Conformational Intermediate of the Amyloidogenic Protein β2-Microglobulin at Neutral pH

Heegaard, Niels H. H.; Sen, Jette W.; Kaarsholm, Niels C.; Nissen, Mogens Holst

doi:10.1074/jbc.m104452200

Cited by 56 publications

(67 citation statements)

References 41 publications

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“…The characteristic spectrum of wild-type ␤ 2 m is compatible with ϳ50% ␤-structures (strands and turns), 50% random structure, and practically no helix structures as reported previously (4,27). The changes in the far-UV spectrum of the cleaved forms, most pronounced for the Lys 58 -␤ 2 m species, are similar to the changes in the CD spectra of wild-type ␤ 2 m obtained in 10 -15% trifluoroethanol or 20 -30% acetonitrile where there is a mixture of the native fold and a solvent-induced species with less ␤-structure and more helix structure (27). Thus the far-UV spectra of Electrophoretic Separation of ␤ 2 m and Its Cleaved Variants-When the three purified ␤ 2 m species were separated by CE, more than one peak was observed in both the Lys 58 -␤ 2 m and the des-Lys 58 -␤ 2 m preparations (Fig.…”

Section: Resultssupporting

confidence: 50%

“…Recent data from other groups support the notion that this folding intermediate may be on the pathway to amyloid formation (25). In addition, we observed by CE that purified Lys 58 -␤ 2 m is also heterogeneous in the absence of organic solvent (27). In the present study, we characterize the Lys 58 -␤ 2 m and des-Lys 58 -␤ 2 m molecules using circular dichroism, CE, and affinity CE with heparin and Congo red.…”

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confidence: 57%

“…2 Using capillary electrophoresis (CE), we were able to demonstrate the existence of two wild-type ␤ 2 m conformations in equilibrium in the presence of acetonitrile and trifluoroethanol and the increased affinity of one of the conformations for the amyloid-specific dye Congo red (27,28). Recent data from…”

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confidence: 99%

“…We and others have recently characterized how solvent and/or pH manipulations can induce variant conformations of wild-type ␤ 2 -microglobulin including long-lived structured intermediates that may be on the folding pathway to amyloid (25)(26)(27)(28). 2 Using capillary electrophoresis (CE), we were able to demonstrate the existence of two wild-type ␤ 2 m conformations in equilibrium in the presence of acetonitrile and trifluoroethanol and the increased affinity of one of the conformations for the amyloid-specific dye Congo red (27,28). Recent data from other groups support the notion that this folding intermediate may be on the pathway to amyloid formation (25).…”

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Cleaved β2-Microglobulin Partially Attains a Conformation That Has Amyloidogenic Features

Heegaard

Roepstorff

Melberg

et al. 2002

Journal of Biological Chemistry

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␤ 2 -Microglobulin, a small protein localized in serum and on cell surfaces, can adopt specific aggregating conformations that generate amyloid in tissues and joints as a complication to long-term hemodialysis. We characterize a proteolytic variant of ␤ 2 -microglobulin (cleaved after Lys 58 ) that as a trimmed form (Lys 58 is removed) can be demonstrated in the circulation in patients with chronic disease. An unexpected electrophoretic heterogeneity of these two cleaved variants was demonstrated by capillary electrophoresis under physiological conditions. Each separated into a fast and a slow component while appearing homogeneous, except for a fraction of oxidized species detected by other techniques. The two components had different binding affinities for heparin and for the amyloid-specific dye Congo red, and the equilibrium between the two forms was dependent on solvent conditions. Together with analysis of the differences in circular dichroism, the results suggest that ␤ 2 -microglobulin cleaved after Lys 58 readily adopts two equilibrium conformations under native conditions. In the cleaved and trimmed ␤ 2 -microglobulin that appears in vivo, the less populated conformation is characterized by an increased affinity for Congo red. These observations may help elucidate why ␤ 2 -microglobulin polymerizes as amyloid in chronic hemodialysis and facilitate the search for means to inhibit this process.1 is a small protein (M r 11,729) localized in the circulation and on cell surfaces, where it constitutes the nonpolymorphic light chain of the class I major histocompatibility complex (1). ␤ 2 m can be cleaved C-terminally to Lys 58 (Lys 58 -␤ 2 m) by activated complement component C1s. In serum, the exposed Lys 58 residue of this cleaved molecule is subsequently removed by a carboxypeptidase B-like activity. This generates a cleaved and trimmed ␤ 2 m variant called desLys 58 -␤ 2 m (␤ 2 -microglobulin cleaved C-terminally to Ser 57 and lacking Lys 58 ) (2). Wild-type ␤ 2 m is a one-chain protein belonging to the immunoglobulin superfamily characterized by two antiparallel ␤-sheets connected by an internal disulfide bridge in a ␤-sandwich topology without helical structures (3, 4). The Lys 58 -␤ 2 m and des-Lys 58 -␤ 2 m variants are disulfide-linked two-chain heterodimers (Fig. 1). des-Lys 58 -␤ 2 m has been demonstrated in sera from patients in hemodialysis treatment (5) and in patients with malignancies and autoimmune and immunodeficiency diseases (2, 6, 7), but, to our knowledge, analyses of the conformational structure of the cleaved variants have not previously been published. In chronic renal failure, plasma concentrations of ␤ 2 m may increase 10 -70 times despite dialysis, and within 2 years after the onset of dialysis, about 20% of patients suffer from complications due to the formation of insoluble deposits of ␤ 2 m (␤ 2 m amyloid) in tissues and joints (8). The events leading to the formation of amyloid in this and other chronic diseases such as Alzheimer's disease are still largely unknown (9). There a...

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Section: Resultssupporting

confidence: 50%

supporting

confidence: 57%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Cleaved β2-Microglobulin Partially Attains a Conformation That Has Amyloidogenic Features

Heegaard

Roepstorff

Melberg

et al. 2002

Journal of Biological Chemistry

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“…In the serum of uremic patients, but not in healthy subjects, the presence of intermediate misfolded forms of b2M was reported by some authors. 9 Uji et al demonstrated poor removal of intermediate forms compared with native b2M among 31 patients on HD treatment, even when the patients were treated with high-flux HD or hemodiafiltration. 10 In vitro studies showed that under physiological conditions, enhanced amyloidogenesis is observed in the presence of an increase in equilibrium concentration of a b2M intermediate form, named intermediate T (I T ), characterized by a trans isomerization of Pro32.…”

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Dialysis-related amyloidosis: challenges and solutions

Scarpioni¹,

Ricardi²,

Albertazzi³

et al. 2016

IJNRD

107

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Amyloidosis refers to the extracellular tissue deposition of fibrils composed of lowmolecular-weight subunits of a variety of proteins. These deposits may result in a wide range of clinical manifestations depending upon their type, location, and the amount of deposition. Dialysis-related amyloidosis is a serious complication of long-term dialysis therapy and is characterized by the deposition of amyloid fibrils, principally composed of b2 microglobulins (b2M), in the osteoarticular structures and viscera. Most of the b2M is eliminated through glomerular filtration and subsequent reabsorption and catabolism by the proximal tubules. As a consequence, the serum levels of b2M are inversely related to the glomerular filtration rate; therefore, in end-stage renal disease patients, b2M levels increase up to 60-fold. Serum levels of b2M are also elevated in several pathological conditions such as chronic inflammation, liver disease, and above all, in renal dysfunction. Retention of amyloidogenic protein has been attributed to several factors including type of dialysis membrane, prolonged uremic state and/or decreased diuresis, advanced glycation end products, elevated levels of cytokines and dialysate. Dialysis treatment per se has been considered to be an inflammatory stimulus, inducing cytokine production (such as interleukin-1, tumor necrosis factor-α, interleukin-6) and complement activation. The released cytokines are thought to stimulate the synthesis and release of b2M by the macrophages and/or augment the expression of human leukocyte antigens (class I), increasing b2M expression. Residual renal function is probably the best determinant of b2M levels. Therefore, it has to be maintained as long as possible. In this article, we will focus our attention on the etiology of dialysis-related amyloidosis, its prevention, therapy, and future solutions.

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Applications of on-line weak affinity interactions in free solution capillary electrophoresis

2002

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The impressive selectivity offered by capillary electrophoresis can in some cases be further increased when ligands or additives that engage in weak affinity interactions with one or more of the separated analytes are added to the electrophoresis buffer. This on-line affinity capillary electrophoresis approach is feasible when the migration of complexed molecules is different from the migration of free molecules and when separation conditions are nondenaturing. In this review, we focus on applying weak interactions as tools to enhance the separation of closely related molecules, e.g., drug enantiomers and on using capillary electrophoresis to characterize such interactions quantitatively. We describe the equations for binding isotherms, illustrate how selectivity can be manipulated by varying the additive concentrations, and show how the methods may be used to estimate binding constants. On-line affinity capillary electrophoresis methods are especially valuable for enantiomeric separations and for functional characterization of the contents of biological samples that are only available in minute quantities.

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Conformational Intermediate of the Amyloidogenic Protein β2-Microglobulin at Neutral pH

Cited by 56 publications

References 41 publications

Cleaved β2-Microglobulin Partially Attains a Conformation That Has Amyloidogenic Features

Cleaved β2-Microglobulin Partially Attains a Conformation That Has Amyloidogenic Features

Dialysis-related amyloidosis: challenges and solutions

Applications of on-line weak affinity interactions in free solution capillary electrophoresis

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