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Chapter 6: The effects of glycosaminoglycans on proIAPP amyloid formation

6.3 DISCUSSION

6.3.1 The effect of different GAGs on proIAPP amyloid formation

The results presented in this chapter show that the GAGs heparin, heparan sulfate and chondroitin sulfate accelerated proIAPP amyloid formation. The GAGs induced a more ordered structure in proIAPP, which is natively unfolded in the absence of GAGs (Figure 6.4). Heparin induced the greatest increase in ordered secondary structure followed by heparan sulfate and chondroitin sulfate (Table 6.1). These processes correlated with the relative affinities of the GAGs for the N-terminal peptide of proIAPP, which had been identified as containing a heparin/heparan sulfate binding site (Park and Verchere, 2001). The more ordered conformations of proIAPP induced by GAGs (Figure 6.4) may represent partially folded intermediates, which are believed to be critical for the formation of a stable nucleus for amyloid formation (Sections 1.5 and 4.1). In this context, the reduced lag time of amyloid formation in the presence of GAGs compared to the absence of GAGs, indicating that nucleation occurred more rapidly in the presence of GAGs (Figure 6.3), is consistent with this notion that the GAGs induced partially folded intermediates critical for the formation of a stable nucleus for amyloid formation.

Furthermore, in the presence of GAGs, the rate of the growth phase was increased compared with the growth rate of amyloid formation in the absence of GAGs (Table 6.1). During the growth phase, elongation of fibrils occurs by addition of soluble protein to fibril ends. In the case of proIAPP, the protein must undergo conformational changes to convert to the cross -sheet of amyloid. The partially folded conformations of proIAPP formed in the presence of GAGs may add onto the fibril ends more rapidly than natively unfolded proIAPP, thus allowing fibril elongation to occur more rapidly in the presence of GAGs.

The different GAGs induced similar amounts of -sheet and -turns but different amounts of -helix, which was approximately 20%, 10% and 7% for heparin, heparin sulfate and chondroitin sulfate, respectively (Table 6.2). As discussed in Section 6.2.2, the high -helical content induced by the presence of heparin relative to that induced by heparan sulfate and chondroitin sulfate may be responsible for the increased rate of amyloid formation in the presence of heparin compared to heparan sulfate and chondroitin sulfate (Figure 6.3 and Table 6.1). Amyloid formation may then occur by an -helix to -strand conversion as has been suggested for A amyloid formation (Kirkitadze et al., 2001, Fezoui and Teplow, 2002).

6.3.2 The minimal subunit of heparin necessary for proIAPP binding

The minimal subunit of heparin necessary for proIAPP binding, based on the abilities of different heparin species to accelerate proIAPP amyloid formation, was greater than a heparin disaccharide (664.4 Da) (Figure 6.13) and less than heparin containing approximately 9 monosaccharides (3 kDa heparin) (Figure 6.12). This is consistent with the minimal heparin subunit necessary to bind to a heparin-binding site on a protein proposed by Margalit et al. being a pentasaccharide (Margalit et al., 1993).

The half-maximal concentration of 4 kDa heparin necessary to induce a conformational change in 100 µg/mL proIAPP as assessed by far-UV CD was determined to be approximately 7 µg/mL, which corresponds to approximately 1.75 µM (Section 6.2.3). If, at the half-maximal concentration of heparin, half the proIAPP (50 µg/mL, 7 µM) is bound to 1.75 µM heparin, this gives a heparin:proIAPP molar ratio of 1:4. If a pentasaccharide is the minimal heparin binding subunit, 4 kDa heparin could at most contain 2 binding sites for proIAPP, suggesting that the 4 kDa heparin may be binding to an oligomer of proIAPP with a partially folded structure (Figure 6.5).

As discussed in Section 6.2.4, the dose-dependence of heparin-induced conformational change is correlated with the dose-dependent enhancement of final ThT fluorescences of proIAPP formed in the presence of heparin. The half-maximal concentrations of heparin necessary for the enhanced final ThT fluorescences for 3 kDa heparin, 4 kDa heparin and 17-19 kDa heparin were 9.2 ± 2.9 µg/mL, 5.6 ± 2.2 and 4.9 ± 1.3 µg/mL, respectively, are the same within error. These concentrations correspond to 3.1 ± 1.0 µM, 1.4 ± 0.6 µM and 0.3 ± 0.1 µM respectively. The decreasing molar half-maximal concentrations with increasing molecular weight of the heparin indicate that the larger molecular weight heparins contain multiple proIAPP binding sites. This could allow the 17-19 kDa heparin to bind to a larger oligomer of proIAPP or bind to multiple smaller oligomers, of the size likely to bind to 4 kDa heparin as discussed above, and by binding multiple small oligomers induce the formation of a larger oligomer.

6.3.3 The effect of buffers of differing ionic strength on heparin-induced conformational changes in proIAPP

The effect of heparin on proIAPP was investigated using both a low ionic strength buffer, (10 mM sodium phosphate, pH 7.4) and two high ionic strength buffers, PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) and 10 mM sodium phosphate, 150 mM NaF, pH 7.4. The binding of proIAPP to heparin, assessed using a heparin-agarose pull-down assay, was weaker in the presence of high ionic strength buffers compared to that in a low ionic strength buffer (Figure 6.20). Increased ionic strength has been shown to similarly reduce the binding of other heparin-binding proteins to heparin (Sections 6.1.6 and 6.1.8). This may be because the increase in ionic strength shields the electrostatic interactions between the heparin-binding protein and heparin (Sections 6.1.6 and 6.1.8).

Figure 6.23 shows a schematic of the proposed effects of heparin on proIAPP in the presence of low and high ionic strength buffers. Almost all the proIAPP was bound to heparin within 15 min, assessed using a heparin-agarose pull-down assay in 10 mM sodium phosphate, pH 7.4 (Figure 6.20A) and this was correlated with an immediate conformational change in proIAPP to a more ordered structure, assessed by far-UV CD (Figures 6.4 and 6.5). In contrast, almost no proIAPP was bound to heparin, assessed using a heparin-agarose pull-down assay in PBS within a 15 min (Figure 6.20B). The absence of proIAPP binding to heparin in a high ionic strength buffer (PBS) was correlated with an absence of an immediate heparin-induced conformational change in proIAPP in 10 mM sodium phosphate, 150 mM NaF, pH 7.4 observed by far-UV CD (Figure 6.21A). This is consistent with the bis-ANS assays that showed that freshly dissolved proIAPP in PBSaz exhibited the same amount of exposed hydrophobic surface regardless of the presence and absence of heparin, suggesting that the conformation of proIAPP had not changed in the presence of heparin (Figure 6.17).

Figure 6.23 Schematic of the effects of heparin on proIAPP in buffers of differing ionic strength.

Heparin induced conformational changes in proIAPP in high ionic strength buffer (10 mM sodium phosphate, 150 mM NaF, pH 7.4) by 8 h as assessed by far-UV CD (Figure 6.21C), which was more slowly than the immediate conformational change seen in low ionic strength buffer (10 mM sodium phosphate, pH 7.4) (Figure 6.5). In the presence of NaF, there was no evidence of a partially folded conformation as had been observed in the absence of NaF, rather a decrease in the intensity of the CD signal was measured (Figure 6.21). As it is unlikely that heparin could interact with a specific heparin-binding site on proIAPP in more than one conformation, heparin is likely to induce partially folded conformation in proIAPP in the presence of NaF, however the increase in ionic strength would increase any hydrophobic interactions and this conformation could progress down the amyloid formation pathway rapidly.

6.3.4 The effect of heparin on the final ThT fluorescence of proIAPP amyloid

Heparin induced an increase in the final ThT fluorescence of proIAPP amyloid (Figure 6.7 and 6.12), which was not due to heparin enhancing the ThT fluorescence because the addition of heparin to fibrils formed in the absence of heparin did not increase the ThT fluorescence (Figure 6.9). Nor could the increase in final ThT fluorescence of proIAPP fibrils formed in the presence of heparin (Figure 6.8) be completely accounted for by increased conversion of soluble proIAPP to fibrils (Figure 6.10) or the incorporation of heparin into the fibrils resulting in fibrils with increased affinity for ThT fluorescence, which was minimal (Figure 6.22).

However the enhancement of final ThT fluorescence of proIAPP fibrils formed in the presence of heparin, compared to that formed in the absence of heparin, is not specific to proIAPP. Amyloid formed in the presence of heparin had enhanced final ThT fluorescences of up to 1.9-fold for A A(Castillo et al., 1999), 6-fold for -synuclein (Cohlberg et al., 2002) and 3.9-fold for IAPP (Castillo et al., 1998), compared to amyloid formed in the absence of heparin from these proteins. As IAPP amyloid formation in the absence of heparin results in almost all the soluble IAPP being converted to amyloid (approximately soluble 1 µM IAPP from an original 25 µM) (Larson et al., 2000), this indicates that the 3.9-fold enhancement of ThT fluorescence observed in the presence of heparin is unlikely to be due to a 3.9-fold increase in amyloid formed in the presence of heparin (Castillo et al., 1998). The fluorescence depends on the number of binding sites, binding affinity and quantum yield. Therefore one possibility is that fibrils formed in the presence of GAGs may have increased numbers of binding sites for ThT than fibrils formed in the absence of GAGs. Thicker fibrils that contain more protofilaments per fibril may contain more ThT binding sites as than fibrils that contain fewer protofilaments per fibril as the regions in contact with one another in a thicker fibril may be inaccessible for ThT binding. However increased final ThT fluorescences do not correlate with thinner fibrils. EM microscopy however shows varied degrees of lateral association of protofilaments for these proteins in the presence of heparin and other GAGs (Section 6.1.4). In the presence of heparin, -synuclein fibrils are thinner and composed of a 3.8 nm protofilament compared to 6.5 nm fibril in the absence of heparin (Cohlberg et al., 2002, Khurana et al., 2003). However A fibrils are more highly aggregated being 20-30 nm wide in the presence of heparin compared to 5-7 nm in the absence of heparin (McLaurin et al., 1999). Moreover, IAPP formed in the presence of heparin was highly associated into large clumps compared to dispersed fibrils formed in the absence of heparin (Castillo et al., 1998).

An alternative possibility is that fibrils formed in the presence of heparin may have an increased affinity for ThT. Given that the heparin concentrations necessary for the half-maximal effect of conformational change were within error, the same as those for the enhanced final fluorescences of ThT, suggesting that the same heparin:proIAPP binding event(s) are responsible for both processes, this possibility appears most likely (Sections 6.2.4 and 6.2.7). The heparin-induced conformational changes in proIAPP may lead to proIAPP being incorporated into amyloid with an altered structure, which results in enhanced ThT fluorescence. The affinity for ThT for different amyloid fibrils depends on the constituent protein however it is not clear why this is the case (LeVine, 1999). The mode of ThT binding to amyloid is unclear at a structural level beyond the fact that it is specific for amyloid making it difficult to propose a reason for the increased final ThT fluorescences of fibrils formed in the presence of heparin.

6.3.5 The role of GAGs in amyloid formation in Type 2 diabetes

In summary, GAGs increased the rate of proIAPP amyloid formation, by inducing the conversion of proIAPP from a natively unfolded conformation to a partially folded oligomeric conformation. The results presented above support the mechanism of IAPP amyloid formation in Type 2 diabetics that proIAPP is secreted from the -cell in higher quantities in Type 2 diabetes, proIAPP then binds to heparan sulfate proteoglycans in the extracellular matrix via its N-terminal heparin/heparan sulfate-binding site and forms a “nidus” for the subsequent accumulation of secreted IAPP, which forms the majority of the deposit (Jaikaran and Clark, 2001, Marzban et al., 2003).

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