Kinetic Analysis of Amyloid Formation in the Presence of Heparan Sulfate: FASTER UNFOLDING AND CHANGE OF PATHWAY*

A number of human diseases are associated with the conversion of proteins from their native state into well defined fibrillar aggregates, depositing in the extracellular space and generally termed amyloid fibrils. Heparan sulfate (HS), a glycosaminoglycan normally present in the extracellular matrix, has been found to be universally associated with amyloid deposits and to promote amyloid fibril formation by all studied protein systems. We have studied the impact of HS on the amyloidogenesis of human muscle acylphosphatase, monitoring the process with an array of techniques, such as normal and stopped-flow far-UV circular dichroism, thioflavin T fluorescence, static and dynamic light scattering, and atomic force microscopy. The results show that HS accelerates the conversion of the studied protein from the native state into the amyloidogenic, yet monomeric, partially folded state. They also indicate that HS does not simply accelerate the conversion of the resulting partially folded state into amyloid species but splits the process into two distinct pathways occurring in parallel: a very fast phase in which HS interacts with a fraction of protein molecules, causing their rapid aggregation into ThT-positive and β-sheet containing oligomers, and a slow phase resulting from the normal aggregation of partially folded molecules that cannot interact with HS. The HS-mediated aggregation pathway is severalfold faster than that observed in the absence of HS. Two aggregation phases are generally observed when proteins aggregate in the presence of HS, underlying the importance of a detailed kinetic analysis to fully understand the effect of this glycosaminoglycan on amyloidogenesis.Deposition of proteins in the form of extracellular amyloid fibrils is a consistent mechanism underlying a group of diverse human diseases, including neurodegenerative disorders and non-neuropathic conditions (1). From a pathogenetic standpoint, these disorders differ by type of aggregated protein and by type of organs involved in amyloid deposition. Among the most prominent neurodegenerative conditions are Alzheimer and Creuzfeldt Jakob diseases, which affect the central nervous system via extracellular deposits. Examples of non-neuropathic systemic amyloidosis are light chain amyloidosis and type II diabetes, where deposits are found in joints, skeletal tissue, and several organs (e.g. heart and kidney). Each of these disorders can be traced back to the aberrant conversion of one specific protein or peptide from its soluble, native state into amyloid structures (1). Numerous biochemical and genetic studies have established a widely accepted causative link between pathological symptoms and amyloid structure formation and deposition (2).Amyloid fibrils are often localized in close proximity to basement membranes, a specialized component of the extracellular matrix that is mainly built of collagens and glycosaminoglycans (GAGs),³ often attached to a protein core to form the proteoglycans (3–5). GAGs are long unbranched polysaccharides that often occur, with the exception of hyaluronic acid existing in a free form, as O- or N-linked side chains of proteoglycans, where they regulate the activity of several proteins. Since they have been found physically associated with all types of amyloid deposits in vivo so far analyzed, they have been attributed fundamental relevance in amyloidogenesis (3, 4). Of the different types of natural GAGs, heparan sulfate (HS) is among the most important cofactors in amyloid deposits. First, it has been established as a universal component of amyloid, since it has been found to be associated with amyloid deposits of different proteins, including the serum amyloid A protein (6), the immunoglobin light chain (7), transthyretin (8), cystatin C (9), the amyloid β peptide (10), the islet amyloid polypeptide (11), and the prion protein (PrP) (12). 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 β peptide (13, 14), mature islet amyloid polypeptide and proislet amyloid polypeptide 1–48 (15), α-synuclein (16), the 173–243 fragment of D187N gelsolin (17), β2-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 β-sheet, presumably aggregated, structure (20, 21) and to convert the prion protein from the PrP^C to the PrP^SC form (22).Despite the large body of data supporting the importance of HS in amyloidogenesis, little is known about the precise mechanism by which HS promotes amyloid formation and the effect that this GAG has on the various phases of the aggregation process and on the overall aggregation pathway. In the current work, human muscle acylphosphatase (mAcP) is utilized to study the impact of HS on amyloid aggregation, with particular attention to the various kinetic phases observed in the presence of this GAG. mAcP represents an enzyme unrelated to any human disease but a particularly suitable model for amyloid aggregation studies for a number of reasons. First, it is small in size (98 residues) and lacks disulfide bridges, trans-peptidyl-prolyl bonds, non-proteinaceous cofactors, and other complexities (23, 24). Second, it can form in the presence of 25% (v/v) trifluoroethanol (TFE) amyloid-like fibrils with extensive β-sheet structure and Congo Red birefringence (25). Third, its aggregation process has been studied using a variety of experimental approaches (25–33) and has been shown to be dramatically influenced by heparin, the highly sulfated form of HS (34).In the presence of 25% (v/v) TFE, mAcP has been shown to unfold rapidly into a denatured state enriched with α-helical structure (25). This partially unfolded state assembles to form, on a time scale of 1–2 h, amyloid-like protofibrils that develop very slowly to form, after a period of several days, long amyloid protofilaments that then associate further to form higher order structures (35). Even the early, protofibrillar aggregates that form within 1–2 h have the ability to bind Congo Red and thioflavin T (ThT) and have an extensive β-sheet structure, as detected with far-UV CD and Fourier transform infrared spectroscopy (25). This indicates that these protofibrillar structures have the essential structural characteristics of amyloid. The unfolding of the native state into a partially unfolded state is required to initiate aggregation, as shown by the need to use denaturing conditions to start aggregation (27, 35), by the finding that mutations destabilizing the native state promote aggregation (26), and by the observation that ligands binding to and stabilizing the native state have the opposite effect (29). Importantly, the TFE-denatured state of mAcP, which is the most commonly used to trigger aggregation of this protein and will also be used here, is not the only aggregation-competent state of mAcP, since other denatured states of mAcP have been shown to be capable of amyloid fibril formation (27).The present study aims at investigating the mechanism through which HS influences mAcP aggregation into amyloid-like aggregates. We will investigate both the unfolding and aggregation phases of mAcP in the presence of HS and will monitor them using a variety of biophysical methods. We will show that HS accelerates unfolding in addition to promoting aggregation of the resulting TFE-denatured state, thus playing a double-faced role in the context of its proaggregating effect. We will also show that HS is responsible for the appearance of parallel phases in the aggregation process of this protein and that its effect is not limited to a simple acceleration of the overall process. Following these findings, we will emphasize that a full understanding of the newly generated kinetics is essential for a correct interpretation of the effects of HS on amyloid formation.