Mechanism of Amylin Fibrillization Enhancement by Heparin

Abstract

We characterized the interaction of amylin with heparin fragments of defined length, which model the glycosaminoglycan chains associated with amyloid deposits found in type 2 diabetes. Binding of heparin fragments to the positively charged N-terminal half of monomeric amylin depends on the concentration of negatively charged saccharides but is independent of oligosaccharide length. By contrast, amylin fibrillogenesis has a sigmoidal dependence on heparin fragment length, with an enhancement observed for oligosaccharides longer than four monomers and a leveling off of effects beyond 12 monomers. The length dependence suggests that the negatively charged helical structure of heparin electrostatically complements the positively charged surface of the fibrillar amylin cross-␤ structure. Fluorescence resonance energy transfer and total internal reflection fluorescence microscopy experiments indicate that heparin associates with amylin fibrils, rather than enhancing fibrillogenesis catalytically. Short heparin fragments containing two-or eight-saccharide monomers protect against amylin cytotoxicity toward a MIN6 mouse cell model of pancreatic ␤-cells.Type 2 diabetes accounts for ϳ90% of adult diabetes. The disease currently affects 200 million people worldwide, and its incidence is projected to rise to 300 million by 2025 (1). Like many complex diseases, type 2 diabetes has multifactorial origins (1-4). The disease is characterized by insulin resistance, which causes the pancreas to synthesize more of the hormones insulin and amylin (5). The late stages of the disease are associated with ␤-cell dysfunction and a loss of ␤-cell mass (3, 5).Amylin (also known as islet amyloid polypeptide) is a 37-residue (4-KDa) endocrine hormone that is synthesized by pancreatic ␤-cells and co-secreted with insulin. In its normal state, amylin works together with insulin to control blood sugar (5). Additional amylin functions include suppressing appetite, slowing down the emptying of the stomach, and inhibiting glucagon secretion (5, 6). Like some 30 other polypeptides associated with human amyloid pathologies (6), amylin can undergo aggregative misfolding into fibrils with a cross-␤ structure (5,7,8). Although it is still uncertain whether fibrils or soluble oligomeric precursors are responsible for adverse affects (8 -12), extracellular amylin aggregates have been implicated in the destruction of pancreatic ␤-cells during the progression of type 2 diabetes (5, 9, 13). Amylin is the main component of fibrillar amyloid deposits found in the interstitial fluid between pancreatic islet cells of type 2 diabetes patients tested post-mortem (3,5,14), and extracellular aggregates of amylin are toxic when added to cultures of ␤-cells (9, 13). Overexpression of human amylin has been found to correlate with ␤-cell apoptosis and diabetes-like symptoms in several transgenic mouse and rat models whose endogenous amylin does not fibrillize (15). Genetic evidence that amylin is involved in pathology comes from the familial S20G mutation, which leads...

Heparan Sulfate Dissociates Serum Amyloid A (SAA) from Acute-phase High-density Lipoprotein, Promoting SAA Aggregation

“…Accumulated evidence shows that the molecular size of heparin is critical for aggregation of different types of amyloid proteins (43,44). It is believed that long HS/heparin structures provide a scaffold for the assembly of amyloid peptides, thereby promoting their polymerization into fibrillar structures.…”

Modulation of amyloid assembly by glycosaminoglycans: from mechanism to biological significance

“…At intermediate lengths (dp6 -dp12), the lag phase of fibril formation for most proteins is markedly reduced (Fraser et al 2001). Generally, fibrillization is accelerated with increasing oligosaccharide size, although for some systems, the effect on fibril assembly plateaus when the chain length becomes sufficiently long (dp ≫ 18) (Jha et al 2011;Takase et al 2016). In addition, the density and distribution of sulfated groups along the polysaccharides also appear to be critical.…”

“…Additionally, NMR studies showed that heparin interacts with the positively charged N-terminal domain of IAPP (Jha et al 2011) whereas isothermal titration calorimetry indicated a moderate affinity between IAPP and heparin (De Carufel et al 2013). Considering that sulfated GAGs interact avidly with IAPP and modulate its fibrillization, the influence of GAGs on IAPP cytotoxicity has been addressed.…”

“…The degree of polymerisation (dp) is of primary importance for fibrillogenesis (Bourgault et al 2011b;Jha et al 2011;Stewart et al 2016;Takase et al 2016). At short lengths (dp ≤ 4), GAGs exert minimal effect on the rate of amyloid formation.…”

“…When applied to isolated pancreatic β-cells, IAPP induces cell death by mecha-D r a f t nisms that are still not clearly understood . Strikingly, soluble heparin and its synthetic fragments reduced the cytotoxicity induced by IAPP (De Carufel et al 2013;Jha et al 2011). The role of cell surface GAGs in IAPP toxicity was investigated using CHO pgs-A-745 cells, which lack GAGs on the outer leaflet of their plasma membrane due to a deficiency in xylosyltransferase, a key enzyme in PG biosynthesis.…”