How Glycosaminoglycans Promote Fibrillation of Salmon Calcitonin

Abstract

Glycosaminoglycans (GAGs) bind all known amyloid plaques and help store protein hormones in (acidic) granular vesicles, but the molecular mechanisms underlying these important effects are unclear. Here we investigate GAG interactions with the peptide hormone salmon calcitonin (sCT). GAGs induce fast sCT fibrillation at acidic pH and only bind monomeric sCT at acidic pH, inducing sCT helicity. Increasing GAG sulfation expands the pH range for binding. Heparin, the most highly sulfated GAG, binds sCT in the pH interval 3-7. Small angle x-ray scattering indicates that sCT monomers densely decorate and pack single heparin chains, possibly via hydrophobic patches on helical sCT. sCT fibrillates without GAGs, but heparin binding accelerates the process by decreasing the otherwise long fibrillation lag times at low pH and accelerates fibril growth rates at neutral pH. sCT⅐heparin complexes form ␤-sheet-rich heparin-covered fibrils. Solid-state NMR reveals that heparin does not alter the sCT fibrillary core around Lys 11 but makes changes to Val 8 on the exterior side of the ␤-strand, possibly through contacts to Lys 18. Thus GAGs significantly modulate sCT fibrillation in a pH-dependent manner by interacting with both monomeric and aggregated sCT.

Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument

“…Hence, it would be of great value to the field to be able to perform SEC-SAXS on an in-house instrument which is close to the home laboratory and where experiments are not affected by radiation damage or time limitations to the same extent as on synchrotron BioSAXS beamlines. Over the past decade, advances in the development of laboratory SAXS instruments optimized for solution measurements have enabled investigation of biological samples using static (not coupled to in-line chromatography) SAXS at the home laboratory (Mortuza et al, 2014;Sibillano et al, 2014;Dupont et al, 2015;Bruetzel et al, 2016;Malmos et al, 2016;Mortensen et al, 2017). To our knowledge, however, there are to date no reports of SEC-SAXS having been implemented on any laboratory instruments.…”

Molecular Origins of the Compatibility between Glycosaminoglycans and Aβ40 Amyloid Fibrils

“…2 a). Although several studies of GAG binding to amyloid proteins have been performed to date [12] , [13] , [21] , [56] , few have investigated the specificity of the GAG structure on the binding affinity to its target protein [37] , [38] . Heparin contains alternating α-D-glucosamine and uronic acid substituents, with sulfate modifications commonly at the 2- O position on α-L-iduronate and the 2-(amino) and 6- O positions on d -glucosamine ( Fig.…”

“…These sulfate groups are proposed as a requirement for molecular recognition via electrostatic interactions between the GAG and basic amino acid side chains of binding partners [36] . However, few models of an amyloid–GAG structure have been determined to date [37] , [38] , leaving unresolved the question of how GAGs recognize the cross-β structure of amyloid. Studies of soluble (non-amyloid) proteins bound to GAGs suggest that there is a complex relationship between protein binding and charge disposition, conformation, and flexibility of the polysaccharide [39] .…”

Release of Pharmaceutical Peptides in an Aggregated State: Using Fibrillar Polymorphism to Modulate Release Levels

“…The release is in agreement with the release behavior reported for gonadotropin-releasing hormone aggregates [28], albeit with much faster kinetics (minutes rather than days). The in vivo release profile may be altered by various serum components known to interact with proteins and protein aggregates, such as glycosaminoglycans [46] and amyloid P protein [47]. We did not address potential immunogenicity of 3474 aggregates, but note that there is large variance between different aggregates [48,49].…”