Beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro.

Amyloid fibrils formed by incubation of recombinant wild-type human beta(2)-microglobulin (beta(2)M) ab initio in vitro at low pH and high ionic strength are short and highly curved. By contrast, fibrils extracted from patients suffering from haemodialysis-related amyloidosis and those formed by seeding growth of the wild-type protein in vitro with fibrils ex vivo are longer and straighter than those previously produced ab initio in vitro. Here we explore the effect of growth conditions on morphology of beta(2)M fibrils formed ab initio in vitro from the wild-type protein, as well as a variant form of beta(2)M in which Asn17 is deamidated to Asp (N17D). We show that deamidation results in significant destabilisation of beta(2)M at neutral pH. Despite this, acidification is still necessary to form amyloid from the mutant protein in vitro. Interestingly, at low pH and low ionic strength long, straight fibrils of recombinant beta(2)M are formed in vitro. The fibrils comprise three distinct morphological types when examined using electron microscopy (EM) and atomic force microscopy (AFM) that vary in periodicity and the number of constituent protofibrils. Using kinetic experiments we suggest that the immature fibrils observed previously do not represent intermediates in the assembly of fully mature amyloid, at least under the conditions studied here.

[1]  U Aebi,et al.  Polymorphic fibrillar assembly of human amylin. , 1997, Journal of structural biology.

[2]  B. Frangione,et al.  Polymerization of intact beta 2-microglobulin in tissue causes amyloidosis in patients on chronic hemodialysis. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Peter T. Lansbury,et al.  Assembly of Aβ Amyloid Protofibrils: An in Vitro Model for a Possible Early Event in Alzheimer's Disease† , 1999 .

[4]  M. Schiffer,et al.  Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains , 2008, Protein science : a publication of the Protein Society.

[5]  J. Kelly,et al.  The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid. , 1996, Biochemistry.

[6]  M. Saraiva,et al.  Ultrastructure of familial amyloid polyneuropathy amyloid fibrils: examination with high-resolution electron microscopy. , 1998, Journal of structural biology.

[7]  F. Gejyo,et al.  Collagen-binding affinity of beta-2-microglobulin, a preprotein of hemodialysis-associated amyloidosis. , 1989, Nephron.

[8]  H. Puchtler,et al.  ON THE BINDING OF CONGO RED BY AMYLOID , 1962 .

[9]  S. Aota,et al.  Formation of amyloid-like fibrils by self-association of a partially unfolded fibronectin type III module. , 1998, Journal of molecular biology.

[10]  C. Ionescu-Zanetti,et al.  Monitoring the assembly of Ig light-chain amyloid fibrils by atomic force microscopy. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[11]  F. Gejyo,et al.  Electron-microscopic and immunohistochemical study of beta-2-microglobulin-related amyloidosis. , 1990, Nephron.

[12]  J. Kelly,et al.  Transthyretin mutation Leu-55-Pro significantly alters tetramer stability and increases amyloidogenicity. , 1993, Biochemistry.

[13]  N. Guex,et al.  SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modeling , 1997, Electrophoresis.

[14]  M. Hurle,et al.  A role for destabilizing amino acid replacements in light-chain amyloidosis. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. Skinner,et al.  In vitro formation of amyloid fibrils from intact beta 2-microglobulin. , 1985, Biochemical and biophysical research communications.

[16]  P. Lansbury,et al.  Seeding “one-dimensional crystallization” of amyloid: A pathogenic mechanism in Alzheimer's disease and scrapie? , 1993, Cell.

[17]  Peter T. Lansbury,et al.  Observation of metastable Aβ amyloid protofibrils by atomic force microscopy , 1997 .

[18]  Satoru Suzuki,et al.  Establishment of a kinetic model of dialysis-related amyloid fibril extension in vitro , 1997 .

[19]  R. Duthie,et al.  Dialysis Arthropathy of the Hip , 1993, Clinical orthopaedics and related research.

[20]  Elena Orlova,et al.  Cryo‐electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing , 1999, The EMBO journal.

[21]  C. Robinson,et al.  Beta2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils. , 1998, European journal of biochemistry.

[22]  M. A. Saper,et al.  Structure of the human class I histocompatibility antigen, HLA-A2 , 1987, Nature.

[23]  D. Vučelič,et al.  1H NMR assignments and secondary structure of human beta 2-microglobulin in solution. , 1992, Biochemistry.

[24]  I. Berggård,et al.  Isolation and properties of a low molecular weight beta-2-globulin occurring in human biological fluids. , 1968, The Journal of biological chemistry.

[25]  L. Serpell,et al.  The protofilament substructure of amyloid fibrils. , 2000, Journal of molecular biology.

[26]  R. Wetzel,et al.  Specificity of abnormal assembly in immunoglobulin light chain deposition disease and amyloidosis. , 1996, Journal of molecular biology.

[27]  M Arakawa,et al.  A new form of amyloid protein associated with chronic hemodialysis was identified as beta 2-microglobulin. , 1985, Biochemical and biophysical research communications.

[28]  I D Campbell,et al.  Amyloid fibril formation by an SH3 domain. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[29]  C M Dobson,et al.  Ultrastructural organization of amyloid fibrils by atomic force microscopy. , 2000, Biophysical journal.

[30]  D. Stuart,et al.  Production and crystallization of MHC class I B allele single peptide complexes , 1996, FEBS letters.

[31]  F. Uchino,et al.  Formation of amyloid-like substance from beta-2-microglobulin in vitro. Role of serum amyloid P component: a preliminary study. , 1994, Nephron.

[32]  K. Ohashi,et al.  Ultrastructural organization of hemodialysis-associated beta 2-microglobulin amyloid fibrils. , 1997, Kidney international.

[33]  P. Lansbury,et al.  Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. W. Bolen,et al.  Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. , 1988, Biochemistry.

[35]  C. Ionescu-Zanetti,et al.  Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates. , 2001, Biochemistry.

[36]  P. Lansbury,et al.  Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid. , 2000, Biochemistry.

[37]  Louise C. Serpell,et al.  Synchrotron X-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous β-sheet helix , 1996 .

[38]  W. Burchert,et al.  Recombinant versus natural human 111In-β2-microglobulin for scintigraphic detection of Aβ2m amyloid in dialysis patients , 2000 .

[39]  D. Walsh,et al.  Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate. , 1997, The Journal of biological chemistry.

[40]  E. Ritz,et al.  Lysine-specific cleavage of beta 2-microglobulin in amyloid deposits associated with hemodialysis. , 1989, Kidney international.

[41]  H. Levine.  Thioflavine T interaction with amyloid β-sheet structures , 1995 .

[42]  U. Aebi,et al.  Watching amyloid fibrils grow by time-lapse atomic force microscopy. , 1999, Journal of molecular biology.

[43]  A. Miranker,et al.  Kidney dialysis-associated amyloidosis: a molecular role for copper in fiber formation. , 2001, Journal of molecular biology.

[44]  J. Philo,et al.  A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions. , 2000, Analytical biochemistry.

[45]  S. Radford,et al.  Partially unfolded states of beta(2)-microglobulin and amyloid formation in vitro. , 2000, Biochemistry.

[46]  S. Radford,et al.  Role of the single disulphide bond of β2‐microglobulin in amyloidosis in vitro , 2001 .

[47]  F. Gejyo,et al.  Serum levels of beta 2-microglobulin as a new form of amyloid protein in patients undergoing long-term hemodialysis. , 1986, The New England journal of medicine.

[48]  Christopher M. Dobson,et al.  Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis , 1997, Nature.

[49]  K. Titani,et al.  Purification and complete amino acid sequence of novel β2-microglobulin , 1990 .

[50]  À. Argilés,et al.  Beta 2 microglobulin isoforms in healthy individuals and in amyloid deposits. , 1995, Kidney international.

[51]  P. Lansbury,et al.  Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-beta protein. , 1997, Chemistry & biology.