Radioimmunoassay for detecting abnormal prealbumin in the serum for diagnosis of familial amyloidotic polyneuropathy (Japanese type)

In the serum of a Japanese patient with familial amyloidotic polyneuropathy (FAP), we demonstrated the presence of a prealbumin variant having a single amino acid substitution of a methionine residue for a valine at position 30. We have developed a highly sensitive and specific method for quantitative analysis of the prealbumin variant in the sera of FAP patients by using radioimmunoassay for a nonapeptide corresponding to subsequence [22-30] of the prealbumin variant. This peptide is produced from the prealbumin variant by cyanogen bromide cleavage followed by tryptic digestion. The serum concentration of the prealbumin variant in five Japanese FAP patients ranges from 4.0 mg/dl to 7.8 mg/dl, which is 100 times or even higher than normal controls. This method should be helpful for an early diagnosis of this hereditary disease.     

Identification of a prealbumin variant in the serum of a Japanese patient with familial amyloidotic polyneuropathy

Serum prealbumin isolated from a Japanese patient with familial amyloidotic polyneuropathy (FAP) has been found to consist of a mixture of normal prealbumin and a prealbumin variant which contains a methionine for valine substitution at position 30. The prealbumin variant in the serum is identical to the prealbumin variant derived from amyloid fibrils of a Japanese FAP patient. FAP likely results from the deposition of abnormal serum prealbumin in various organs as amyloid fibrils.     

Molecular genetics of familial amyloidotic polyneuropathy

We established a diagnosis of familial amyloidotic polyneuropathy (FAP) based on DNA and demonstrated a direct link between prealbumin gene mutation and FAP. The individuals with FAP, so far examined, were heterozygous for the prealbumin gene, carrying one normal and one mutant gene. To investigate the molecular pathogenesis of FAP, we isolated a normal prealbumin gene (7 kb in length) and also a mutant prealbumin gene associated with FAP. We compared their nucleotide sequences and found that they matched except for a single-base substitution present in the second exon. In an effort to construct mouse model systems for the FAP, we developed strains of transgenic mice carrying human mutant prealbumin genes.     

Genetic basis for familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is an inherited systemic amyloidosis, characterized by the extracellular deposition of fibrillar amyloid protein, i.e. a variant type of prealbumin, and by prominent peripheral nerve involvement. We recently established the basis of FAP, using a cloned human prealbumin cDNA, restriction endonuclease(s) and Southern blot procedures. This approach clearly revealed a direct link between mutation in the prealbumin gene and FAP; individuals with FAP are heterozygous for the prealbumin gene, carrying one normal and one mutant gene. Molecular analysis of the prealbumin gene yielded pertinent data on the genetic basis for FAP.     

Identification of carriers of mutant prealbumin gene associated with familial amyloidotic polyneuropathy type I by Southern blot procedures: study of six pedigrees in the Arao district of Japan

Summary Fifty-six Japanese individuals from six pedigrees with familial amyloidotic polyneuropathy (FAP), together with 2 individuals with symptomatic FAP from an unknown pedigree were analyzed, using the Southern blot procedures for the prealbumin gene structure. A human prealbumin cDNA was used as the probe. Altogether, these individuals included 20 with symptomatic FAP, 30 who were asymptomatic, and 8 disease-free spouses. Twenty individuals with symptomatic FAP were all heterozygous for the prealbumin genes, carrying one normal and one mutant gene. We confirmed the direct linkage between the mutant prealbumin gene and the Japanese type of FAP. Moreover, 10 of the 30 asymptomatic individuals from pedigrees with FAP were also heterozygous for the prealbumin gene. The number of asymptomatic individuals with the mutant prealbumin gene showed age-related decreases, and none was over 40 years. A linkage between the mutant prealbumin gene and serum levels of the prealbumin variant was also evident.     

Tetramer formation of a variant type human transthyretin (prealbumin) produced by Escherichia coli expression system

A variant of human transthyretin(TTR, prealbumin) with methionine for valine substitution at position 30 is a major component of amyloid fibrils found in patients of familial amyloidotic polyneuropathy(FAP) type I, an autosomal dominant genetic disease. But the molecular nature of the variant TTR has been obscure, because most of plasma TTR from FAP patients is a mixture of variant and wild type TTR and no pure preparation of the variant has been available. For this reason, we constructed a system in which the variant type TTR was efficiently synthesized. In this system, the recombinant variant TTR was first synthesized as a fusion protein with E. coli outer membrane protein A (ompA) signal peptide, processed to eliminate the signal peptide and finally secreted to the culture medium. The final concentration of the recombinant variant TTR in the medium was about 5 mg/l. SDS polyacrylamide gel electrophoresis and gel filtration analysis suggested that the recombinant variant TTR can form tetramer as seen for native one. Purification of the protein was accomplished by only two steps of chromatography.     

A DNA test for Indiana/Swiss hereditary amyloidosis (FAP II).

Autosomal dominant amyloidosis of the Indiana/Swiss type is a late-onset disorder characterized by carpal tunnel syndrome, progressive peripheral neuropathy, vitreous deposits, and cardiomyopathy. This disorder was originally described in a large Indiana family of Swiss descent and is also known as familial amyloidotic polyneuropathy (FAP) type II. In the Indiana family, the genetic basis of the disease is a variant of plasma prealbumin (transthyretin), which has a serine-for-isoleucine substitution at amino acid 84 of the 127-residue prealbumin molecule. We predicted that the corresponding mutation in the prealbumin gene consisted of a T-to-G change in codon 84 (which created an AluI recognition site) and then demonstrated the extra AluI site in the DNA of patients by Southern blot analysis with a genomic prealbumin probe. This verifies the protein findings at the DNA level and provides a direct, reliable DNA test for the Ser-84 prealbumin gene associated with Indiana/Swiss hereditary amyloidosis.     

Role of variant prealbumin in the pathogenesis of familial amyloidotic polyneuropathy: fate of normal and variant prealbumin in the circulation

According to recent studies on protein chemistry and genetic engineering, replacement of the Val30 residue of prealbumin by methionine is believed to play a critical role in the formation of amyloid deposit and the pathogenesis of familial amyloidotic polyneuropathy (FAP). However, only limited information is available concerning the behavior of prealbumin in the circulation. To obtain the molecular insight into the mechanism of amyloid deposition, it is indispensable to know the fates of normal and variant prealbumin in vivo. Thus, the fates of prealbumin samples from normal and FAP patients were studied in normal rats as well as in animals that were challenged with acute inflammation induced by turpentine. The effect of in vitro photooxidation of prealbumin samples on their behavior was also examined in vivo. Kinetic analysis revealed no appreciable difference between prealbumin samples from normal and FAP patients. These results suggest that factors other than the rate of transfer of the variant form prealbumin from plasma to an extravascular compartment may play a critical role in the pathogenesis of amyloid deposition in FAP patients.     

Revised analysis of amino acid replacement in a prealbumin variant (SKO-III) associated with familial amyloidotic polyneuropathy of Jewish origin

Amyloid fibril protein (SKO-III) of 14K daltons associated with familial amyloidotic polyneuropathy of Jewish type was identified by Pras et al. as a prealbumin variant with a single amino acid substitution of a glycine for a threonine at position 49, mainly based on data obtained by automated sequence analyses. Structural re-investigation of SKO-III was performed by comparing tryptic peptide maps of SKO-III and normal human prealbumin. The present analysis reveals that the reported replacement at position 49 is not present in the molecule of SKO-III. SKO-III should be revised to be a prealbumin variant with one amino acid substitution of an isoleucine for a phenylalanine at position 33.     

Molecular analyses of an acidic transthyretin Asn 90 variant.

A mutation in transthyretin (TTR Asn 90) has been identified in the Portuguese and German populations. This variant has a lower pI and was found by screening analyses in 2/4,000 German subjects and in 4/1,200 Portuguese by using either double one-dimensional (D1-D) electrophoresis with isoelectric focusing (IEF) or hybrid isoelectric focusing in immobilized pH gradient (HIEF) as the final separation step. The Portuguese population sample was from the area where TTR Met 30-associated familial amyloidotic polyneuropathy (FAP) prevails, and it was divided into (a) a group of 500 individuals belonging to FAP kindreds and (b) a group of 700 collected at random. HIEF showed two particular situations: (1) one case, from an FAP kindred, was simultaneously carrier of the Met 30 substitution and the acidic variant, and (2) one individual, from the randomly selected Portuguese sample, had only the acidic monomer. Comparative peptide mapping, by HPLC, of the acidic variant carriers and of normal TTR showed the presence of an abnormal tryptic peptide, not present in the normal TTR digests, with an asparagine-for-histidine substitution at position 90 explained by a single base change of adenine for cytosine in the histidine codon. This was confirmed at the DNA level by RFLP analyses of PCR-amplified material after digestion with SphI and BsmI. In all carriers of the Asn 90 substitution, no indicators were found for an association with traits characteristic for FAP.     

The prealbumin nature of the amyloid protein in familial amyloid polyneuropathy (FAP)-Swedish variety

Familial amyloid polyneuropathy (FAP) is a dominant hereditary type of amyloidosis affecting kinships originating in many countries. We have isolated a 15,000 dalton protein from the amyloid laden tissue of a patient of Swedish origin with familial amyloid polyneuropathy. By N-terminal sequence analysis it is homologous to the normal plasma protein, prealbumin. An antiserum prepared to the isolated protein confirms this by reacting identically with the amyloid protein and prealbumin. The normal plasma protein, prealbumin, is linked to a disease syndrome for the first time.     

Haplotype analysis of familial amyloidotic polyneuropathy

Summary Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant genetic disease characterized by systemic accumulation of amyloid fibrils. A major component of FAP anyloid has been identified as variant transthyretin (TTR, also called prealbumin). In particular, a variant with the substitution ³⁰ValMet has been commonly found in FAP of various ethnic groups. To understand the origin and spread of the ValMet mutation, we analyzed DNA polymorphisms associated with the TTR gene in six Japanese FAP families and several Portuguese FAP patients. Three distinct haplotypes associated with the ValMet mutation were identified in Japanese FAP families, one of which was also found in Portuguese patients. On the other hand, it was found that the ValMet mutation can be explained by a C-T transition at the C_pG dinucleotide sequence of a mutation hot spot. Thus, our findings indicate that the ValMet mutation has probably recurred in the human population, to generate FAP families of independent origin.     

Chromosomal localization of the mouse prealbumin gene (Ttr) by in situ hybridization.

Prealbumin is a serum protein which plays an important role in plasma transport of retinol and thyroxine. The accumulation of a variant prealbumin is associated with a hereditary disorder, familial amyloidotic polyneuropathy (FAP). In situ hybridization with a mouse prealbumin gene cDNA probe was carried out in mouse fibroblasts. Analysis of 114 R-banded metaphases showed that 13% of the total grains were located on chromosome 4 and 46% of the grains on this chromosome were in the region C6-D1. Linkage and syntenic group analysis showed that the prealbumin gene (Ttr) is located between two syntenic groups on mouse chromosome 4, which corresponded to two syntenic groups present on human chromosomes 1 and 9.     

Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY are novel substrates of fibroblast activation protein-α

Fibroblast activation protein-α (FAP) is a cell surface-expressed and soluble enzyme of the prolyl oligopeptidase family, which includes dipeptidyl peptidase 4 (DPP4). FAP is not generally expressed in normal adult tissues, but is found at high levels in activated myofibroblasts and hepatic stellate cells in fibrosis and in stromal fibroblasts of epithelial tumours. FAP possesses a rare catalytic activity, hydrolysis of the post-proline bond two or more residues from the N-terminus of target substrates. α(2)-antiplasmin is an important physiological substrate of FAP endopeptidase activity. This study reports the first natural substrates of FAP dipeptidyl peptidase activity. Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY were the most efficiently hydrolysed substrates and the first hormone substrates of FAP to be identified. In addition, FAP slowly hydrolysed other hormone peptides, such as the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are efficient DPP4 substrates. FAP showed negligible or no hydrolysis of eight chemokines that are readily hydrolysed by DPP4. This novel identification of FAP substrates furthers our understanding of this unique protease by indicating potential roles in cardiac function and neurobiology.     

Evidence that the amyloid fibril protein in senile systemic amyloidosis is derived from normal prealbumin

Familial amyloidosis in different kindreds is associated with a variety of point mutations in the prealbumin gene, resulting in prealbumin variants which are believed to be amyloidogenic, i.e. prone to form amyloid fibrils. In the most common amyloid-associated variant, there is a methionine for valine substitution in position 30. We have studied the prealbumin-derived amyloid protein ASc1 in the common age-related senile systemic amyloidosis. Evidence is presented that there is no abnormality in the primary structure of prealbumin in this disease and that, in addition to complete prealbumin, fibrils contain prealbumin fragments lacking a significant part of the N-terminus.     

Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites

A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-alpha (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest Km of 21 microM. Overall the Km values were relatively similar for both high and low ranked substrates, whereas the kcat values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporation into FAP-activated prodrugs.     

Activatable Near-Infrared Fluorescent Probe for In Vivo Imaging of Fibroblast Activation Protein-alpha

Fibroblast activation protein-alpha (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein, we report an activatable near-infrared (NIR) fluorescent probe (ANP(FAP)) for in vivo optical imaging of FAPα. The ANP(FAP) consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANP(FAP), high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vitro assay on ANP(FAP) indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANP(FAP) showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα expression, demonstrating the in vivo targeting specificity of the ANP(FAP). Ex vivo imaging also demonstrated ANP(FAP) had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANP(FAP) could serve as a useful NIR optical probe for early detection of FAPα expressing tumors.     

Proline at position 36: a new transthyretin mutation associated with familial amyloidotic polyneuropathy.

Familial amyloidotic polyneuropathy (FAP) is associated with the deposition of an abnormal transthyretin (TTR) molecule. We have studied DNA from a family of Greek descent with FAP. The proband's TTR gene was asymmetrically amplified by using PCR and then was sequenced directly, to reveal a cytosine-for-guanine substitution in codon 36. This substitution removes a recognition site for endonuclease Fnu4HI. Allele-specific PCR was employed for diagnosis of the mutation. The predicted amino acid change of alanine to proline at position 36 was confirmed by protein sequencing of the proband's plasma TTR.     

A rare variant in human fibroblast activation protein associated with ER stress,loss of enzymatic function and loss of cell surface localisation

Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the β propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the β-propeller domain, inactive, unfolded FAP can be tolerated by cells.