ß(2)-microglobulin amyloidosis.

Dialysis-related amyloidosis (DRA) is a clinical syndrome of pain, loss of function and other symptoms due to the deposition of amyloid consisting of ß(2)-microglobulin (ß(2)m) in the musculoskeletal system. The condition is seen in patients who suffer from chronic kidney disease and are treated with hemodialysis for a long time. Even though ß(2)m easily can be manipulated to form amyloid in laboratory experiments under non-physiological conditions the precise mechanisms involved in the formation of ß(2)m-amyloid in patients with DRA have been difficult to unravel. The current knowledge which is reviewed here indicates that conformational fluctuations centered around the D-strand, the DE-loop, and around the cis-configured Pro32 peptide bond are involved in ß(2)m amyloidosis. Also required are highly increased concentrations of circulating ß(2)m and possibly various post-translational modifications mediated by the pro-inflammatory environment in uremic blood, together with the influence of divalent metal ions (specifically Cu(2 +)), uremic toxins, and dialysis-enhanced redox-processes. It seems plausible that domain-swapped ß(2)m dimers act as building blocks of ß-spine cross-ß -sheet fibrils consisting of otherwise globular, roughly natively folded protein. An activated complement system and cellular activation perpetuate these reactions which due to the affinity of ß(2)m-amyloid for the collagen of synovial surfaces result in the DRA syndrome.

Glycosaminoglycans enhance the fibrillation propensity of the ß2-microglobulin cleavage variant--ΔK58-ß2m.

Dialysis related amyloidosis (DRA) is a serious complication to long-term hemodialysis treatment which causes clinical symptoms such as carpal tunnel syndrome and destructive arthropathies. The disease is characterized by the assembly and deposition of ß2-microglobulin (ß2m) predominantly in the musculoskeletal system, but the initiating events leading to ß2m amyloidogenesis and the molecular mechanisms underlying amyloid fibril formation are still unclear. Glycosaminoglycans (GAGs) and metal ions have been shown to be related to the onset of protein aggregation and to promote de novo fiber formation. In this study, we show that fibrillogenesis of a cleavage variant of ß2m, ΔK58-ß2m, which can be found in the circulation of hemodialysis patients and is able to fibrillate at near-physiological pH in vitro, is affected by the presence of copper ions and heparan sulfate. It is found that the fibrils generated when heparan sulfate is present have increased length and diameter, and possess enhanced stability and seeding properties. However, when copper ions are present the fibrils are short, thin and less stable, and form at a slower rate. We suggest that heparan sulfate stabilizes the cleaved monomers in the early aggregates, hereby promoting the assembly of these into fibrils, whereas the copper ions appear to have a destabilizing effect on the monomers. This keeps them in a structure forming amorphous aggregates for a longer period of time, leading to the formation of spherical bodies followed by the assembly of fibrils. Hence, the in vivo formation of amyloid fibrils in DRA could be initiated by the generation of ΔK58-ß2m which spontaneously aggregate and form fibrils. The fibrillogenesis is enhanced by the involvement of GAGs and/or metal ions, and results in amyloid-like fibrils able to promote the de novo formation of ß2m amyloid by a scaffold mechanism.

A beta2-microglobulin cleavage variant fibrillates at near-physiological pH.

Beta2-microglobulin (beta2m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of beta2m are still largely unknown. In vitro, acidic conditions, pH < 4.5, induce amyloid fibrillation of native beta2m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of beta2m--found in the circulation of many dialysis patients--is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native beta2m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of beta2m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native beta2m. This may explain the apparent lack of detectable amounts of the variant beta2m in extracts of amyloid from DRA patients.

The influence of Cu(2+) on the unfolding and refolding of intact and proteolytically processed beta(2)-microglobulin.

Human beta(2)-microglobulin (beta(2)m) is an amyloidogenic protein in patients suffering from chronic kidney disease and especially in those patients that need intermittent hemodialysis for longer periods, e.g., when awaiting transplantation. While many in vitro conditions induce beta(2)m-amyloid formation from wild-type (wt) beta(2)m and while a number of structurally altered beta(2)m molecules are known to be conformationally unstable and amyloidogenic on their own, it is not known why beta(2)m-amyloid is generated in some dialysis patients. For many amyloid proteins it is known that divalent metal ions, especially Cu(2+), display strong binding and distinct destabilizing effects on protein conformation. The present study uses CE to assess conformational states of wt and cleaved beta(2)m (dK58-beta(2)m, beta(2)m cleaved at lysine-58, a modification found in the circulation of hemodialysis patients) in the presence of divalent metal ions. The experiments provide both qualitative and quantitative data showing the specific destabilizing effects of Cu(2+)-ions on the folding of wt beta(2)m. Both refolding after acid denaturation and solution structure of beta(2)m under otherwise native conditions are severely influenced by Cu(2+). An increased unfolding, aggregation, and induction of Congo red-reactive molecular species in Cu(2+)-incubated wt-beta(2)m could be demonstrated while the refolding kinetics of dK58-beta(2)m, already slower than the wt molecule, appeared not to be further decreased by Cu(2+). Given the interest in the actions of metal ions in other types of amyloidosis, including, e.g., Alzheimer's disease and the prion encephalopathies, the use of microelectrophoretic methods to monitor unfolding and refolding of biomolecules available in scarce amounts as shown in this study is an attractive option.

Variants of beta-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature.

Cleavage of the small amyloidogenic protein beta2-microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related beta2-microglobulin amyloidosis, since elevated levels of cleaved beta2-microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved beta2-microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine beta2-microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of beta2-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type beta2-microglobulin and is strongly dependent on temperature and protein concentration. 1H-NMR spectroscopy and amide hydrogen (1H/2H) exchange monitored by MS show that the overall three-dimensional structure of the variants is similar to that of wild-type beta2-microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E beta-strands close to the D-E loop cleavage site at lysine-58, and the experiments suggest conformational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered beta2-microglobulin molecules. This is the first single-residue resolution study of a specific beta2-microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in beta2-microglobulin amyloidogenicity in vivo.

Quantification of cleaved beta2-microglobulin in serum from patients undergoing chronic hemodialysis.

BACKGROUND: Patients on chronic hemodialysis are prone to develop amyloid deposits of misfolded beta(2)-microglobulin (beta(2)M) in osteoarticular tissues. beta(2)M with various deletions/truncations and chemical modifications has been found together with structurally intact beta(2)M in extracts of beta(2)M amyloid fibrils. The state of the circulating population of beta(2)M molecules has not been characterized previously with high-resolution methods. METHODS: We used immunoaffinity-liquid chromatography-mass spectrometry analysis of serum samples to examine whether structurally modified beta(2)M is generated in the circulation. In addition, we developed an immunoassay for the quantification of a cleaved beta(2)M variant in biological fluids based on novel monoclonal antibodies and applied this assay to patient and control sera. RESULTS: A specific alteration compatible with the generation of lysine-58-cleaved and truncated beta(2)M (DeltaK58-beta(2)M) was found in the sera of many (20%-40%) dialysis patients but not in control sera or sera from patients with cerebral amyloidosis (Alzheimer disease). Applied to patient sera, specific immunoassays revealed that dialysis, as expected, significantly lowered the total beta(2)M concentration, but the concentrations of DeltaK58-beta(2)M remained unchanged after dialysis. The results also show that patients dialyzed with less biocompatible membranes have higher serum concentrations of cleaved beta(2)M (mean, 8.5, 1.8, and 0.7 mg/L in cuprophane membrane-dialyzed, polysulfone membrane-dialyzed, and control sera, respectively). CONCLUSIONS: This study for the first time demonstrates and assigns the structure of a specific beta(2)M variant in sera from dialysis patients. Because this variant is conformationally unstable in vitro, it may be involved in in vivo amyloidogenesis.

Unfolding, aggregation, and seeded amyloid formation of lysine-58-cleaved beta 2-microglobulin.

Beta(2)-microglobulin (beta(2)m) is the amyloidogenic protein in dialysis-related amyloidosis, but the mechanisms underlying beta(2)m fibrillogenesis in vivo are largely unknown. We study a structural variant of beta(2)m that has been linked to cancer and inflammation and may be present in the circulation of dialysis patients. This beta(2)m variant, DeltaK58-beta(2)m, is a disulfide-linked two-chain molecule consisting of amino acid residues 1-57 and 59-99 of intact beta(2)m, and we here demonstrate and characterize its decreased conformational stability as compared to wild-type (wt) beta(2)m. Using amide hydrogen/deuterium exchange monitored by mass spectrometry, we show that DeltaK58-beta(2)m has increased unfolding rates compared to wt-beta(2)m and that unfolding is highly temperature dependent. The unfolding rate is 1 order of magnitude faster in DeltaK58-beta(2)m than in wt-beta(2)m, and at 37 degrees C the half-time for unfolding is more than 170-fold faster than at 15 degrees C. Conformational changes are also reflected by a very prominent Congo red binding of DeltaK58-beta(2)m at 37 degrees C, by the evolution of thioflavin T fluorescence, and by changes in intrinsic fluorescence. After a few days at 37 degrees C, in contrast to wt-beta(2)m, DeltaK58-beta(2)m forms well-defined high molecular weight aggregates that are detected by size-exclusion chromatography. Atomic force microscopy after seeding with amyloid-beta(2)m fibrils under conditions that induce minimal fibrillation in wt-beta(2)m shows extensive amyloid fibrillation in DeltaK58-beta(2)m samples. The results highlight the instability and amyloidogenicity under near physiological conditions of a slightly modified beta(2)m variant generated by limited proteolysis and illustrate stages of amyloid formation from early conformational variants to overt fibrillation.