Heparin Strongly Enhances the Formation of β2-Microglobulin Amyloid Fibrils in the Presence of Type I Collagen

Abstract

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of ␤ 2 -microglobulin (␤ 2 -m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of ␤ 2 -m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes ␤ 2 -m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7-99 truncated form of ␤ 2 -m (⌬N6␤ 2 -m) that is a ubiquitous constituent of the natural ␤ 2 -m fibrils. The formation of this ␤ 2 -m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition. Dialysis-related amyloidosis (DRA),2 a severe disease arising as a complication of long term hemodialysis, involves the deposition of ␤ 2 -microglobulin (␤ 2 -m) amyloid fibrils in bones and ligaments. ␤ 2 -m constitutes the light chain of the major histocompatibility complex class I and CD1 (1), and in normal catabolism, it is continuously released in the serum and cleared from the circulation by the kidney. The replacement of renal function by hemodialysis does not efficiently remove ␤ 2 -m. The persistent increase of its plasma concentration is associated with ␤ 2 -m deposition in the osteotendineous system, which is the specific target tissue of this type of amyloidosis. Among extra-cerebral amyloidoses, DRA represents the most striking case of tissue-specific targeting. Although other organs can be involved, bones and ligaments never escape amyloid deposition. Homma (2) first pointed out that collagen might be involved in determining this tissue specificity and demonstrated a collagen/␤ 2 -m interaction.We have recently determined the binding properties governing the collagen/␤ 2 -m interaction, and we found that the latter is quite weak but is enhanced when ␤ 2 -m is truncated at the N-terminal end, and the pH is reduced from 7.4 to 6.4 (3). We subsequently demonstrated that fibrillar collagen (type I), which is abundant in skeletal...

A Common β-Sheet Architecture Underlies in Vitro and in Vivo β2-Microglobulin Amyloid Fibrils

“…While subtle differences in the fibril architecture (for example in the precise stacking of the ␤-strands, the organization of non-␤-sheet components, and the arrangement of the side chains) cannot be discerned by FTIR spectroscopy and would require more detailed elucidation, for example, by solid state NMR spectroscopy (48), the ability of FTIR spectroscopy to distinguish the structures of long straight versus worm-like ␤ 2 m fibrils and different types of aggregates (amyloid-like fibrils versus amorphous aggregates) supports the concept of a common ␤-sheet organization for the ␤ 2 m amyloid fibrils studied here. These results are consistent with previous findings whereby limited proteolysis revealed a common structural core of ex vivo and in vitro ␤ 2 m amyloid involving residues ϳ10 -99 (27,49,50).…”

Effect of Tetracyclines on the Dynamics of Formation and Destructuration of β2-Microglobulin Amyloid Fibrils

“…For ␤2-m, we have recently set up a method of in vitro fibrillogenesis that generates ␤2-m fibrils under conditions reproducing a physiological milieu in the presence of two ubiquitous constituents of amyloid fibrils: collagen and heparin (31,32). Although the method exhibits some variability in the time of onset of fibril growth, probably because of the complexity of the aggregation conditions, AFM analysis is extremely sensitive and specific for monitoring fibril growth on the surface of collagen fibers.…”

Ultrasonication: An Efficient Agitation for Accelerating the Supersaturation-Limited Amyloid Fibrillation of Proteins

“…Recently, the mechanism of amyloid fibrillation under physiological pH conditions has been focused on. 18,[25][26][27][28] Amyloid fibrillation consists of nucleation and growth. 16,24,29,30) The nucleation process, in which a number of monomers associate to form a minimal fibril unit, does not readily occur.…”