Crystal-storing histiocytosis (CSH) with massive accumulation of particulate immunoglobulins is a rare phenomenon accompanying B-cell dyscrasias. In the reported case (M51), the disease presented as systemic CSH and later was proved to be a frank multiple myeloma. The aggregates of crystal-laden histiocytes were demonstrated in the bone marrow, lungs, kidney, and liver. Additionally, the crystalline immunoglobulin particles were identified in renal stromal cells and in hepatocytes. The patient developed lung adenocarcinoma and died 12 months after the presentation, shortly after the lobectomy. In this paper, we report the results of morphological (including electron microscopy), immunohistochemical, and biochemical analysis. The tendency for aggregation of the IgG kappa monoclonal protein was due to the abnormal physicochemical properties of its heavy chain. Massive accumulation of crystal-storing histiocytes surpassed the myeloma tumor burden and markedly contributed to the severity of the disease.

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The self-assembling tendency and protein complexation capability of dyes related to Congo red and also some dyes of different structure were compared to explain the mechanism of Congo red binding and the reason for its specific affinity for beta-structure. Complexation with proteins was measured directly and expressed as the number of dye molecules bound to heat-aggregated IgG and to two light chains with different structural stability. Binding of dyes to rabbit antibodies was measured indirectly as the enhancement effect of the dye on immune complex formation. Self-assembling was tested using dynamic light scattering to measure the size of the supramolecular assemblies. In general the results show that the supramolecular form of a dye is the main factor determining its complexation capability. Dyes that in their compact supramolecular organization are ribbon-shaped may adhere to polypeptides of beta-conformation due to the architectural compatibility in this unique structural form. The optimal fit in complexation seems to depend on two contradictory factors involving, on the one hand, the compactness of the non-covalently stabilized supramolecular ligand, and the dynamic character producing its plasticity on the other. As a result, the highest protein binding capability is shown by dyes with a moderate self-assembling tendency, while those arranging into either very rigid or very unstable supramolecular entities are less able to bind.

The ordered amyloid-like organization of protein aggregates was obtained using for their formation the rigid fibrillar nanostructures of Congo red as the scaffolding. The higher rigidity of used dye nanoparticles resulted from the stronger stacking of molecules at low pH (near the pK of the dye amino group) because of the decreased charge repulsion. The polylysine, human globin, and immunoglobulin L chain were arranged in this way to form deposits of amyloid properties. The scaffolding was introduced simply by mixing the dye and proteins at a low pH or the dye was used in the preorganized form by maintaining it in the electric field before and during protein addition. The polarization and electron microscopy studies confirmed the unidirectional organization of the complex. The precipitate of the complex was used for studies directly or after the partial or complete removal of the dye. The results suggest that the process of formation of amyloid-like deposits may bypass the nucleation step. It is possible if the protein aggregation occurs in unidirectionally organized (because of scaffolding) assembly of molecules, arranged prior to self-association. The recognition of the structure of amphoteric Congo red nanoparticles used for the scaffolding was based on the molecular dynamics simulation.

Congo red, a dye of high self-assembling tendency, has been found to form complexes with proteins by adhesion of the ribbon-like supramolecular ligand to polypeptide chains of beta-conformation. Complexation is allowed by local or global protein instability, facilitating penetration of the dye to the locus of its binding. At elevated temperatures, L chain lambda of myeloma origin was found to form two distinct complexes with Congo red, easily differentiated in electrophoresis as slow- and fast-migrating fractions, bearing four- and eight-dye-molecule ligands, respectively, in the V domain of each individual chain. The slow-migrating complex is formed after displacement of the N-terminal polypeptide chain fragment (about 20 residues) from its packing locus, thereby exposing the entrance to the binding cavity. In this work the formation and stability of this complex was studied by molecular dynamics (MD) simulations. The effect of three- and five-molecule ligands introduced to the site binding the dye was also analyzed in an attempt to understand the formation of fast-migrating complexes. The wedging of the ligand containing five dye molecules, hence longer than established experimentally as the maximum for the slow-migrating complex, was found to generate significant structural changes. These changes were assumed to represent the crossing of the threshold on the way to forming a fast-migrating complex more capacious for dyes. They led to almost general destabilization of the V domain, making it susceptible to extra dye complexation. Theoretical studies were designed in close reference to experimental findings concerning the number of dye molecules in the ligand inserted to the site binding the dye, the location of the site in the domain, and the conditions of formation of the complexes. The results of the two kinds of studies appeared coherent.