Sulfate content and specific glycosaminoglycan backbone of perlecan are critical for perlecan's enhancement of islet amyloid polypeptide (amylin) fibril formation.

Abstract

Islet amyloidosis is characterized by the deposition and accumulation of amylin in pancreatic beta-cells and is observed in 90% of patients with type 2 diabetes. Previous studies have also revealed the presence of the specific heparan sulfate proteoglycan, perlecan, colocalized to islet amyloid deposits, similar to perlecan's known involvement with other amyloid proteins. In the present study, perlecan purified from the Engelbreth-Holm-Swarm (EHS) tumor was used to define perlecan's interactions with amylin (i.e., islet amyloid polypeptide) and its effects on amylin fibril formation. Using a solid phase-binding immunoassay, human amylin, but not rat amylin, bound immobilized EHS perlecan with a single dissociation constant (Kd) = 2.75 x 10(-6) mol/l. The binding of human amylin to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans (GAGs), and was completely abolished by 10 micromol/l heparin. Using thioflavin T fluorometry, Congo red staining, and electron microscopy methodology, intact perlecan was found to enhance amylin fibril formation in a dosage-dependent manner, with the majority of these effects attributed to the heparan sulfate GAG chains of perlecan. Other sulfated GAGs and related macromolecules were also effective in the enhancement of amylin fibril formation in the order of heparin > heparan sulfate > chondroitin-4-sulfate = dermatan sulfate = dextran sulfate > pentosan polysulfate, implicating the importance of the specific GAG/carbohydrate backbone. The sulfate content of heparin/heparan sulfate was also important for the enhancement of amylin fibril formation in the order of heparin > N-desulfated N-acetylated heparin > completely desulfated N-sulfated heparin > completely desulfated N-acetylated heparin. These studies suggest that the enhancement effects of perlecan on amylin fibril formation are mediated primarily by both specific GAG chain backbone and GAG sulfate content, and implicate perlecan as an important macromolecule that is likely involved in the pathogenesis of islet amyloidosis.

Small molecule inhibitors of Aβ‐aggregation and neurotoxicity

“…Similarly, highly sulfated GAGs, such as carrageenan-t pentosan sulfate and heparin sulfate protect against decreased cell viability caused by Ab in PC-12 cells, while less sulfated GAGs were less effective [Pollack et al, 1995a]. The importance of sulfate group number and distribution on the GAG backbone for the effectiveness of its interaction with Ab [Fraser et al, 1992;Leveugle et al, 1994], was confirmed by reports that heparin-induced increases in Ab fibril formation were impaired following removal of O-and N-sulfates and were completely lost upon removal of all sulfate moieties from heparin [Castillo et al, 1998[Castillo et al, , 1999.…”

Role of Glycosaminoglycan Sulfation in the Formation of Immunoglobulin Light Chain Amyloid Oligomers and Fibrils

“…Kisilevsky and co-workers (12,13) hypothesized that the deposition of truncated serum amyloid A (SAA) was preceded by the up-regulation of HS and chondroitin sulfate (CS) proteoglycans. Interestingly, other investigators demonstrated that HS promoted the aggregation of SAA (14) and hypothesized that HS was a stabilizer of amyloid (15). The association of GAGs with other fibrillar deposits was demonstrated by Cohlberg et al (16), whose studies showed that heparin increased the rate of fibrillation of ␣-synuclein and actually became an integral part of the fibril.…”

Kinetic Analysis of Amyloid Formation in the Presence of Heparan Sulfate

“…mAcP was incubated as described in the legend to Fig. 1A promote amyloid fibril formation in vitro by all studied protein systems (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). In particular, it is generally reported that HS causes an increase of the rate by which fibrils are formed (13)(14)(15)(16)(17)22) and of the final ThT fluorescence (14 -17).…”

“…More importantly, it has been attributed an active role in amyloidogenesis. Its ability to promote fibrillogenesis has been reported for both the 42-and 40-residue forms of the amyloid ␤ peptide (13,14), mature islet amyloid polypeptide and proislet amyloid polypeptide 1-48 (15), ␣-synuclein (16), the 173-243 fragment of D187N gelsolin (17), ␤2-microgloblulin (18), and the tau protein (19). HS has also been found to shift the secondary structure of a subtype of serum amyloid A protein from a random coil to a ␤-sheet, pre-sumably aggregated, structure (20,21) and to convert the prion protein from the PrP C to the PrP SC form (22).…”