Systemic light chain (LC) amyloidosis (AL) is a disease where organs are damaged by an overload of a misfolded patient-specific antibody-derived LC, secreted by an abnormal B cell clone. The high LC concentration in the blood leads to amyloid deposition at organ sites. Indeed, cryogenic electron microscopy (cryo-EM) has revealed unique amyloid folds for heart-derived fibrils taken from different patients. Here, we present the cryo-EM structure of heart-derived AL amyloid (AL59) from another patient with severe cardiac involvement. The double-layered structure displays a u-shaped core that is closed by a β-arc lid and extended by a straight tail. Noteworthy, the fibril harbours an extended constant domain fragment, thus ruling out the variable domain as sole amyloid building block. Surprisingly, the fibrils were abundantly concatenated with a proteinaceous polymer, here identified as collagen VI (COLVI) by immuno-electron microscopy (IEM) and mass-spectrometry. Cryogenic electron tomography (cryo-ET) showed how COLVI wraps around the amyloid forming a helical superstructure, likely stabilizing and protecting the fibrils from clearance. Thus, here we report structural evidence of interactions between amyloid and collagen, potentially signifying a distinct pathophysiological mechanism of amyloid deposits.
Introduction
Systemic AL amyloidosis is a rare plasma cell dyscrasia with an annual incidence of about 12-15 new cases per million people1. AL amyloidosis is due to the overexpression of an amyloidogenic LC that misfolds and forms amyloid deposits in several organs2. The circulating LC molecules exert proteotoxicity which concurs with the mass effect produced by amyloid deposits to fatal organ dysfunction1. Due to genomic recombination and somatic mutations every AL patient bears a virtually unique amyloidogenic LC sequence, originating from either the λ− or κ−gene locus3,4. Most patients are affected by deposits in multiple organs, but heart manifestation dictates the prognosis in ~75% of cases5,6,7,8,9. Without its associated heavy chain, free LCs fold into homodimers where each monomer consists of an N-terminal variable domain (VL) and a C-terminal constant domain (CL) connected by a flexible joining region10,11,12,13. While free LCs are eliminated rapidly under healthy conditions, abnormal levels of an amyloidogenic LC cause vast accumulations of cross-β amyloid fibrils in AL amyloidosis1,14. Cryo-EM has emerged as a powerful method to determine molecular structures of ex vivo amyloids, retrieved from patients affected by various amyloidoses15,16,17,18,19,20,21. The structures of fibrils from cardiac tissue of four AL patients, denoted as λ6-AL55, λ3-FOR005, λ1-FOR001, and λ1-FOR006, display distinct folds3,15,16,17,18,19. So far, only residues belonging to VL were found in the structured core of the AL amyloid, resulting in high sequence variability in the core of the deposited fibrils3,15,16,17,18,19. Structures of sequence-identical amyloid from the heart and kidney of the same patient are well superposable, indicating a crucial role of the VL sequence in determining the fibrillar structure19. Other sources of variability in this disease are post-translational modifications (PTM), and in particular proteolytic processing and N-glycosylation hotspots. The latter were shown to correlate with AL onset for κ LCs22,23,24. To date, glycosylation in λ LCs is not considered a risk factor for AL24,25, but the cryo-EM structure of λ1-FOR001 shows a covalently linked glycan that may impact the resulting amyloid fold18.
Ancillary proteins are reproducibly found in amyloid deposits, including heparan sulphate proteoglycan, serum amyloid P-component and various extracellular matrix elements (ECM) such as collagen1,26,27,28,29,30. The ECM provides structural support for organs and tissues and is dynamically remodelled, controlling tissue homoeostasis and modulating immune cell responses31,32,33. As most prominent ECM component, collagens are frequently detected in deposits extracted from different amyloidosis types27. Collagen interactions seem to affect directly amyloid formation and disease progression27,34,35,36,37,38,39,40. Collagens facilitate misfolding of native human β2-microglobulin (β2 m) into amyloid, leading to fibril deposition in the joints of haemodialyses patients36,37,38,39. Increased collagen content was found in cerebral microvessels of patients with Alzheimer’s disease, and a neuron-protective role was put forward for COLVI41,42,43. Recent evidence suggests that binding of collagens in general, but particularly COLI and COLIV, protects AL amyloid against phagocytic clearance in experimental mouse models34,35. Thus, collagen/amyloid interactions modulate the progression of various amyloidoses.
Here we report the 3.6 Å resolution cryo-EM structure of N-glycosylated AL amyloid, which was extracted from the heart of a patient with cardiac AL amyloidosis and is referred to as AL59. AL59 is, to the best of our knowledge, the first AL amyloid structure with an extended constant domain fragment in its fibril core. The fold is related to that of a previously reported AL amyloid structure, belonging to the same λ3-gene subfamily. Ex vivo AL59 fibrils display the unique ability to interact with a polymer from the extracellular matrix, which was identified as COLVI. While COLVI was not resolved in the helical reconstruction of the amyloid, we applied IEM and cryo-ET to reveal that COLVI wraps helically around the fibril, potentially stabilising and protecting AL59 amyloids from macrophage recognition.