Amyloidosis

The Mass Spectrometry Resource at Boston University School of Medicine (BUSM) has established on-going collaborative projects with the Amyloid Treatment and Research Program at BUSM/Boston Medical Center. The main purpose of these endeavors is the clinical application of mass spectrometry in amyloidosis.

Amyloidosis is a term used to describe a group of diseases that are associated with abnormal protein deposition in tissues and organs. A few well-known examples of these diseases are Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob diseases. Currently, at least 18 human proteins are known to be amyloidogenic. The sequences and structures of these amyloidogenic proteins are diverse, yet they all undergo a conformational change into abnormal forms that give rise to disease-related amyloid fibrils. These fibrils appear to share the same characteristics. All amyloid fibrils are unbranched and are approximately 60 to 120 Å in diameter, stain with Congo red and display a green birefringence, and exhibit a cross-ß structure. These properties are depicted on the cover of the special amyloid issue of the Journal of Structural Biology.

Amyloidosis associated with transthyretin. Transthyretin (TTR, formerly called prealbumin) is a homotetramer with each subunit consisting of 127 amino acid residues. Although it is synthesized predominantly in the liver and secreted into plasma, TTR is also produced in the choroid plexus and the eye. In plasma, TTR circulates as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex (link to the crystal structure of TTR). TTR is associated with senile systemic amyloidosis (SSA) and familial transthyretin amyloidosis (ATTR) (Benson, 1995; Falk et al., 1997). SSA, also referred to as senile cardiac amyloidosis (SCA), is a nonhereditary disorder that affects about 25% of individuals over 80 years old (Cornwell et al., 1983). In SSA, the amyloid fibrils are usually composed of wild type (wt) TTR and/or its fragments and are found mainly in the heart.

In contrast, ATTR is an autosomal dominant disorder involving the deposition of TTR variants, the wt protein, and/or their fragments as amyloid fibrils in various tissues and organs. Although the incidence of ATTR is unknown, the gene frequency for TTR variants has been estimated to be between 1:100,000 and 1:1,000,000 in the United States (Benson, 1995). Certain amino acid substitutions in TTR are hypothesized to affect the stability of the tetramer and cause the TTR to form intermediates that may self-associate into amyloid fibrils (Lai et al., 1996). More than 80 TTR variants have been identified, with the majority being amyloidogenic (Connors et al., 2000; Saraiva, 2001) (linked to TTR database). The clinical features of ATTR appear to be related to specific mutations that are characteristic of certain ethnic groups. However, in general, the main clinical characteristics of ATTR are peripheral and autonomic neuropathy, cardiomyopathy, and vitreous opacities. The age of onset depends mainly on the mutation involved. The disease is usually fatal within 7?15 years after the appearance of symptoms (Bergethon et al., 1996). Since the most effective treatment of ATTR is liver transplantation (Bergethon et al., 1996), correct diagnosis is crucial. A definitive diagnosis of ATTR depends on the detection and identification of pathological TTR variants. To aid the clinical diagnosis of ATTR, we have developed a mass spectrometry-based approach for the detection and identification of TTR variants (Théberge et al., 1999, 2000; Lim et al., 2002).

Since 1997, we have analyzed TTR samples from 182 patients and have found that 65 of these exhibited 28 different TTR variants, including eight that were previously unreported (shown in bold lettering).

Asp18Gly, Val20Ile, Ser23Asn, Val30Ala, Val30Gly, Val30Met, Phe33Cys, Lys35Asn, Asp38Ala, Trp41Leu, Glu42Gly/His90Asn, Phe44Ser, Gly47Glu, Leu58His, Thr59Lys/Arg104His, Tyr69His, Thr60Ala, Phe64Ser, Phe64Leu, Val71Ala, Ser77Tyr, Ala81Thr, Ala97Ser, Gly6Ser/Tyr114Cys, Thr119Met, Gly6Ser/Val122Ala, and Val122Ile.

Both wt and variant TTR are usually post-translationally modified by S-cysteinylation (Kishikawa et al., 1996) and S-sulfation (Kishikawa et al., 1999; Théberge et al., 1999) on the single Cys10 residue. In addition, the Phe33Cys variant was found to be both S-cysteinylated and S-sulfated at the variant Cys33 residue (Lim et al., 2001a).

Amyloidosis associated with light chain. Amyloid-deposited light chain (AL) amyloidosis is linked to the overproduction of a monoclonal immunoglobulin light chain by a particular B-lymphocyte clone. Approximately 1,275 to 3,200 new cases of AL amyloidosis are diagnosed every year in the United States (Kyle et al., 1989). The outcome of this disease depends on which organ is being affected. The disease is usually fatal within 24 months of onset. However, recent treatments using high-dose chemotherapy and autologous stem cell transplantation have begun to extend survival time for some patients (Comenzo et al., 1998). The underlying factors responsible for initiating the deposition of the light chain are not yet fully understood, but in vitro studies have shown that amino acid substitutions in the variable region of the light chain could cause the protein to undergo a conformational change leading to amyloid fibril formation. While the fibril deposits most often consist of the N-terminal (variable region) fragments of the light chain, they may also be composed of the constant region and/or the intact light chain itself. Mass spectrometry is used in conjunction with cDNA analysis to establish the gene and protein sequences and to identify and locate post-translational modifications in the overexpressed light chains. We have recently observed the S-cysteinylation at Cys214 of a k1 light chain isolated from the urine of a patient diagnosed with AL amyloidosis (Lim et al., 2001b). The novelty of this S-cysteinylation is not yet known since many laboratories have routinely used reducing agents in the initial purification of the light chain. Ongoing research is aimed at achieving a more complete understanding of the mechanism of amyloid fibril deposition and the structural features that cause some sequences to favor this process.

References
Benson, M. D. Amyloidosis. In The Metabolic and Molecular Bases of Inherited Disease;
Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., Eds.; McGraw-Hill: New York, 1995; pp 4159-4191.

Bergethon, P. R., Sabin, T. D., Lewis, D., Simms, R. W., Cohen, A. S., and Skinner, M. (1996)
Improvement in the polyneuropathy associated with familial amyloid polyneuropathy after liver transplantation, Neurology 47, 944-951.

Connors, L. H., Richardson, A. M., Théberge, R., and Costello, C. E. (2000) Tabulation of
transthyretin (TTR) variants as of 1/1/2000, Amyloid: Int. J. Exp. Clin. Invest. 7, 54-69.

Cornwell, G. G., Murdoch, W. L., Kyle, R. A., Westermark, P., and Pitkänen, P. (1983)
Frequency and distribution of senile cardiovascular amyloid: A clinicopathologic correlation, Am. J. Med. 75, 618-623.

Falk, R. H., Comenzo, R. L., and Skinner, M. (1997) The systemic amyloidoses, N. Engl. J. Med.
337, 898-909.

Lai, Z., Colón, W., and Kelly, J. W. (1996) The acid-mediated denaturation pathway of
transthyretin yields a conformational intermediate that can self-assemble into amyloid, Biochemistry 35, 6470-6482.

Lim, A., McComb, M. E., O’Connor, P. B., Prokaeva, T., Connors, L. H., Skinner, M.,
and Costello, C. E. (2001a) Identification of
novel transthyretin variants in familial amyloidosis by MALDI peptide mapping and ESI tandem mass spectrometry, 49th Annual Conference on Mass Spectrometry and Allied Topics, Chicago, IL, USA: May 27-31.

Lim, A., Wally, J., Walsh, M. T., Skinner, M., and Costello, C. E. (2001b) Identification and
location of a cysteinyl posttranslational modification in an amyloidogenic k1 light chain protein by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry, Anal. Biochem. 295, 45-56.

Lim, A., Prokaeva, T., McComb, M. E., O’Connor, P. B., Théberge, R., Connors, L. H., Skinner,
M., and Costello, C. E. (2002) Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis, Anal Chem, in press.

Kishikawa, M., Nakanishi, T., Miyazaki, A., Shimizu, A., Nakazato, M., Kangawa, K., and
Matsuo, H. (1996) Simple detection of abnormal serum transthyretin from patients with familial amyloidotic polyneuropathy by high-performance liquid chromatography/electrospray ionization mass spectrometry using material precipitated with specific antiserum, J. Mass Spectrom. 31, 112-114.

Kishikawa, M., Nakanishi, T., Miyazaki, A., and Shimizu, A. (1999) A simple and reliable
method of detecting variant transthyretins by multidimensional liquid chromatograpy coupled to electrospray ionization mass spectrometry, Amyloid: Int. J. Exp. Clin. Invest. 6, 48-53.

Saraiva, M. J. M. (2001) Transthyretin mutations in hyperthyroxinemia and amyloid diseases,
Hum. Mutat. 17, 493-503.

Théberge, R., Connors, L., Skinner, M., Skare, J., and Costello, C. E. (1999) Characterization of
transthyretin mutants from serum using immunoprecipitation, HPLC/electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry, Anal. Chem. 71, 452-459.

Théberge, R., Connors, L. H., Skinner, M., and Costello, C. E. (2000) Detection of transthyretin
variants using immunoprecipitation and matrix-assisted laser desorption/ionization bioreactive probes: A clinical application of mass spectrometry, J. Am. Soc. Mass Spectrom. 11, 172-175.

Primary teaching affiliate
of BU School of Medicine