8.

<p>These images show the expected ‘deflated football’ structure for these vesicles.</p

Western blot showing free light chain immuno-reactive proteins for MM patients.

<p>15% SDS-PAGE gels under reducing conditions were used. The monoclonal proteins found in each patient are as follows: MM-ex1 = IgGκ; MM-ex2 = IgAκ; MM-ex3 = κ; MM-ex4 = IgGλ; MM-ex5 (also called MCN-ex5) = IgGλ+λ.</p

Western blot showing free light chain immuno-reactive proteins for AL patients.

<p>15% SDS-PAGE gels under reducing conditions were used. The monoclonal proteins found in each patient are as follows: AL-ex1 = IgGλ + λ; AL-ex2 =  IgGλ; AL-ex3 = λ; AL-ex4 = IgGλ; AL-ex5 = IgMκ; AL-ex6 = λ; AL-ex7 = λ; AL-ex8 = κ.</p

Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils—A metric for predicting amyloid propensity

<div><p>Background</p><p>Monoclonal free light chain (LC) proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL) amyloidosis and multiple myeloma (MM). Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10–30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death. The overall survival of patients with amyloidosis is worse than for those with early stage MM.</p><p>Methods and findings</p><p>We have developed a sensitive binding assay quantifying the recruitment of full length, patient-derived LC proteins by synthetic amyloid fibrils, as a method for studying their amyloidogenic potential. In a survey of eight urinary LC, both AL and MM-associated proteins were recruited by synthetic amyloid fibrils; however, AL-associated LC bound significantly more efficiently (p < 0.05) than did MM LCs. The LC proteins used in this study were isolated from urine and presumed to represent a surrogate of serum free light chains.</p><p>Conclusion</p><p>The binding of LC to synthetic fibrils in this assay accurately differentiated LC with amyloidogenic propensity from MM LC that were not associated with clinical amyloid disease. Notably, the LC from a MM patient who subsequently developed amyloid behaved as an AL-associated protein in the assay, indicating the possibility for identifying MM patients at risk for developing amyloidosis based on the light chain recruitment efficacy. With this information, at risk patients can be monitored more closely for the development of amyloidosis, allowing timely administration of novel, amyloid-directed immunotherapies—this approach may improve the prognosis for these patients.</p></div