Nils Eriksen

The major proteins of human and monkey amyloid substance: Common properties including unusual N-terminal amino acid sequences

Amyloid substance is a complex proteinaceous material found in the tissues of patients with the disease amyloidosis. Recently we have presented evidence that there are two chemically distinct kinds of amyloid substance, one associated with the classical inflammation-related amyloidosis and another, frequently designated atypical or paramyloid, occurring with tumors such as multiple myeloma or without evident pre-existing disease [l] . The classical, or inflammation-associated, substance is distinguished by: a) instability to alkali of its characteristic Congo red binding capacity; b) its amino acid composition and c) the presence of a major protein constituent, amyloid protein A. The family of proteins comprising amyloid protein A has a molecular weight range of 6000-8000 and a characteristic amino acid composition [2] ; in addition, human amyloid protein A has the capacity to bind Congo red and exhibits the characteristic hyperchromism and spectral changes previously described for amyloid substance [ 1] . In this communication we compare the chromatographic and electrdphoretic properties, the amino acid composition and the 24 amino acid N-terminal sequence of the humanand monkeyderived protein A of amyloid substance.

SAA, AN APOPROTEIN OF HDL: ITS STRUCTURE AND FUNCTION*

The putative precursor of amyloid protein AA appears in serum as an apoprotein (apoSAA) of heavier HDL fractions of lipoproteins found in humans and mice. ApoSAA is found by precipitation with specific anti-AA antibodies to be on a particle that also carries apoA-I and some C-apoproteins. In endotoxin-treated mice a lipoprotein fraction with about 2 moles of apoSAA to 1 mole of apoA-I can be isolated, an apoSAA-carrying subset of HDL being thus suggested. In mice, rapid clearance from plasma of native apoSAA compared to apoA-I suggests a special function for apoSAA. The C-terminal amino acid portion of apoSAA may play a role in this function.

Primary structure of duck amyloid protein A The form deposited in tissues may be identical to its serum precursor

The amino acid sequence has been determined for the major protein that accumulates in amyloid fibrils in tissues of the Pekin duck. With the exception of 16 residues at the amino terminus, this 106‐residue protein is homologous with human serum amyloid protein A (104‐residue apoSAA), which is the putative precursor of the 76‐residue protein that accumulates in human patients with amyloidosis. Duck serum is shown to contain a protein that is immunologically related and approximately equal in size (12 kDa) to the deposited form in ducks. These results indicate that proteolytic processing of the precursor is not a necessary step in the deposition of amyloid fibrils, at least in the duck.

Enzymic oxidation of the indole nucleus of 5-hydroxytryptamine: properties of an enzyme in human serum and of the products of oxidation.

Abstract 5-Hydroxytryptamine (5-HT) consumes oxygen in the presence of pooled serum from pregnant humans and ceruloplasmin concentrates. The properties of the catalyst in serum were those of an enzyme. 5-HT oxidase may be identical with the enzyme which oxidizes p -phenylenediamine. Chromatography of incubation mixtures containing 5-HT and serum or 5-HT and ceruloplasmin concentrates revealed several indolic products as well as a pigment having properties similar to the melanins. These products appear to be identical with those resulting from 5-HT oxidation by air in the presence of copper or by AgNO 3 ; the major product of the latter reaction has been identified as a dimer resulting from dehydrogenative coupling of the indole nuclei of two molecules of 5-HT (1).

[16] Serum amyloid A (ApoSAA) and lipoproteins

Publisher Summary The discovery of a unique protein (AA) characteristic of amyloid deposits in tissues of patients with chronic inflammatory diseases have led to the immunological recognition of a structurally related protein (SAA) in human serum. This chapter describes the preparation of ApoSAA, physical and chemical characteristics of ApoSAA, and assay of SAA. ApoSAA might participate in a variety of molecular associations, any one of which could interfere to some extent with its detection. Limited proteolysis of apo- SAA in vivo or during storage of specimens, for example, could result in the appearance of AA, which itself could participate in various associations and thus further complicate the quantification of apoSAA. It has been suggested that apoSAA must serve an important function in vertebrate physiology, inasmuch as human and murine apoSAA show perfect amino acid sequence homology with residues 33 through 45 of AA proteins from man, monkey, mink, and duck. However, what role apoSAA plays among the variety of normal metabolic functions served by the plasma apolipoproteins and whether it is an essential determinant of structure in a particular subset of HDL particles are questions that remain to be answered.

Mouse SAA3: Detection in Mouse Tissues with Specific Antibody

Antibodies to a protein A-SAA3 fusion protein were generated in rabbits. Immunochemical studies using SAA3 antiserum were performed on tissues from LPS treated mice and revealed positive reactions to liver hepatocytes and other tissues.

Protein AA and Associated Proteins in Type-AA Amyloid Substance

Type-AA amyloid substance was isolated from human amyloidotic liver by the water-extraction method of Pras. Pellet material collected by 105 g centrifugation of the water extracts was dialyzed against water and lyophilized. The lyophilized product was extracted with 6 M urea at pH 3, and the soluble portion was fractionated by passage through a Sephadex G-100 column in the acid-urea solvent. The intact pellet material, the acid-urea-insoluble portion, and the chromatographic fractions were subjected to SDS/polyacrylamide gel electrophoresis, electrotransfer of the electrophoretically resolved samples to nitrocellulose, and subsequent reaction with antibodies to human AA, AP, prealbumin, albumin, fibronectin and kappa and lambda L-chains.

Serum Amyloid A (SAA) Induction in the Serum High Density Lipoproteins of the Syrian Hamster

SAA elevations in the serum lipoproteins of hamsters were induced by injection of lipopolysaccharide intraperitoneally or turpentine subcutaneously. Lipoprotein fractions isolated by sequential density centrifugation of serum samples obtained 20-hours post-stimulus were examined by SDS-PAGE followed by electroimmunoblotting. We observed a marked enhancement of a 12-kDa doublet in the electrophoretic pattern of the acute-phase HDL3. Antibodies to human or monkey AA reacted preferentially with the leading member of the doublet, whereas antibodies to mouse AA reacted preferentially with the trailing member. Antibodies to human and mouse SAA and to duck AA reacted with both members. The different reactions displayed by the members of the hamster SAA doublet against the several antibody preparations suggest that at least two of the three proteins predicted by a study of SAA-gene expression in the hamster may circulate in the HDL.