Gelsolin-derived familial amyloidosis caused by asparagine or tyrosine substitution for aspartic acid at residue 187.

Dominantly inherited familial amyloidosis, Finnish type (FAF) is caused by the accumulation of a 71-amino acid amyloidogenic fragment of mutant gelsolin (GSN). FAF is common in Finland but is very rare elsewhere. In Finland and in two American families, the mutation is a G654A transition leading to an Asp to Asn substitution at residue 187. We found the same mutation in a Dutch family but a Danish FAF family had a G654T mutation, predicting Asp to Tyr at residue 187. We also found the G654T transversion in a Czech family. Using GSN polymorphisms, different haplotypes were found in the Danish and Czech families. We conclude that substitution of the uncharged Asn or Tyr for the acidic Asp at residue 187 creates a conformation that may be preferentially amyloidogenic for GSN.

Raised serum levels of cachectin/tumor necrosis factor alpha in renal allograft rejection.

A sensitive radioimmunoassay was used for monitoring serum levels of endogenous cachectin/tumor necrosis factor alpha (TNF) in 10 renal transplant recipients. Acute allograft rejections were associated with marked elevations of circulating TNF. The peak levels of TNF (median 140 pg/ml) were in the same concentration range as previously reported in parasitic infections. The results show that the release of TNF into circulation is an early event in renal allograft rejection and that raised levels of TNF in man can also be induced by noninfectious stimuli.

The emerging concept of functional amyloid.

Although amyloid has usually been considered a pathological structure, growing evidence indicates that amyloid may also be a productive part of cell biology contributing to normal physiology. In fact, amyloid formation seems to be an intrinsic propensity of polypeptides in general and the amyloid beta-fold an evolutionary highly conserved structure. Functional amyloids have been found in a wide range of organisms, from bacteria to mammals, with functions as diverse as biofilm formation, development of aerial structures, scaffolding, regulation of melanin synthesis, epigenetic control of polyamines and information transfer. Obviously, organisms have evolved taking advantage of the canonical amyloid beta-sheet fold, a conformation that possesses both high resistance to proteolysis, self-replicative properties and capability to function as a molecular memory.

Self-propagating beta-sheet polypeptide structures as prebiotic informational molecular entities: the amyloid world.

The idea is advanced that under the extreme earth conditions for ~3.9 billions years ago, protein-based beta-sheet molecular structures were the first self-propagating and information-processing biomolecules that evolved. The amyloid structure of these aggregates provided an effective protection against the harsh conditions known to decompose both polyribonucleotides and natively folded polypeptides. In the prebiotic amyloid world, both the replicative and informational functions were carried out by structurally stable beta-sheet protein aggregates in a prion-like mode involving templated self-propagation and storage of information in the beta-sheet conformation. In this amyloid (protein)-first, hybrid replication-metabolism view, the synthesis of RNA, and the evolvement of an RNA-protein world, were later, but necessary events for further biomolecular evolution to occur. I further argue that in our contemporary DNA<-->RNA-->protein world, the primordial beta-conformation-based information system is preserved in the form of a cytoplasmic epigenetic memory.

Gelsolin-related amyloidosis. Identification of the amyloid protein in Finnish hereditary amyloidosis as a fragment of variant gelsolin.

The Finnish type of familial amyloidosis is a systemic disease characterized by progressive cranial neuropathy, corneal lattice dystrophy, and distal sensimotor neuropathy. Amyloid fibrils were isolated from the kidney and heart of a patient with Finnish amyloidosis. After solubilization, the amyloid proteins were fractionated by gel filtration and purified by reverse-phase HPLC. Complete amino acid sequence analyses show that the two amyloid components obtained are fragments of gelsolin, an actin-modulating protein occurring in plasma and the cytoskeleton. The larger component represents residues 173-243 and the minor component residues 173-225, respectively, of mature gelsolin. When compared with the predicted primary structure of human gelsolin a single amino acid substitution is present in amyloid: at position 15 of the amyloid proteins an asparagine is found instead of an aspartic acid residue at the corresponding position (187) in gelsolin. Antibodies to a dodecapeptide of the amyloidogenic region of gelsolin specifically stain the tissue amyloid deposits in Finnish hereditary amyloidosis. The results show that the amyloid subunit protein in Finnish hereditary amyloidosis represents a new type of amyloid that is derived from an actin filament-binding region of a variant gelsolin molecule by limited proteolysis.

Isolation and characterization of cardiac amyloid in familial amyloid polyneuropathy type IV (Finnish): relation of the amyloid protein to variant gelsolin.

Amyloid subunit protein was isolated from familial amyloid polyneuropathy type IV (Finnish type) cardiac tissue and purified to homogeneity. N-terminal amino acid sequence analysis shows that the amyloid protein is a fragment of the inner region of human gelsolin. When compared with the predicted sequence of human plasma gelsolin, the amyloid protein contains an asparagine-for-aspartic acid substitution at position 15 corresponding to residue 187 of the secreted protein. Antibodies raised against the amyloidogenic region of gelsolin specifically stained the amyloid deposited in tissues in familial amyloidosis type IV. The results show that the subunit amyloid protein in familial amyloid polyneuropathy type IV represents a unique type of amyloid derived from a variant (Asn-187) gelsolin molecule by limited proteolysis.

No evidence of amyloidosis in type I diabetics treated with continuous subcutaneous insulin infusion.

A recent study has demonstrated secondary amyloidosis in dogs treated with continuous intravenous insulin infusion. Since elevated levels of serum amyloid A protein (SAA) and diminished amyloid fibril degrading activity (AFDA) are associated with amyloidosis, we measured SAA and AFDA in ten type I diabetics treated with continuous subcutaneous insulin infusion and in five conventionally treated patients. Only one pump- and one conventionally treated patient had detectable but low SAA levels, comparable with these seen in healthy controls. In patients with secondary amyloidosis the mean SAA level was 24-fold higher than in controls (P less than 0.001). Similarly, in both diabetic groups, AFDA was normal whereas it was reduced by 41% in patients with amyloidosis (P less than 0.001). Furthermore, no local amyloidosis was seen at the infusion site in any of the patients studied. Thus, our data fail to provide any evidence of secondary amyloidosis in patients treated for 3-40 mo with continuous subcutaneous insulin infusion.

Finnish hereditary amyloidosis. Amino acid sequence homology between the amyloid fibril protein and human plasma gelsoline.

Amyloid fibrils were isolated from the kidney of a patient with Finnish hereditary amyloidosis. After solubilization of the fibrils in guanidine-HCl, fractionation by gel filtration, and purification by reverse-phase high-performance liquid chromatography, a homogeneous amyloid protein with an apparent Mr of 9000 was obtained. The protein was subjected to enzymatic digestion by trypsin and endoproteinase Lys-C. The amino acid sequences were determined for 6 of the released peptides and they were all found to be identical to the reported, deduced primary structure of human plasma gelsoline in the region of amino acids 235-269. The results show that the amyloid fibril protein in Finnish hereditary amyloidosis represents a new type of amyloid protein that shows amino acid sequence homology with gelsoline, an actin-modulating protein.

Finnish hereditary amyloidosis is caused by a single nucleotide substitution in the gelsolin gene.

The amyloid protein in Finnish hereditary amyloidosis is a fragment of the actin-filament binding region of a variant gelsolin molecule. Here we demonstrate, using polymerase chain reaction and allele-specific oligonucleotide hybridization analyses of genomic DNA, a single base mutation (G654----A654) in the gelsolin gene segment encoding the amyloid protein. The mutation is responsible for the expression of the variant (Asn187) gelsolin molecule in Finnish hereditary amyloidosis. The nucleotide substitution was found in all five unrelated patients with Finnish amyloidosis studied, but not in 45 unrelated control subjects. The mutation co-segregated with the disease phenotype in a family with Finnish amyloidosis. The results show that a single substitution in the gelsolin gene causes Finnish hereditary amyloidosis. The allele-specific oligonucleotide hybridization method provides a simple and accurate means of detecting this mutation.

Variant plasma gelsolin responsible for familial amyloidosis (Finnish type) has defective actin severing activity.

Familial amyloidosis, Finnish type is caused by a single base mutation in gelsolin, an actin filament severing and capping protein that is present in most tissues and in blood plasma. The mutation replaces aspartic acid with asparagine at residue 187 of the plasma sequence. This renders the gelsolin susceptible to proteolysis as a consequence of which amyloid protein is formed. Here it is shown that the mutant protein in plasma from a patient homozygous for this mutation lacks both actin severing and nucleating activities. Evidence is presented that the cleaved mutant gelsolin has dissociated under non-denaturing conditions and that the resultant 65,000 and 55,000 M(r) C-terminal fragments aggregate.

Enzyme immunoassay of antibodies to epithelial glycoprotein: increased level of antibodies in coeliac disease.

From the papules of a patient with massive cutaneous hyalinosis, we earlier isolated a mannose-rich glycoprotein that, by immunohistochemical methods, was also shown to be present in epithelial cells of small intestine and normal skin. A solid-phase enzyme immunoassay of IgG and IgM antibodies to this epithelial glycoprotein is described, in which polystyrene tubes are coated with the purified antigen. The antibodies are allowed to bind, and are detected with alkaline phosphatase-conjugated anti-human IgG and IgM. IgG, IgA and IgM antibodies were determined in the sera of patients with pemphigus, pemphigoid, dermatitis herpetiformis, several autoimmune diseases, in a patient with massive cutaneous hyalinosis, in coeliac disease, and in control subjects. Very high values were found in the patient with massive cutaneous hyalinosis, and significantly elevated values in coeliac disease. In the other patient groups values were equal to or only slightly higher than in controls.

Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immunodeficiency syndrome.

PURPOSE: In order to evaluate the relationship between cachectin/tumor necrosis factor (TNF) and cachexia in the acquired immunodeficiency syndrome (AIDS), we studied the serum levels of endogenous cachectin/TNF in subjects with human immunodeficiency virus (HIV) infection. PATIENTS AND METHODS: Fifty-three serum samples were obtained from 39 HIV-seropositive patients. The condition of each patient was clinically classified as either asymptomatic, lymphadenopathy syndrome (LAS), AIDS-related complex (ARC), or AIDS. Control sera were obtained from 29 healthy male blood donors. A double antibody radioimmunoassay was used to measure the serum levels of cachectin/TNF. RESULTS: Cachectin/TNF levels were within the reference range of the control values in all (eight of eight) asymptomatic HIV-infected subjects and in 11 of 13 of the patients with LAS. In contrast, all patients with AIDS (nine of nine) and five of nine of the patients with ARC had raised levels of cachectin/TNF. Fluctuation of the levels of cachectin/TNF occurred during follow-up, but initially raised levels remained elevated. CONCLUSION: Since cachectin/TNF suppresses lipoprotein lipase in adipocytes in vitro, and causes weight loss under experimental conditions, the findings of raised levels of cachectin/TNF in patients with AIDS may have relevance to the pathogenesis of cachexia.

Serum immunoreactive interleukin 1 in renal transplant recipients. Association of raised levels with graft rejection episodes.

A solid-phase radioimmunoassay was used for monitoring serum interleukin 1 levels in 12 renal transplant recipients. From 10-fold to 20-fold elevations of interleukin 1 occurred in association with 10 of 12 graft rejection episodes. The interleukin 1 elevation preceded the clinical rejection diagnosis by an average of one day in transplant recipients with initially functioning grafts, and by two days in recipients with delayed onset of graft function. The results show that the release of interleukin 1 into the circulation is an early event in renal allograft rejection and demonstrate the potential utility of interleukin 1 antigenic assays in the early diagnosis of rejection.

Serum amyloid A protein: a sensitive indicator of renal allograft rejection in humans.

Acute human renal allograft rejection induces a dramatic elevation of serum amyloid A protein (SAA). To evaluate the clinical significance of this finding we monitored 31 consecutive recipients of cadaveric renal allografts by daily SAA measurements. SAA increased significantly during 37/38 rejection episodes. Mean peak SAA level during the reversible rejections was 271 +/- 31 mg/L (SE, median 220 mg/L, n = 35) and during the irreversible rejections 680 +/- 29 mg/L (median 705 mg/L, n = 3). Excluding the predictable operation-induced SAA elevations that peaked on the second post-operative day, there were seven out of 42 SAA elevations (greater than or equal to 100 mg/L) not due to rejection. They were all caused by severe infections, and in one instance by a surgical complication. In 17 of the 35 SAA-positive rejections the SAA elevation (greater than or equal to 100 mg/L) preceded the clinical diagnosis by 1-5 days; in 11 it occurred on the same day; and in 7 one day later. Rejection episodes in recipients with initially nonfunctioning grafts were all also characterized by significant SAA elevations. We conclude that daily monitoring of SAA concentrations offers a valuable aid in the early diagnosis of acute allograft rejection. The SAA test is not a renal function test, so it can also be carried out in transplant patients who are anuric or oliguric in the postgrafting stage.

Serum amyloid A levels in acute graft-versus-host disease in bone marrow transplant recipients.

We studied serum amyloid A levels of 31 bone marrow transplant recipients with and without acute graft-versus-host disease. Before transplantation the mean SAA concentration was 5.1 +/- 0.8 mg/l (mean +/- SEM). It remained low in patients with no signs of aGVHD but increased significantly during aGVHD to 54.0 +/- 8.2 mg/l (p less than 0.001 compared to pre-BMT value). Severe infections also induced high SAA levels.

Massive cutaneous hyalinosis. Identification of the hyalin material as monoclonal kappa light chains, adhesive 90 kD glycoprotein, and type I collagen.

The hyalin material in massive cutaneous hyalinosis, a disease characterized by extensive tumorous periodic acid-Schiff-(PAS) positive extracellular cutaneous deposits, has been elucidated by biochemical and immunologic methods. Three major components were found: kappa light chains, a mannose-rich glycoprotein, and type I collagen. Trace amounts of fibrinogen, fibronectin, laminin, IgG, pregnancy-specific glycoprotein, albumin, and keratan sulfate, but not keratin, were also present. The kappa light chains were monoclonal, cryoprecipiting, and more basic than the kappa chains from two myeloma patients. The glycoprotein, which could not be identified as any known glycoprotein, had an apparent molecular weight of 90,000 D. Amino acid analysis showed that glutamic acid, aspartic acid, leucine, and threonine were abundant, whereas hydroxyproline, hydroxylysine, and sulfhydryl amino acids were absent. The carbohydrate content of the protein was approximately 20%. The major monosaccharides were mannose and N-acetylglucosamine. Galactose, N-acetylneuraminic acid and fucose also were present. The third major component of the hyalin material was identified as type I collagen. A humoral immune response to the storage material was found: the patient's serum contained IgM and IgG class antibodies against the mannosylglycoprotein (90 kD glycoprotein) and against type I collagen.

Immunohistochemical demonstration of hyalinosis-associated 90 kD glycoprotein in amyloid deposits of lichen amyloidosus.

Immunohistochemical methods were used to study the nature of the amyloid deposits in lichen amyloidosus, in nodular amyloidosis, and in the cutaneous amyloid deposits found in Finnish-type systemic amyloidosis. In every case the anti-keratin serum stained the epidermis and sweat ducts but not the amyloid itself. None of the amyloids stained with anti-sera to prealbumin, to serum amyloid A protein, or to the free kappa or lambda light chains of immunoglobulins. In lichen amyloidosus but not in the other types of amyloidosis the amyloid substance stained intensely with the anti-serum to 90 kD glycoprotein. This glyco-protein, which is present in the basal cells of the hair follicles of normal skin, was first isolated from the extensive cutaneous deposits of a patient with a nonamyloid disease. The demonstration of this glycoprotein in lichen amyloidosus but not in nodular amyloidosis suggest a difference in pathogenesis between the two diseases. Tests for 90 kD glycoprotein may prove to be of value in the differential diagnosis of cutaneous amyloidosis.

Amyloid fibril protein in familial amyloidosis with cranial neuropathy and corneal lattice dystrophy (FAP type IV) is related to transthyretin.

Immunocytochemical methods were used to study the nature of the amyloid deposits in the Finnish type-familial amyloid polyneuropathy (FAP) type IV, which is characterized by cranial neuropathy and corneal lattice dystrophy. Commercial antisera to human plasma transthyretin (prealbumin) did not stain the amyloid deposits, but in every case a positive staining was obtained with antibodies raised against transthyretin-related amyloid fibril whole protein isolated from the myocardium of a patient with familial amyloid polyneuropathy from the state of New York. The FAP type IV amyloid deposits stained also with antiserum to serum amyloid P component, but did not stain with antisera to retinol-binding protein, amyloid A protein, gamma-trace protein, beta 2-microglobulin, or immunoglobulin light chains. The serum level of serum transthyretin was significantly decreased in FAP type IV patients (256 +/- 75 (SD) mg/L, n = 15) as compared with Finnish control subjects (360 +/- 56 mg/L, n = 30, P less than 0.001), whereas the level of retinol-binding protein was within the normal range. The results of this study strongly suggest that the amyloid fibril protein in FAP type IV amyloidosis is related to transthyretin.

Fibrillogenesis in gelsolin-related familial amyloidosis.

To clarify the mechanisms involved in amyloid formation in Finnish-type familial amyloidosis (FAF), we have tested the in vitro fibrillogenicity of synthetic wild-type and mutated gelsolin peptide analogs and studied the fragmentation patterns of gelsolin in the circulation of FAF patients with the Asn-187 or Tyr-187 gelsolin mutation. Fibril formation of synthetic peptides having sequence homology with wild-type or mutant gelsolins was monitored by Congo-red staining and polarization microscopy, negative staining electron microscopy and quantitative thioflavine-T fluorometry. Immunoblotting with anti-gelsolin and amyloid-specific antibodies and sequence analyses were used to study the fragmentation pattern of gelsolin. Ultrastructurally amyloid-like fibrils were formed from mutant Asn-187 and Tyr-187 gelsolin peptides. Fluorometric analysis revealed highly accelerated fibril formation from the mutant peptides as compared with the corresponding wild-type peptides. Addition of mercaptoethanol alone or in combination with dithiotreitol tended to enhance fibril formation of the 9-mer and 11-mer Asn peptides. Blocking of the C-terminal carboxyl of the mutant Asn-187 gelsolin182-192 peptide by amidation increased amyloidogenicity. The Tyr-187 gelsolin mutation, corresponding to the naturally occurring mutation in the Danish subtype of FAF, required acidic conditions to form fibrils meeting the criteria of amyloid. In FAF patients, in addition to the full-sized gelsolin, a series of lower-molecular mass C-terminal fragments of gelsolin (70,000-45,000 Da) was found in the circulation. In homozygous FAF(Asn-187) the 65-kDa fragment containing the amyloid forming region and the 55-kDa fragment, devoid of that region, was the major gelsolin species in the plasma. The results indicate that the 65-kDa gelsolin fragment derived by alpha-gelsolinase cleavage at the mutation-induced novel proteolysis site Arg172-Ala173 represents the putative circulating precursor protein of tissue amyloid in FAF and that the Asp187Asn/Tyr substitution in gelsolin creates a conformation that is highly fibrillogenic.