Binding of nascent collagen by amyloidogenic light chains and amyloid fibrillogenesis in monolayers of human fibrocytes

Light (L) chain dimers expressed by multiple myeloma cells and collected as Bence-Jones proteins from the urine of human subjects were tested for thei r ability to form deposits in fibroblast monolayer cell cultures, Bence-Jon es proteins from subjects with primary amyloidosis associated with L chains were shown to form fibrillar deposits by the in vitro assay introduced in this report. Filaments interspersed with nascent collagen could be detected after only 48 h, Deposition of L chains continued over a period of 72 h cu lminating in the appearance of dense fibrils with widths of 80-100 Angstrom and a variety of lengths. Formation of amyloid-like fibrils was accompanie d by interference with the maturation of the collagen produced by the fibro blast cells. Fibrils composed of the Meg lambda-type L chain were deposited between collagen fibers, thus expanding them later-ally and leading to the ir partial disintegration. Mature collagen was completely missing from fibr oblast monolayers exposed to the Sea lambda chain and the Jen kappa chain. Collagen with the characteristic striped pattern matured normally in contro l samples, such as those not dosed with amyloid precursors or those treated with a non-amyloidogenic Bence-Jones protein (e.g., the Hud gamma chain di mer), By immunochemical techniques using fluorescein- and gold-labeled anti -L chain antibodies, amyloidogenic L chains were shown to decorate the stra nds of nascent collagen. This observation suggests that amyloidogenic L cha ins are concentrated in the extracellular matrix by monovalent antigen-anti body type reactions. The capacity of the Meg L chain dimer to bind collagen -derived sequences was tested by soaking crystals with a collagenase substr ate, PZ-Pro-Leu-Gly-Pro-D-Arg. Difference Fourier analysis at 2.7 Angstrom resolution indicated that the PZ-peptide is a site-filling ligand, It could not be removed from the active site by perfusion of the crystal with ammon ium sulfate crystallizing media. Similar experiments with the collagen-deri ved peptide (Pro-Pro-Gly)(5) showed substantial hysteresis effects extendin g from one end of the Meg dimer to the other. After the ligand was withdraw n, the active site of the Meg dimer could no longer bind the PZ-peptide, Ho wever, if the active site was first blocked by the PZ-peptide and subsequen tly exposed to the (Pro-Pro-Gly)(5) peptide, the difference Fourier map was indistinguishable from that obtained with the PZ-peptide alone. We conclud ed that amyloidogenic L chains such as the Meg dimer could be concentrated in the perivascular space by binding to normal tissue constituents. These c omponents include nascent collagen, which can be deterred from maturing as a result of this binding. Participation in such pathological activity is al so self-destructive to the amyloidogenic L chains, which lose their binding capabilities for collagen-derived peptides and also become susceptible to irreversible conversion to amyloid fibrils. All of these events may be prev ented by prior treatment of the amyloidogenic L chains with site-filling li gands, Copyright (C) 2000 John Wiley & Sons, Ltd.