Contents Into the Extracellular Environment (Westermark Et Al

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Chapter 6: The effect of glycosaminoglycans on proIAPP amyloid formation

contents into the extracellular environment (Westermark et al., 1996). EDTA was used to chelate any trace amounts of metal cations present in buffer solutions. The effect of heparin on

proIAPP was the same regardless of the presence or absence of metal cations indicating that metal cations were not required for heparin induced amyloid formation (Figure 6.11). EDTA appeared to slightly enhance the effect of heparin on proIAPP. Since electrostatic interactions are important in the binding of heparin to heparin-binding sites, sequestration of trace cations from the phosphate buffer by EDTA, may remove a slight shielding effect from divalent cations and slightly increase the effects of heparin. All subsequent experiments were done without added divalent cations.

Figure 6.11 The effect of heparin on proIAPP amyloid formation is not dependent on metal cations at pH 7.4 and 5.5. 125 µg/mL ProIAPP was incubated in the absence of heparin (red) or in the presence of 31 µg/mL heparin (pink), with 1 mM CaCl2 (green), 1 mM MgCl2 (black), 1 mM ZnCl2 (orange) and 1 mM EDTA (blue) at pH 7.4 (A) and 5.5 (B). Aliquots of the proIAPP were diluted to 37.5 µg/mL in 5 µM ThT, 100 mM HEPES, 150 mM NaCl, pH 7.4 (A) or 5 µM ThT, 100 mM HEPES, 150 mM 150 mM, pH 6.5 (B) and the fluorescence measured as described in Section 2.2.15a.

6.2.7 The effect of heparins of different molecular weights on formation of amyloid from proIAPP

The effect of heparins of known molecular weight on proIAPP amyloid formation was investigated to try to define a minimal heparin unit responsible for the heparin-induced acceleration of this process. A low molecular weight heparin (3 kDa) and heparin with a molecular weight of between 17 and 19 kDa, were tested. The effect of a smaller heparin subunit, a heparin disaccharide (molecular weight 664.4 Da), was also investigated. The 3 kDa heparin (Figure 6.12A), 17-19 kDa heparin (Figure 6.12B) had very similar effects to that seen previously with the approximately 4 kDa heparin (Figure 6.5): a shortening of the lag time to approximately 10 h and a growth phase that was approximately 25 h long. The fibrils formed in the presence of heparins all had increased ThT fluorescences compared to fibrils formed in the absence of heparin. The similarity of the effects of the different heparin of different molecular weights indicates that minimal heparin unit is smaller than 3 kDa. In contrast heparin disaccharides, did not increase the rate of amyloid formation, indicating that the minimal unit is greater than greater than 664.4 and 3 kDa or less (Figure 6.13).

Figure 6.12 The effect of heparins of different molecular weights on proIAPP amyloid formation. The effect of a 3 kDa (A) or a 17-19 kDa heparin (B) on the amyloid formation of 125 µg/mL proIAPP in PBSaz, pH 7.4 was measured using a ThT assay as described in Section 2.2.16a. The heparin concentrations used were: 0 (red), 4 µg/mL (orange), 8 µg/mL (yellow), 16 µg/mL (aqua), 31 µg/mL (pink), 63 µg/mL (black), 125 µg/mL (green) and 250 µg/mL (blue). The sigmoidal curve described by equation 3 in Section 2.2.16a was fitted to the ThT fluorescences.

Figure 6.13 The effect of heparin disaccharides on proIAPP amyloid formation. 125 µg/mL proIAPP was incubated alone (red) and with 4 µg/mL (black), 16 µg/mL (green) and 63 µg/mL (blue) heparin disaccharides. 4 Amyloid formation was measured using ThT assays as described in Section 2.2.16a

The sigmoidal equation 3 described in Section 2.2.16a was fitted to the ThT fluorescences shown in Figure 6.12 to allow the final ThT fluorescences to be calculated. The concentrations of heparin were expressed in molar units for 3 kDa and 17-19 kDa heparin and the concentration of heparin necessary for the half-maximal effect of enhancing ThT fluorescence was determined by fitting equation 4 to the final ThT fluorescences as described in Section 2.2.16a. The results of these analyses are shown in Figure 6.14. The concentration of heparin necessary for the half-maximal effect was calculated to be 3.1 ± 1.0 µM for the 3 kDa heparin and 0.3 ± 0.1 µM for the 17-19 kDa heparin. In comparison, the concentration of 4 kDa heparin necessary for the half-maximal effect of the enhancement of ThT fluorescence was 5.7 ± 2.2 µg/mL at pH 7.4 and 5.8 ± 3.9 µg/mL at pH 5.5 (Section 6.2.4), which correspond to 1.4 ± 0.6 µM and 1.4 ± 1.0 µM, respectively. These values suggest that the heparins with larger molecular weights contain more binding sites for proIAPP per heparin molecule than the 3 kDa heparin. Therefore, in addition to a role for heparin in inducing a partially folded intermediate that is likely to favour oligomer formation (Sections 6.2.2 and 6.2.3), the binding of multiple proIAPP molecules on a single heparin molecule would bring these proIAPP molecules in close proximity to one another, favouring oligomer formation.

Figure 6.14 The heparin concentration-dependence of final ThT fluorescence of proIAPP amyloid induced by 3 kDa and 17-19 kDa heparin. The final ThT fluorescences of 3 kDa heparin data (red) and 17-19 kDa heparin data (blue) was determined by fitting equation 3 in Section 2.2.16a to the ThT fluorescences presented in Figure 6.12. Equation 4 in Section 2.2.16a was then fitted to the final ThT fluorescences.

The minimal heparin subunit necessary for heparin-induced proIAPP amyloid formation is greater than 664.4 Da but less than 3 kDa. A 3 kDa heparin would correspond to approximately 9 monosaccharides. The model of a heparin-binding site, which accommodates a pentasaccharide of heparin, proposed by Margalit and co-workers, is consistent with our data (Margalit et al., 1993) (Section 6.1.6). If a pentasaccharide is the minimal heparin subunit, the 3 kDa heparin could contain 1 or two proIAPP binding sites, the 4 kDa heparin could contain 2 proIAPP binding sites and the 17-19 kDa heparin could contain 10 proIAPP binding sites.

6.2.8 The effect of heparin disaccharides on heparin-induced proIAPP amyloid formation

Since heparin disaccharides did not induce amyloid formation, we investigated whether heparin disaccharides could act as competitors of heparin-induced proIAPP amyloid formation. The heparin disaccharides were tested at 4, 16 and 63 µg/mL in mixtures with proIAPP and 16 µg/mL heparin. In the absence of heparin or heparin disaccharides, the lag phase was 20 h (Figure 6.15). In the presence of 16 µg/mL heparin without heparin disaccharides, the lag phase had decreased to approximately 10 h, which is consistent with previous findings (Figure 6.7). The lag phase was also approximately 10 h in the presence of 16 µg/mL heparin and at all concentrations of heparin disaccharide, which at the highest concentration of heparin disaccharide, 63 µg/mL, represents 4-fold more heparin disaccharide by weight than heparin (Figure 6.15). This indicated that the heparin disaccharides had no effect on the heparin-induced shortening of the lag phase.

The heparin disaccharides reduced the final ThT fluorescences when amyloid formation has gone to completion in the presence of heparin. This may indicate that the heparin disaccharides reduced the total amount of amyloid formed in the presence of heparin. Protein concentration assays to determine the final concentration of proIAPP remaining in solution as has been done in Figure 6.10 for heparin-induced proIAPP amyloid formation, were unsuccessful as the heparin disaccharides strongly cross-reacted with the BCA assay reagents (results not shown). ThT fluorescence assays using fibrils, formed in the presence of heparin, which had been spiked with heparin disaccharides were carried out see if the ThT fluorescence was quenched by heparin disaccharides. It was found that heparin disaccharides did not alter the ThT fluorescence of the fibrils (Figure 6.16). The heparin disaccharides may interfere with the presumptive changed fibril morphology discussed in Section 6.2.4 that causes the enhanced ThT fluorescence seen for amyloid formed in the presence of heparin. This possibility may be more likely than inhibition of amyloid formation by the heparin disaccharides because the heparin disaccharides did not block the heparin-induced shortening of the growth phase.

Figure 6.15 The effect of heparin disaccharides on heparin-induced proIAPP amyloid formation. 125 µg/mL proIAPP in absence of heparin and heparin disaccharides (red) and in the presence of 16 µg/mL heparin alone (blue) and in the presence of 16 µg/mL heparin and 4 µg/mL heparin disaccharides (pink), in the presence of 16 µg/mL heparin and 16 µg/mL heparin disaccharides (black) and in the presence of 16 µg/mL heparin and 63 µg/mL heparin disaccharides (green). Amyloid formation was measured using ThT assays as described in Section 2.2.16a. The sigmoidal curve described by equation 3 in Section 2.2.16a was fitted to the ThT fluorescences.

Figure 6.16 The addition of heparin disaccharides to proIAPP amyloid fibrils formed in the presence of heparin does not quench ThT fluorescence. Amyloid fibrils were formed by incubation of 125 µg/mL proIAPP, 31 µg/mL heparin at 37 oC. The fibrils were incubated for 1 h in the absence of heparin disaccharide (red) or in the presence of 63 µg/mL heparin disaccharide (blue), 18 µg/mL heparin disaccharide (green) and 4 µg/mL heparin dissacharide (black). 4 Amyloid formation was measured using ThT assays as described in Section 2.2.16a.