Biochemical characterization of vitreous and cardiac amyloid in Ile84Ser transthyretin amyloidosis

Abstract

Plasma transthyretin (TTR) is synthesized in the liver and is the source for visceral amyloid deposits in TTR amyloidosis. However, TTR is also synthesized in the retinal pigment epithelium of the eye and choroid plexus of the brain. It has been postulated that vitreous amyloid, which is associated with approximately 20% of the known amyloidogenic TTR mutations, results from local synthesis of TTR in the eye. In order to elucidate if differences in amyloid between organs exists, we have analyzed vitreous and cardiac amyloid fibrils in Ile84Ser TTR patients for comparison. Analysis of guanidine hydrochloride solubilized protein from isolated vitreous and cardiac amyloid fibrils indicated that the amyloid TTR in both organs is highly proteolyzed with minor amounts of intact TTR present. While vitreous protein was amenable to direct Edman sequence analysis, cardiac protein gave low yields indicating it was mostly N-terminally blocked or inaccessible to Edman degradation. While vitreous contained major 11 kDa and minor 9 kDa fragments, cardiac contained at least three major fragments of 7-11 kDa. Vitreous protein was cleaved between Lys48-Thr49, while cardiac protein was cleaved at multiple sites in the residue 46-52 region. While deposits in both tissues were enriched in variant TTR, vitreous fibrils contained more variant protein than cardiac fibrils (80-89% vs. 60-65% Ser84TTR). These differences suggest that the mechanism or pathway of fibril formation may differ in various tissues.

Progression of cardiac amyloid deposition in hereditary transthyretin amyloidosis patients after liver transplantation

“…Ratios of variant to normal TTR were calculated by recovered amounts of tryptic peptides containing the variant or normal residue [11].…”

“…Amyloid fibrils were isolated from autopsy heart tissue stored at 7208C or 7808C by a modified procedure of Pras et al, as previously described [10,11]. Cardiac muscle from the left ventricular wall was repeatedly homogenized and centrifuged in 0.1 M sodium citrate, 0.15 M sodium chloride (4 times), 0.15 M sodium chloride (4-8 times), and water (4 times).…”

The molecular biology and clinical features of amyloid neuropathy

“…Although full-length transthyretin protein may be present in amyloid fibril deposits, a large percentage of the protein has been proteolyzed to give fragments of the carboxyl-terminal portion starting at amino acid positions 49 and 52. 8,17,41 Is this relevant to amyloid pathogenesis or an epiphenomenon? It should be noted that TTR amyloid fibrils isolated from tissues of patients heterozygous for a transthyretin gene mutation contain normal as well as the variant TTR protein.…”

“…The mechanisms involved in amyloid deposition in the vitreous may be similar to peripheral nerve or cardiac pathology, but an intriguing variation is that amyloid fibrils in the vitreous are greatly enriched (approximately 90%-95%) in variant TTR compared to the proportion (60%-65%) in nerve and cardiac tissue of heterozygotes. 41 Vitreous opacities are found with approximately 20% of the known amyloid-producing TTR mutations and may be seen in conjunction with leptomeningeal amyloidosis. 6 This may, however, be found with other mutations that primarily cause peripheral neuropathy or cardiomyopathy.…”

Genetic analysis of hypertrophic cardiomyopathy phenocopies

“…His88 is also located in the EF-loop (amino acids 95-101) in TTR. Mutations within the EF-helix (amino acids 102-110, where 108His is located) and the EF-loop are disposed to TTR monomer aggregation into fibrils (60)(61)(62)(63). Several amino acid residues, including His88, in this region are highly affected by conformational changes at acidic pH.…”