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

Annalisa Relini,Silvia De Stefano,Silvia Torrassa,Ornella Cavalleri,Ranieri Rolandi,Alessandra Gliozzi,Sofia Giorgetti,Sara Raimondi,Loredana Marchese,Laura Verga,Antonio Rossi,Monica Stoppini,Vittorio Bellotti

doi:10.1074/jbc.m702712200

Abstract

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of beta2-microglobulin (beta2-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 beta2-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 beta2-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 beta2-m (DeltaN6beta2-m) that is a ubiquitous constituent of the natural beta2-m fibrils. The formation of this beta2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition.

Highlights

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, which is abundant in skeletal tissues, is a potent promoter of ␤2-m fibrillation at 37 °C and pH 6.4 [4]; we chose this pH because it was compatible with pathophysiological conditions including the presence of inflammatory processes frequently associated with this amyloid deposition
In vivo studies have demonstrated the coincident deposition of amyloidogenic protein and GAGs [13], and in a mouse model of amyloid protein A (AA) amyloidosis a resistance against the amyloid deposition can be achieved through the overexpression of heparanase, which degrades heparan sulfate [14]

Summary

Introduction

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 performed light scattering measurements to test the effect of heparin on the aggregation process of ␤2-m.

Results

Conclusion