Identification of amyloid prealbumin variant in familial amyloidotic polyneuropathy (Japanese type)

Structural studies on an amyloid fibril protein of 14 K daltons (AFj(INO] isolated from a Japanese patient who suffered from familial amyloidotic polyneuropathy were carried out to unambiguously identify its difference from normal human serum prealbumin. Sequence analyses performed by comparing peptide maps prepared from cyanogen bromide fragments and tryptic peptides of purified RCM-amyloid protein with those from RCM-prealbumin indicate that only a valine residue at position 30 in prealbumin is replaced by a methionine residue. Furthermore, it was also proved that AFj(INO) consists of four components; the prealbumin variant and its three related proteins, which are derived by successively accumulated deletion of the N-terminal three amino acid residues (Gly1, Pro2 and Thr3) from the prealbumin variant.     

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.     

Complete amino acid sequence of a unique protein related to the variable domain of lambda light chain from a case with Fanconi syndrome

The complete amino acid sequence has been determined of a unique protein from a 55-years-old female with multiple myeloma associated with Fanconi syndrome. It existed in a monomer form with an apparent molecular weight of 10K daltons, and was consisted of 106 amino acid residues. The sequence was characteristic of the V-region of lambda light chains and was highly homologous with that of the first 106 residues of V lambda III subgroup. The presence of an intact light chain as well as a 13K daltons fragment, corresponding to the entire C-region, strongly suggests that the unique component is a catabolic product from the intact light chain rather than an aberrant product of synthesis.     

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.     

Proteomic characterization of novel serum amyloid P component variants from human plasma and urine

Serum amyloid P component (SAP) is a human plasma protein that has been widely studied for its influence on amyloid plaque formation and stabilization. SAP was characterized directly from human plasma and urine samples via novel affinity mass spectrometry-based proteomic technology that is able to readily discriminate between mass-altered protein variants. These analyses were able to identify several variants of SAP that have not been previously reported. These variants include microheterogeneity of the glycan structure, from the loss of one or both terminal sialic acid residues, as well as the loss of the C-terminal valine residue. Moreover, the analysis of urine allowed for the consistent identification of serum amyloid P component as a normal constituent of the urine proteome.     

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.     

A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA

Amyloid fibrils from a patient with diffuse amyloid disease are dissociated in 6 m guanidine hydrochloride and fractionated by gel chromatography. Two major components are separated on Sepharose 6B. Both proteins are characterized by chromatography, immunodiffusion, discontinuous gel electrophoresis, amino acid tryptic peptide mapping and amino acid sequence analysis. The smaller of the two components is typical of the known protein AA by size (8400 daltons), amino acid composition and a 30-residue N-terminal sequence. The larger of the components (25,000 daltons) undergoes electrophoresis as a single band and appears unaffected by thiol reduction. It differs from protein AA in amino acid content and by its tryptic peptide map, although it contains an N-terminal amino acid sequence identical to protein AA when carried to 20 residues. Treatment of this larger component by mild acid hydrolysis results in the release of the 8400-dalton protein AA. Fractionation after guanidine hydrochloride treatment of this particular amyloid fibril preparation is compared to the fractionation of a typical secondary amyloid preparation that contains only protein AA as the major component. The origin and relationship of the 8,400- and 25,000-dalton protein components is discussed.     

Vitamin A transport in plasma of the non-mammalian vertebrates: isolation and partial characterization of piscine retinol-binding protein

Studies were conducted to explore vitamin A transport in the non-mammalian vertebrates, especially Pisces, Amphibia, and Reptilia, and to isolate and partially characterize piscine retinol-binding protein. Retinol-containing proteins in fresh plasma obtained from bullfrogs and a turtle exhibited similar properties to those found in mammalian and chicken plasma: i.e., molecular weight of about 60,000-80,000 as estimated by gel filtration and binding affinity to prealbumin on human prealbumin-Sepharose affinity chromatography. In sharp contrast, vitamin A-containing proteins in plasma from larvae of bullfrogs as well as three fishes (carp, blue sharks, and young yellowtails) appeared to be present in plasma as monomeric retinol-binding proteins without any affinity to human prealbumin. On the other hand, plasma vitamin A in the lamprey (Cyclostomes) was found to exist exclusively as an ester form in association with the lipoproteins of hydrated density less than 1.21 g/ml. Piscine retinol-binding protein was isolated from pooled plasma of young yellowtails and was converted (1000-fold purification) to a homogeneous component by a procedural sequence that included gel filtration on Sephadex G-100, chromatography on SP-Sephadex, gel isoelectric focusing, and, finally, polyacrylamide gel electrophoresis. Purified piscine retinol-binding protein showed physico-chemical properties distinctly different from the mammalian and chicken retinol-binding proteins examined, i.e., a smaller molecular weight of approximately 16,000, a lower isoelectric point of 4.3, a prealbumin mobility on analytical polyacrylamide gel electrophoresis, and a lack of binding affinity for human prealbumin; however, it displayed similar characteristics in two ways: a 1:1 molar complex with retinol, and a high content of tryptophan (four residues). These results strongly suggest that the piscine retinol-binding protein is a prototype of the specific vitamin A-transporting protein in plasma of the vertebrates, being modified later in evolution, during phylogenetic development of the vertebrates, to acquire a binding site for prealbumin on the molecule.     

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.     

Pipecolic acid pathway: the major lysine metabolic route in the rat brain.

A peptide of 6,700–8,000 daltons has been isolated from an ethanolic KOH digest of bovine ligamentum nuchae elastin by a combination of ion exchange chromatography and gel filtration techniques. The peptide is non-polar and is composed almost entirely of glycine, valine, and proline (or hydroxyproline) in the ratio 2:2:1. Edman degradation of the first 17 residues gave the sequence: NH₂-Gly-Phe-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val. These results suggest that the peptide is made up of repeating pentapeptide units with the sequence: Pro-Gly-Val-Gly-Val.     

A new transthyretin mutation associated with amyloid cardiomyopathy.

In transthyretin (TTR) a new mutation (TTR-Thr45) has been identified in a patient with familial amyloidosis characterized clinically by prominent cardiomyopathy and the absence of peripheral neuropathy. Comparative peptide mapping by high-performance liquid chromatography of the patient's plasma TTR together with normal TTR showed the presence of an abnormal tryptic peptide in the patient's TTR. The sequence of this peptide (peptide 6, residues 36-48) demonstrated the presence of a threonine-for-alanine substitution at position 45. This change can be explained by a single base change of adenine for guanine in the Ala-45 codon and was demonstrated directly by DNA sequence analysis of PCR-amplified exon 2 of the TTR gene; allele-specific oligonucleotide hybridization both in the patient and in fixed heart tissue from his aunt confirmed the base change. The TTR-Thr45 mutation is a new variant TTR found associated with cardiomyopathy.     

N-terminal amino acid sequences of acid proteases: acid proteases from Penicillium roqueforti and Rhizopus chinensis and alignment with penicillopepsin and mammalian proteases.

The amino-terminal sequence (33 residues) of the acid protease from Penicillium roqueforti has been determined with an automated sequencer. The amino-terminal sequence of Rhizopus pepsin (published by Sepulveda, P., Jackson, K. W. & Tang, J. (1975) Biochem. Biophys. Res. Commun. 63, 1106-1112) has been extended from 27 residues to 39 residues. Also, it was found that two forms of Rhizopus pepsin differ in position 15, where Rhizopus pepsin I has an isoleucine and Rhizopus pepsin II a valine residue. The new sequences have been aligned with the amino-terminal sequences of penicillopepsin (EC 3.4.23.7), pig pepsin (EC 3.4.23.1), calf chymosin (EC 3.4.23.4), human pepsin (EC 3.4.23.2), human gastricsin (EC 3.4.23.3), and cow pepsin (EC 3.4.23.1). Residues 31-35 (numbering based on pig pepsin, Tang, J., Sepulveda, P., Marciniszyn, Jr., J., Chen, K.S.C., Huang, W.-Y. , Tao, N., Liu, D. & Lanier, P. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3437-3739) are identical in all enzymes. This section contains one of the two aspartic acids (Asp-32) implicated in the active site. The similarity of the sequences provides strong evidence for the homology of these acid proteases.     

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.     

Designing heterodimeric two-stranded alpha-helical coiled-coils. Effects of hydrophobicity and alpha-helical propensity on protein folding,stability, and specificity

The E/K coil, a heterodimeric coiled-coil, has been designed as a universal peptide capture and delivery system for use in applications such as biosensors and as an expression and affinity purification tag. In this design, heterodimer formation is specified through the placement of charged residues at the e and g positions of the heptad repeat such that the E coil contains all glutamic acid residues at these positions, and the K coil contains all lysine residues at these positions. The affinity and stability of the E/K coil have been modified to allow a greater range of conditions for association and dissociation. Increasing the hydrophobicity of the coiled-coil core, by substituting isoleucine for valine, gave increases in stability of 2.81 and 3.73 kcal/mol (0.47 kcal/mol/substitution). Increasing the alpha-helical propensity of residues outside the core, by substituting alanine for serine, yielded increases in stability of 2.68 and 3.28 kcal/mol (0.41 and 0.45 kcal/mol/substitution). These sequence changes yielded a series of heterodimeric coiled-coils whose stabilities varied from 6.8 to 11.2 kcal/mol, greatly expanding their scope for use in protein engineering and biomedical applications.     

Oxidation inhibits amyloid fibril formation of transthyretin

The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated. Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on protein structures. Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing protein structures. Transthyretin (TTR) is a soluble human plasma protein. The wild-type (WT) and several of its variants are prone to fibril formation, which leads to amyloidosis associated with many clinical syndromes. The effects of amino acid side chain oxidations were investigated by comparing the kinetics of fibril formation of oxidized and unoxidized proteins. The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species. In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins. Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments. Side chain modification of methionine residues at position 13 and 30 (the latter for V30M TTR only) were dominant oxidative products. Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach. Oxidation inhibited the initial rates and extent of fibril formation for both the WT and V30M TTR proteins. In the case of WT TTR, oxidation inhibited fibril growth by approximately 76%, and for the V30M TTR by nearly 90%. These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures.     

Eukaryotic precursor proteins are processed by Escherichia coli outer membrane protein OmpP.

A new specific endopeptidase that cleaves eukaryotic precursor proteins has been found in Escherichia coli K but not in E. coli B strains. After purification, protein sequencing and Western blotting, the endopeptidase was shown to be identical with E. coli outer membrane protein OmpP [Kaufmann, A., Stierhof, Y.-D. & Henning, U. (1994) J. Bacteriol. 176, 359-367]. Further characterization of enzymatic properties of the new peptidase was performed. Comparison of the cleavage specificities of the newly found endopeptidase and that of rat mitochondrial processing peptidase (MPP) showed that patterns of proteolytic cleavage on the investigated precursor proteins by both enzymes are similar. By using three mitochondrial precursor proteins, the specificity assigned to OmpP previously, a cleavage position between two basic amino-acid residues, was extended to a three amino-acid recognition sequence. Positions +1 to +3 of this extended recognition site consist of an amino-acid residue with a small aliphatic side chain such as alanine or serine, a large hydrophobic residue such as leucine or valine followed by an arginine residue. Additionally, structural motifs of the substrate seem to be required for OmpP cleavage.     

Characterization of an archaeal multidrug transporter with a unique amino acid composition

The Smr family of multidrug transporters consists of small membrane proteins that extrude various drugs in exchange with protons rendering cells resistant to these drugs. Smr proteins identified to date have been found only in Eubacteria. In this work we present the cloning and characterization of an Smr protein from the archaeon Halobacterium salinarum, the first Smr in the archaeal kingdom. The protein, named Hsmr, was identified through sequence similarity to the Smr family, and the DNA sequence was cloned into an Escherichia coli expression system. Hsmr is heterologously expressed in a functional form despite the difference in lipid composition of the membrane and the lower salt in the cell and its environment. Cells harboring the Hsmr plasmid transport ethidium bromide in an uncoupler-sensitive process and gain resistance to ethidium bromide and acriflavine. Hsmr binds tetraphenylphosphonium (TPP(+)) with a relatively low affinity (K(D) approximately 200 nm) at low salt concentration that increases (K(D) approximately 40 nm) upon the addition of 2 m of either NaCl or KCl. The Hsmr protein contains many of the signature sequence elements of the Smr family and also a high content of negative residues in the loops, characteristic of extreme halophiles. Strikingly, Hsmr is composed of over 40% valine and alanine residues. These residues are clustered at certain regions of the protein in domains that are not important for activity, as judged from lack of conservation and from previous studies with other Smr proteins. We suggest that this high content of alanine and valine residues is a reflection of a "natural" alanine and valine scanning necessitated by the high GC content of the gene. This phenomenon reveals significant sequence elements in small multidrug transporters.     

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.     

Purification and amino acid sequence of lactocin S, a bacteriocin produced by Lactobacillus sake L45.

Lactocin S, a bacteriocin produced by Lactobacillus sake L45, has been purified to homogeneity by ion exchange, hydrophobic interaction and reverse-phase chromatography, and gel filtration. The purification resulted in approximately a 40,000-fold increase in the specific activity of lactocin S and enabled the determination of a major part of the amino acid sequence. Judging from the amino acid composition, lactocin S contained approximately 33 amino acid residues, of which about 50% were the nonpolar amino acids alanine, valine, and leucine. Amino acids were not detected upon direct N-terminal sequencing, indicating that the N-terminal amino acid was blocked. By cyanogen bromide cleavage at an internal methionine, the sequence of the 25 amino acids (including the methionine at the cleavage site) in the C-terminal part of the molecule was determined. The sequence was Met-Glu-Leu-Leu-Pro-Thr-Ala-Ala-Val-Leu-Tyr-Xaa-Asp-Val-Ala-Gly-Xaa-Phe- Lys-Tyr-Xaa-Ala-Lys-His-His, where Xaa represents unidentified residues. It is likely that the unidentified residues are modified forms of cysteine or amino acids associated with cysteine, since two cysteic acids per lactocin S molecule were found upon performic acid oxidation of lactocin S. The sequence was unique when compared to the SWISS-PROT data bank.     

Purification and amino acid sequence of lactocin S, a bacteriocin produced by Lactobacillus sake L45

Lactocin S, a bacteriocin produced by Lactobacillus sake L45, has been purified to homogeneity by ion exchange, hydrophobic interaction and reverse-phase chromatography, and gel filtration. The purification resulted in approximately a 40,000-fold increase in the specific activity of lactocin S and enabled the determination of a major part of the amino acid sequence. Judging from the amino acid composition, lactocin S contained approximately 33 amino acid residues, of which about 50% were the nonpolar amino acids alanine, valine, and leucine. Amino acids were not detected upon direct N-terminal sequencing, indicating that the N-terminal amino acid was blocked. By cyanogen bromide cleavage at an internal methionine, the sequence of the 25 amino acids (including the methionine at the cleavage site) in the C-terminal part of the molecule was determined. The sequence was Met-Glu-Leu-Leu-Pro-Thr-Ala-Ala-Val-Leu-Tyr-Xaa-Asp-Val-Ala-Gly-Xaa-Phe- Lys-Tyr-Xaa-Ala-Lys-His-His, where Xaa represents unidentified residues. It is likely that the unidentified residues are modified forms of cysteine or amino acids associated with cysteine, since two cysteic acids per lactocin S molecule were found upon performic acid oxidation of lactocin S. The sequence was unique when compared to the SWISS-PROT data bank.