Journal of Clinical & Experimental Nephrology

Keywords

Dialysis-related amyloidosis; β₂-microglobulin; Haemodialysis

Introduction

Amyloid is a fibrillar protein and is primarily a conformational variant of an originally globular precursor protein. β₂M is an essential protein for normal conformation of major histocompatibility complex class I molecules on cell membranes and is degraded mainly in renal proximal tubules. β₂M passes freely across vascular walls. Therefore, serum levels of β₂M are extremely high in patients undergoing HD. Although HD can clear as much as 70% of circulating β₂M, β₂M in the interstitial space can accumulate and progressively lead to development of DRA [1]. The amyloid processes may occur in the interstitial space. We have studied the amyloidogenicity of β₂M and found a previously unknown amyloidogenic process, which we describe in this short review.

Specific information about the different β₂M structures follows here:

The three-dimensional structure (conformation) of β₂M: β₂M consists of 99 amino acids. The three-dimensional structure of native β₂M is formed by the folding of seven peptide segments (strands) into a globular conformation, with both the N-terminal and the C-terminal segments folded inward from the molecular surface. McParland et al. demonstrated that unfolding in β₂M started first in the N-terminal segment and then continued in the C-terminal segment [2].

The intermediate structure of β₂M: Several basic studies described a conversion process in which amyloid protein is formed. In this process, the folded conformation, i.e., native β₂M is unfolded into a partially unfolded conformer, i.e., the intermediate species. The amyloid protein is insoluble, but the intermediate species is soluble and can be identified directly, with native β₂M, via capillary electrophoresis. By using this technique [3], we confirmed that β₂M in serum consisted of two forms, the folded native β₂M (N-β₂M) and the partially unfolded intermediate β₂M (I-β₂M) in both healthy persons and patients undergoing HD (Figure 1) [4,5].

Figure 1: Native β₂M (N- β₂M) and intermediate β₂M (I- β₂M) as determined with capillary electrophoresis. Unpublished data for n=30 HD patients.

C-terminal unfolding and β₂M 92-99: Stoppini et al. first implicated the C-terminal region of β₂M in amyloidogenicity by using a monoclonal antibody specific for the C-terminal eight amino acids [6].We then reported on β₂M with an unfolded C-terminal region, i.e., β₂M 92-99, in amyloid tissues from patients undergoing HD [7]. However, we did not detect β₂M 92-99 in serum from patients undergoing HD because the C-terminal region in I-β₂M may be not completely unfolded as is the case for β₂M 92-99 [8].

ΔN6β₂M: In 1987, Linke et al. first reported a variant of β₂M lacking the six N-terminal amino acids, i.e., ΔN6β₂M, in patients undergoing HD [9]. Esposito et al. and others then confirmed that ΔN6β₂M was a highly amyloidogenic variant [10,11]. We recently reported that the C-terminal region was completely unfolded in ΔN6β₂M as well as in β₂M 92-99 [8].

Glycosaminoglycans (GAGs) and heparin: GAGs including heparin are an essential component of interstitial tissue. In addition, heparin is most often used as an anti-coagulant in the clinical setting of HD. Several studies showed that GAGs facilitate the amyloidogenicity of β₂M [12,13], and we also found that heparin promoted β₂M unfolding [14].

The working hypothesis: N-β₂M coexists with I-β₂M at a ratio of 10:1 in body fluids, and the HD procedure causes a shift from N-β₂M toward I-β₂M. GAGs in the interstitial space promote unfolding of the C-terminal region of I-β₂M to generate β₂M 92-99. β₂M 92-99 cannot be refolded or returned to the vascular space. An accumulation of β₂M 92-99 leads to the generation of amyloid β₂M in the interstitial space (Figure 2).

Figure 2: Unfolding processes that leads to formation of amyloid β₂M.

ΔN6 β₂M aptamer: We recently showed that an aptamer for ΔN6 β₂M has a domain for the C-terminal region and could block amyloid fibril formation in-vitro [15].

Conclusion

Amyloidosis, including DRA and Alzheimer disease, is becoming a major clinical entity. Amyloidosis is a disease of misfolded precursor proteins. Thus far, we have demonstrated that misfolding of β₂M is initiated by an unfolding of the C-terminal segment of β₂M, and GAGs in interstitial tissue are intimately associated with the completion of the C-terminal unfolding of β₂M. We believe that an intermediate β₂M with an unfolded C-terminal may be a key intermediate molecule for amyloid β₂M, as (Figure 2) illustrates. The data related to the ΔN6 β₂M aptamer support our belief.

References

1. Strihou YVC, Floege J, Jadoul M (1994) Amyloidosis and its relationship to different dialysis. Nephrol Dial Transplant 9: 156-161.
2. McParland VJ, Kalverda AP, Homans SW, Radford SE (2002) Structural properties of an amyloid precursor of β2-microglobulin. Nat Struct Biol 9: 326-331.
3. Heegaard NH, Sen JW, Nissen MH (2000) Congophilicity (Congo red affinity) of different β2-microglobulin conformations characterized by dye affinity capillary electrophoresis. J Chromatogr A 894: 319-327.
4. Uji Y, Motomiya Y, Ando Y (2009) A circulating β2-microglobulin intermediate in hemodialysis patients. Nephron Clin Pract111: c173-c181.
5. Motomiya Y, Uji Y, Ando Y (2012) Capillary electrophoretic profile of β2-microglobulin intermediate associated with hemodialysis. Ther Apher Dial 16: 350-354.
6. Stoppini M, Bellotti V, Mangione P, Ferri G (1997) Use of anti- β2-microglobulin mAb to study formation of amyloid fibrils. Eur J Biochem 249: 21-26.
7. Motomiya Y, Ando Y, Haraoka K, Sun X, Morita H, et al. (2005) Studies on unfolded β2-microglobulin at C-terminal in dialysis-related amyloidosis. Kidney Int 67: 314-320.
8. Motomiya Y, Higashimoto Y, Uji Y, Suenaga G, Ando Y (2015) C-terminal unfolding of an amyloidogenic β2-microglobulin fragment: DN6 β2M-microglobulin. Amyloid 22: 54-60.
9. Linke RP, Hampl H, Lobeck H, Ritz E, Bommer J, et al. (1989) Lysine-specific cleavage of β2-microglobulin in amyloid deposits associated with hemodialysis. Kidney Int 36: 675-681.
10. Esposito G, Michelutti R, Verdone G, Viglino P, Hernández H, et al. (2009) Removal of the N-terminal hexapeptide from human β2-microglobulin facilitates protein aggregation and fibril formation. Protein Sci 9: 831-845.
11. Myers SL, Jones S, Jahn TR, Morten JI, Tennent GA, et al. (2006) A systematic study of the effect of physiological factors on β2-microglobulin amyloid formation at neutral pH. Biochem 45: 2311-2321.
12. Yamamoto S, Yamaguchi I, Hasegawa K, Tsutsumi S, Goto Y, et al. (2004) Glycosaminoglycans enhance the trifluoroethanol-induced extension of β2-microglobulin-related amyloid fibrils at a neutral pH. J Am Soc Nephrol 15: 126-133.
13. Borysik AJ, Morten IJ, Radford SE, Hewitt EW (2007) Specific glycosaminoglycans promote unseeded amyloid formation from β2-microglobulin under physiological conditions. Kidney Int 72: 174-181.
14. Fukasawa K, Higashimoto Y, Motomiya Y, Uji Y, Ando Y (2016) Influence of heparin molecular size on the induction of C-terminal unfolding in β2-microglobulin. Mol Biol Res Commun 5: 225-232.
15. Fukasawa K, Higashimoto Y, Ando Y, Motomiya Y (2017) Selection of DNA aptamer that blocks the fibrillogenesis of a proteolytic amyloidogenic fragment β2M. Ther Apher Dial 22: 61-66.