Two alloalbumins with identical electrophoretic mobility are produced by differently charged amino acid substitutions.

We describe the amino acid substitutions of albumins Sondrio and Paris 2, two slow moving variants of human serum albumin, which show an identical electrophoretic mobility on cellulose acetate at three different pH values. These variants have been found in several instances in a wide geographic area including Northern Italy and France. Both alloalbumins were isolated from the sera of heterozygous subjects. Isoelectric focusing analysis of CNBr fragments from the purified variants allowed us to localize the mutation of albumin Sondrio in fragment CNBr V (residues 330-446) and that of albumin Paris 2 in CNBr VII (residues 549-585). Sequential analysis of the variant CNBr VII established the molecular defect of albumin Paris 2 as 563 Asp----Asn. Fragments CNBr V from normal and Sondrio albumins were isolated on a preparative scale and subjected to tryptic and V8 proteinase digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution of glutamic acid 333 by lysine. Thus, a +1 change in the C-terminal region of the albumin molecule produces a variant with the same electrophoretic mobility as an alloalbumin with a +2 substitution in the central domain, suggesting a higher degree of exposure to the solvent of the C-terminal tailpiece. Both amino acid substitutions are consistent with a G----A transition in the first position of the corresponding codon in the structural gene.     

The molecular defect of albumin Castel di Sangro: 536 Lys----Glu

Albumin Castel di Sangro is a rare fast-moving variant of human serum albumin which has been discovered in heterozygous form in the serum of an 85-year-old woman living in Castel di Sangro (Abruzzo, Italy). Isoelectric focusing analysis of CNBr fragments from the purified variant allowed us to localize the mutation in fragment CNBr VI (residues 447-548). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by reverse-phase and cation-exchange HPLC, revealed that the variant arises from the substitution of lysine 536 by glutamic acid. This amino acid replacement, probably due to a single-base substitution in the structural gene, causes a change in the net charge of -2 units, which is in keeping with both the increased electrophoretic mobility of the native protein and the isoelectric point of the modified CNBr fragment.     

Detection of a genetic variant, lysine→glutamic acid at position 372 of human serum albumin, by capillary electrophoresis and structural identification

A genetic variant of human serum albumin (alloalbumin) is detected by capillary electrophoresis (CE). Two albumin peaks, which were in the ratio of approximately one, were clearly separated. One of the peaks had the same migration time as normal albumin (Alb A) and the other (Alb X) had a longer migration time. SDS-polyacrylamide gel electrophoresis of CNBr fragments (CB) of Alb X indicated that the amino acid substitution was localized in the CB5 fragment (residue 330–446) of the molecule, because of anomalous migration of CB5 in the gel. The CE mapping of the tryptic peptides from the variant CB5 revealed clearly the existence of a new peptide, and the lack of two normal peptides. The sequence analysis of the variant peptide collected by CE micropreparation showed that the N-terminus of the variant peptide corresponded to that of T49 in Alb A. The substitution site, lysine→glutamic acid at the position 372, was revealed by sequence determination of the variant peptide purified by reversed-phase HPLC.     

Alloalbuminemia in North India.

Electrophoretic screening of sera from 550 individuals from Punjab, North India, revealed four cases of alloalbuminemia. Two albumin variants migrated slower and two migrated faster than the common albumin A. These variants were further analyzed by electrophoresis of their cyanogen bromide fragments to localize their molecular differences. One of the slow variants appears similar to, if not identical with, albumin B, with an altered cyanogen bromide fragment CNBr VII. The other slow variant appears to be a new variant (proposed name albumin Punjab) differing from albumin A in an altered fragment CNBr VI (which also occurs in albumins Kashmir and Adana) and in an altered fragment CNBr I. Among the fast variants, one has the same altered fragment CNBr V as albumin Naskapi, while the other appears to be a new variant (proposed name albumin Patiala) having an altered fragment CNBr VI. The presence of albumin Naskapi in Punjabis, North American Indians, and Eti Turks (previously reported) is consistent with the existence of a common ancestral population in which the mutation to Naskapi occurred before the migrations eastward and westward.     

The molecular defect of albumin Tagliacozzo: 313 Lys----Asn

Albumin Tagliacozzo is a fast-moving genetic variant of human serum albumin found in 19 unrelated families. The protein was isolated from the serum of a heterozygous healthy subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to CNBr fragment IV (residues 299-329). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by RP-HPLC, revealed the variant was caused by 313 Lys----Asn substitution, probably due to a point mutation in the structural gene. The lack of a lysine residue accounts for the electrophoretic behavior of albumin Tagliacozzo.     

Structural characterization of a chain termination mutant of human serum albumin

Mutant forms of human serum albumin have been detected on the basis of their abnormal electrophoretic mobility which is either faster or slower than that of normal albumin. In the present work we have studied the structure of a slow variant, referred to as albumin Ge/Ct, in order to define the cause of its genetic abnormality. The protein was isolated from the serum of a young healthy woman homozygous for the variant. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585). This fragment was isolated on a preparative scale and subjected to tryptic digestion. All tryptic peptides were purified by reverse-phase high performance liquid chromatography and characterized. Sequential analysis of three abnormal peptides revealed that albumin Ge/Ct has a shortened chain with the following COOH-terminal sequence: Leu-Val-Ala-Ala-Ser-Lys580-Leu-Pro. The presence of an additional lysine residue accounts for the electrophoretic behavior of the variant. It is likely that the variant may be caused by a single base deletion in the structural gene, a Cyt in mRNA codon 580, and the consequent shift in reading frame.     

A nucleotide insertion and frameshift cause albumin Kénitra, an extended and O-glycosylated mutant of human serum albumin with two additional disulfide bridges.

Albumin Kenitra is a new type of genetic variant of human serum albumin that has been found in two members of a family of Sephardic Jews from Kenitra (Morocco). The slow-migrating variant and the normal protein were isolated by anion-exchange chromatography and, after treatment with CNBr, the digests were analyzed by two-dimensional electrophoresis in a polyacrylamide gel. The CNBr peptides of the variant were purified by reverse-phase high performance liquid chromatography and submitted to sequence analysis. Albumin Kenitra is peculiar because it has an elongated polypeptide chain, 601 residues instead of 585, and its sequence is modified beginning from residue 575. DNA structural studies showed that the variant is caused by a single-base insertion, an adenine at nucleotide position 15 970 in the genomic sequence, which leads to a frameshift with the subsequent translation to the first termination codon of exon 15. Mass spectrometric analyses revealed that the four additional cysteine residues of the variant form two new S-S bridges and showed that albumin Kenitra is partially O-glycosylated by a monosialylated HexHexNAc structure. This oligosaccharide chain has been located to Thr596 by amino-acid sequence analysis of the tryptic fragment 592-597.     

Protein and DNA sequence analysis of a ‘private’ genetic variant: albumin Ortonovo (Glu-505 → Lys)

Albumin Ortonovo is a slow moving variant of human serum albumin which has been found only in people coming from the small villages of Ortonovo and Nicola (Liguria, Italy) and reaches polymorphic frequency (≥1%) in the poorly admixed population group living in that area. This is the first report of a ‘private’ varint detected in a Caucasin population. It probably originated as a mutation in a founder individual many generations ago. Isoelectric focusing analysis of CNBr fragments from the purified variant localized the mutation in fragment CNBr (residues 447–548). This fragment was isolated on a preparative scale by reversed-phase HPLC and subjected to V8 proteinase digestion. Sequence analysis of the abnormal V8 peptide revealed that the variant arises from a previously unreported substitution at position 505 where glutamic acid has been replaced by lysine. The protein data were confirmed by DNA sequence analysis which indicated a single nucleotide change of $\text{G}\text{AA}\text{→}\text{A}\text{AA}$ in the corresponding codon of the structural gene. Since the amino acid substitution found in albumin Ortonovo accords with its electrophoretic mobility on cellulose acetate, residue 505 is probably exposed to the solvent. The clustering of the mutations in the intersubdomain connection linking subdomains IIIA and IIIB (residues 492–511) accords with the fact that this region lies on the molecular surface and is accessible to solvent.     

Structural characterization, stability and fatty acid-binding properties of two French genetic variants of human serum albumin

Four bisalbuminemic, unrelated persons were found in Bretagne, France, and their variant and normal albumins were isolated by DEAE ion exchange chromatography, reduced, carboxymethylated and treated with CNBr. Comparative two-dimensional electrophoresis of the CNBr digests showed that three of the variants were modified in fragment CB4, whereas the fourth had an abnormal fragment CB1. These fragments were isolated, digested with trypsin and mapped by reverse-phase HPLC. Sequencing of altered tryptic peptides showed that the three variants modified in CB4 were caused by the same, previously unreported, amino acid substitution: Asp314-->Val (albumin Brest). The fourth, however, was a proalbumin variant with the change Arg-2-->Cys (albumin Ildut). Both amino acid substitutions can be explained by point mutations in the structural gene: GAT-->GTT (albumin Brest) and CGT-->TGT (albumin Ildut). The proalbumin Ildut is very unstable and already in vivo it is to a large extent cleaved posttranslationally to Arg-Albumin and normal albumin. Furthermore, we observed that during a lengthy isolation procedure the remaining proalbumin was changed to Arg-Albumin or proalbumin lacking Arg-6. In addition, part of normal albumin had lost Asp1. Gas chromatographic investigations using isolated proteins indicated that albumin Brest has improved in vivo fatty acid-binding properties, whereas the structural modification(s) of albumin Ildut does not affect fatty acid binding.     

Isolation and properties of a fragment of human serum albumin demonstrating the absence of a methionine residue from some of the albumin molecules.

1. A fragment designated D was isolated from human serum albumin degraded by CNBr. Its properties show that it is made up of the B and C fragments isolated by McMenamy et al. (1971) (J. Biol. Chem., 246, 4744-4750). 2. Reduction of fragment D gives rise to two chains, one of which consists of the second subfragment of reduced fragment B linked to fragment C by an amino acid different from methionine. It thus demonstrates the existence of albumin molecules from which the second methionine residue located between fragments B and C is missing.     

Membrane glycophorins in Sta blood group erythrocytes

Structural and immunochemical studies of glycophorins isolated from erythrocytes of an individual homozygous for the M Sta blood group phenotype are described. Reactivities with specific monoclonal antibodies indicated that two major M and N glycophorins were present. The M and N Sta glycophorins were resolved by Lens culinaris lectin affinity chromatography. The N species was not held on the lectin but the M species, like control alpha glycophorins, was retained and could be eluted with alpha-methylmannoside. The two proteins were present in almost equimolar amounts. Studies of the CNBr fragments provided evidence that the structure of M Sta glycophorin is the same as that of the usual M alpha glycophorin but that the N Sta glycophorin is a variant. The amino-terminal octapeptides of the M and N species were similar in amino acid and carbohydrate composition to those isolated, respectively, from M and N alpha glycophorins. The studies focused on CNBr glycopeptide B that, in control alpha glycophorins, extends from amino acid residues 9 to 81. The fragment from the M species exhibited properties identical to those of the corresponding fragment of control alpha glycophorins in terms of size, chromatographic behavior, amino acid and carbohydrate contents and compositions, the presence of O-glycosidically linked saccharides and a single Asn-linked carbohydrate unit. The structures of the O-linked units were inferred experimentally to be NeuAc(alpha 2,3)Gal-(beta 1,3)GalNAc and NeuAc(alpha 2,3)Gal(beta 1,3) [NeuAc(alpha 2,6)]GalNAc, present in a ratio similar to that found in controls; and the Asn-linked unit also appeared to be as in the control. The tryptic glycopeptide pattern of the M Sta glycophorin CNBr fragment B was identical to the pattern of the corresponding control fragment, and the composition of the tryptic peptides suggested sequence identity with the control fragment. In contrast, the N Sta glycophorin yielded two CNBr glycopeptides B; both contained fewer amino acid residues and virtually lacked Man and GlcNAc, indicating the absence of the Asn-linked carbohydrate. The much decreased levels of these carbohydrates in the intact N protein, corroborated the latter finding. The O-glycosidic saccharides appeared similar to those found in control alpha glycophorins. However, the tryptic glycopeptide pattern of the variant differed from control M or N alpha glycophorins, suggesting a deletion of a large segment of the molecule near residues 40-61 and/or a substitution of methionine for a residue upstream from residue 40.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and partial structural characterization of an equine fibrinogen CNBr fragment that exhibits immunologic cross-reactivity with an A alpha-chain cross-linking region of human fibrinogen

Immunochemical studies of equine fibrinogen were conducted to characterize the structural basis for the immunologic cross-reactivity observed between human and equine A alpha chains when employing an antiserum to the 26K, human cyanogen bromide (CNBr) fragment, A alpha 241-476 (CNBr VIII). A 38K, equine CNBr fragment that reacts with this antiserum was isolated from CNBr-digested equine fibrinogen by Sephadex G-100 gel filtration. It was further purified by sequential hydrophobic chromatography on phenyl-Sepharose CL-4B, followed by reversed-phased (C-8) high-performance liquid chromatography (HPLC). NH2-Terminal analysis of the purified fragment, designated EqA alpha CNBr, identified one major sequence whose first three residues, E-L-E, were identical with those of human CNBr VIII. Tryptic and staphylococcal protease digests of the equine fragment were resolved by reversed-phase HPLC (C-4, C-18), and the separated components were characterized by amino acid analysis and automated Edman degradation. A total of 34 tryptic and 20 staph protease peptides yielded sequence information that permitted the alignment of 271 equine residues with residues A alpha 241-517 from the COOH-terminal two-thirds of the human A alpha chain so that 63% of the possible matches were identical. Other features of interest included (1) an amino acid substitution in which the methionine residue at A alpha 476 in the human A alpha chain was replaced by a valine residue, thus accounting, in part, for the larger EqA alpha CNBr fragment obtained from the equine molecule, and (2) a region of striking homology in which 36 successive residues, corresponding to A alpha 428-464 in the human A alpha chain, were identical in both species. These findings, together with available structural data for the COOH-terminal portion of the rat and bovine A alpha chains, indicate that the region corresponding to (human) A alpha 240-517 represents a conserved portion of the fibrinogen molecule. This may, in turn, explain the difficulties encountered when trying to raise monoclonal antibodies to cross-linking regions that are contained within the COOH-terminal two-thirds of the human A alpha chain.     

The molecular defect in a COOH-terminal-modified and shortened mutant of human serum albumin

Albumin Venezia is a fast migrating genetic variant of human serum albumin which, in heterozygous subjects, represents about 30% of the circulating protein. The molecular defect in this variant was studied in a subject possessing an atypical level of the mutant (80% of the total protein) and in other members of his family. Albumins, isolated from the sera of the propositus and his heterozygous relatives, were treated with CNBr and the resulting fragments analyzed by isoelectric focusing. The peptides were then isolated in a homogeneous form by reverse-phase high performance liquid chromatography and submitted to sequence analysis. The results show that albumin Venezia possesses a shortened polypeptide chain, 578 residues instead of 585, completely variant from residue 572 to the COOH-terminal end: sequence: (see text). This extensive modification may be accounted for by the deletion of exon 14 and translation to the first terminator codon of exon 15, which normally does not code for protein. The absence of a basic COOH-terminal dipeptide in the mature molecule can be explained by the probable action of serum carboxypeptidase N. Additional support for such action comes from examination of the remaining 20% of the total albumin of the propositus, which is found to contain an extra Arg at its COOH terminus, probably due to partial digestion by carboxypeptidase N. The low serum level of the variant in heterozygous subjects suggests that the COOH-terminal end of the molecule is critical for albumin stability.     

Molecular analysis of glycophorin A and B gene structure and expression in homozygous Miltenberger class V (Mi. V) human erythrocytes

In the Miltenberger class V (Mi. V) condition, red cells lack glycophorin A (GPA) and glycophorin B (GPB) but carry instead an unusual glycoprotein thought to be a hybrid molecule produced by the unequal crossing-over between the closely linked genes encoding for GPA and GPB. By Western blot analysis with rabbit anti-GPA antibodies specific for discrete domains of GPA, it was found that the Mi. V glycoprotein (donor F. M.) contains approximately 60 amino acid residues of GPA at its N-terminus. As a preliminary approach to the molecular analysis of this variant the restriction maps of the GPA and GPB genes were established by Southern blot analysis of genomic DNA and from genomic clones isolated from a human leukocyte library constructed in lambda EMBL4. The GPA and GPB genes cover about 30 kb of DNA and are organized into seven exons (A-1-A-7) and five exons (B-1-B-5), respectively. In addition to the normal genes, a third gene (named inv), closely resembling the GPA and GPB genes, was also identified. In the homozygous Mi. V individual the normal GPA and GPB genes were absent, but an unusual form of gene structure was detected by Southern blot analysis. The Mi. V glycoprotein gene was composed of exon B-1 of the GPB gene followed by exons A-2 and A-3 of the GPA gene and the exons B-3, B-4 and B-5 of the GPB gene. Exon B-1 can be distinguished from exon A-1 of GPA since it is located within a different restriction fragment, but both encode the same amino acid sequence (N-terminal region of the signal peptides). Using the polymerase chain reaction, the junction between exon A-3 and exon B-3 was confirmed by amplification of the DNA region where the putative crossing-over has occurred and it was deduced that the Mi. V glycoprotein is a hybrid molecule composed of amino acid residues 1-58 from GPA fused to amino acid residues 27-72 of GPB. In addition, the finding that part of the signal peptide and the 5'-untranslated region are derived from GPB suggests that the genetic background of the Mi. V variant is rather complex and may involve a cascade of recombination or gene conversion events.     

Structural characterization of two genetic variants of human serum albumin

In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.     

Influence of aspirin and iron(III) tetrasulfonated phthalocyanine on bilirubin binding by human serum albumin

The interaction of bilirubin with aspirin-modified human serum albumin (HSA) and the influence of iron tetrasulfonated phthalocyanine on bilirubin binding by the native protein has been studied by difference spectroscopy and circular dichroism measurements. Spectroscopic studies of the systems containing bilirubin and aspirin-modified HSA compared to the analogous systems with the native protein have shown that selective acetylation of albumin at lysine 199 inhibits bilirubin binding by this protein. In both cases, interaction between bilirubin and albumin leads to complex formation at a molar ratio of ligand to protein of 2:1. The studies of the reaction of bilirubin with fragments of albumin produced by reaction with CNBr have demonstrated that one of the strong bilirubin binding sites is located in the M fragment and is close to the high-affinity binding site of aspirin. The other one was found in fragment C. Acetylation of albumin brings about marked conformational change in the protein, which probably accounts for the decrease in its ability to react with anti-HSA antibody. Bilirubin does not change the secondary structure of albumin but, like aspirin, lowers its antigenicity. It has been suggested that the decrease in antigenic properties in this case results from cooperation of the closely neighboring antigenic and bilirubin-binding sites. The studies of the influence of iron(III) tetrasulfonated phthalocyanine on bilirubin binding by HSA suggest that there is no competition between strong sites for iron(III) tetrasulfonated phthalocyanine and bilirubin, but these compounds compete for some of the weaker sites.     

Limited pepsin digestion of human plasma albumin.

Limited pepsin digestion of human plasma albumin at pH 3.5 and 0 degrees in the presence of octanoate caused cleavage at residue 307 of the albumin molecule to yield two fragments. Thw two fragments corresponding to the NH2- and the COOH-terminal halves of the molecule were isolated in yields of about 15%. The COOH-terminal fragment is a mixture in which about 85% of the molecules had an additional cleavage at residue 422 of the albumin molecule. The COOH-terminal fragment with the additional cleavage at residue 422 contains two peptides which are linked by a disulfide bridge at residues 391 and 437 of the albumin molecule. Both the NH2- and the COOH-terminal fragment of human albumin showed no detectable binding of octanoate anions, that is, less than 1/170 of the binding constant of the primary site of human albumin. These findings differ from earlier observations on limited pepsin digestion of bovine plasma albumin where the corresponding COOH-terminal fragment had the octanoate-binding activity, about 1/8 of the primary binding constant of bovine albumin, while the NH2-terminal fragment did not. The COOH-terminal fragment of bovine albumin did not have cleavage at residue 422 as in the corresponding fragment of human albumin. However, it is not clear that the loss of octanoate-binding activity of fragment C of human albumin is a direct consequence of the cleavage at residue 422.     

Evidence for structural dissociation of two biologic actions of growth hormone

The effects of purified growth hormone and its CNBr fragments on somatomedin induction and on the stimulation of hepatic and renal ornithine decarboxylase (l-ornithine carboxylase, EC 4.1.1.17) activity in rats have been investigated. At the doses tested, none of the CNBr fragments induced somatomedin as evidenced by lack of an effect on sulfate, leucine, and thymidine incorporation into cartilage of hypophysectomized rats. However, the largest fragment, consisting of two peptides corresponding to Residues 6–124 and 150–179 linked by a disulfide bridge, stimulated both renal and hepatic ornithine decarboxylase activity in hypophysectomized rats and the activity of the hepatic enzyme in intact animals. A smaller CNBr fragment corresponding to Residues 125–149 slightly stimulated the activity of renal ornithine decarboxylase but failed to increase activity of the hepatic enzyme. A similar slight stimulation of the activity of the renal, but not the hepatic, enzyme was produced by a large carboxyl-terminal fragment (molecular weight 8000) prepared by proteolytic cleavage of partially purified ovine growth hormone. Circular dichroic spectra of the CNBr fragments demonstrated that the largest fragment retained much of the ordered secondary structure of intact growth hormone while two smaller CNBr fragments were devoid of ordered secondary structure. These observation indicate that different biological activities of growth hormone may be dissociated by fragmentation of the parent molecule.     

Physical and binding properties of large fragments of human serum albumin.

Three large fragments of human serum albumin were produced by peptic digestion of the native protein [Geisow & Beaven (1977) Biochem. J. 161, 619-625]. Fragment P44 represents residues 1-386 and fragments P29 and P31 represent residues 49-307 and residues 308-584 respectively of the albumin molecule. The large N-terminal fragment P44 has a similar percentage of alpha-helix to stored defatted albumin, although the alpha-helix content of all the fragments is significantly less than that of freshly prepared albumin. The fragment P44 appears to account for all the binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonate to albumin. N-Acetyl-L-tryptophan binds to this fragment and displaces one of the bound molecules of 8-anilinonaphthalene-1-sulphonate. Bilirubin binds to fragments P44 and P29, and the complexes show similar circular-dichroism spectra to that of the complex between bilirubin and whole albumin. These results are in agreement with affinity-labeling work on albumin with reactive ligands where substitution occurs in the N-terminal region of the molecule. The sharp conformational transitional transition in albumin which is observed between pH4 and 3.5 was absent from the fragments. This isomerization, usually called the N-F transition, probably occurs in intact albumin as a result of the unfolding or separation of the C-terminal third of the protein from the remainder of the molecule.     

Gelsolin variant (Asn-187) in familial amyloidosis, Finnish type.

Familial amyloidosis, Finnish type (FAF), is an inherited form of systemic amyloidosis clinically characterized by cranial neuropathy and lattice corneal dystrophy. We have demonstrated that the protein subunit isolated from amyloid fibrils shows considerable sequence identity with gelsolin, an actin-binding protein. We have purified the amyloid subunit from a second case and further analysed different fractions from the previous one. Sequence analysis shows that, in both cases, the amyloid subunit starts at position 173 of the mature molecule; it has a heterogeneous N-terminus and contains one amino acid substitution, namely asparagine for aspartic acid, at position 15 (gelsolin residue 187), that is due to a guanine-to-adenine transversion corresponding to nucleotide-654 of human plasma gelsolin cDNA. The substitution maps in a fragment with actin-binding activity and is located in a repetitive motif highly conserved among species. Thus FAF is the first human disease known to be caused by an internal abnormal degradation of a gelsolin variant. We designate this variant of gelsolin-associated amyloidosis 'Agel Asn-187'.