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

Abstract

Primary amyloidosis (AL) results from overproduction of unstable monoclonal immunoglobulin light chains (LCs) and the deposition of insoluble fibrils in tissues, leading to fatal organ disease. Glycosaminoglycans (GAGs) are associated with AL fibrils and have been successfully targeted in the treatment of other forms of amyloidosis. We investigated the role of GAGs in LC fibrillogenesis. Ex vivo tissue amyloid fibrils were extracted and examined for structure and associated GAGs. The GAGs were detected along the length of the fibril strand, and the periodicity of heparan sulfate (HS) along the LC fibrils generated in vitro was similar to that of the ex vivo fibrils. To examine the role of sulfated GAGs on AL oligomer and fibril formation in vitro, a 1 LC purified from urine of a patient with AL amyloidosis was incubated in the presence or absence of GAGs. The fibrils generated in vitro at physiologic concentration, temperature, and pH shared morphologic characteristics with the ex vivo 1 amyloid fibrils. The presence of HS and over-O-sulfatedheparin enhanced the formation of oligomers and fibrils with HS promoting the most rapid transition. In contrast, GAGs did not enhance fibril formation of a non-amyloidogenic 1 LC purified from urine of a patient with multiple myeloma. The data indicate that the characteristics of the full-length 1 amyloidogenic LC, containing post-translational modifications, possess key elements that influence interactions of the LC with HS. These findings highlight the importance of the variable and constant LC regions in GAG interaction and suggest potential therapeutic targets for treatment.Amyloid deposition is hypothesized to play a role in many diseases including rheumatoid arthritis and diabetes, as well as the amyloidoses themselves (1). The amyloidoses are a group of systemic and localized diseases that exhibit deposition of insoluble fibrillar proteins in organs and tissues. Primary amyloidosis (AL) 2 is the most common systemic form in the UnitedStates. It results from a plasma cell dyscrasia in which clonal B cells produce an excess of amyloidogenic immunoglobulin light chains (LCs), which circulate and deposit as insoluble fibrils throughout the body (2). A structural rearrangement from nonfibril-prone oligomers to more fibril-prone conformers is required for protofilament formation (3). Nascent protofilaments can nucleate and elongate through the addition of partially folded oligomeric intermediates to the ends of the growing protofilaments. Protofilaments can further intertwine to form protofibrils and fibrils (4, 5). Glycosaminoglycans (GAGs) are one of the major components of the extracellular matrix. They consist of disaccharide repeating units that are unbranched and attached to a serine residue of a core protein through a trisaccharide linker. Each GAG has a unique sequence, and post-assembly modifications, which can be induced by a number of factors, lead to alterations in sulfation, acetylation, or length of chain (6). Recently, Staples et al. (7) showed that the fu...

The Kinetic Stability of a Full-Length Antibody Light Chain Dimer Determines whether Endoproteolysis Can Release Amyloidogenic Variable Domains

“…LC dimers do not efficiently refold from a chaotrope-denatured state; hence, the refolded state could be susceptible to endoproteolysis, although we did not experimentally test this hypothesis herein. It is established that extracellular matrix components hasten the aggregation of amyloidogenic proteins [50, 51], a variable that merits further exploration, although we have not observed such an effect in our experiments to date (data not shown). It is also possible that amyloid fibrils composed of LC fragments can seed or accelerate the aggregation of full-length LCs, although we have not observed this in vitro with these LCs (data not shown).…”

Divergent effect of glycosaminoglycans on the in vitro aggregation of serum amyloid A

“…8). Although sulfation, carboxylation, and charge pattern of GAGs have been shown to be important for their effect on amyloid formation, this GAG structure-function correlation is not obvious in our results and warrants further investigation [42, 43]. However, our AFM data show that further studies are necessary to better understand the intrinsic effects of sulfation and carboxylation patterns, and saccharide conformation and content, on the aggregation propensity of SAA and other amyloidogenic proteins.…”

In vivo molecular imaging of peripheral amyloidosis using heparin-binding peptides

“…Because of the critical role played by heparin in the coagulation cascade and the importance of heparin-like binding motifs expressed by signaltransducing cellular glycosaminoglycans, there has been considerable interest in identifying and characterizing heparin-binding peptides (34). In addition, several recent in vitro studies have shown that highly sulfated heparin catalyzes the fibrillogenesis of amyloid precursor proteins, including Ig light chains, β-2-microglobulin, Aβ, and sAA, relative to less-sulfated glycosaminoglycans (35)(36)(37). Based on our hypothesis that HS in amyloid is heparin-like, we synthesized a panel of heparinbinding peptides and assessed their ability to bind amyloid in vivo.…”