Extrahepatic production of acute phase serum amyloid A

Amyloidosis is a group of diseases characterized by the extracellular deposition of protein that contains non-branching, straight fibrils on electron microscopy (amyloid fibrils) that have a high content of beta-pleated sheet conformation. Various biochemically distinct proteins can undergo transformation into amyloid fibrils. The precursor protein of amyloid protein A (AA) is the acute phase protein serum amyloid A (SAA). The concentration of SAA in plasma increases up to 1000-fold within 24 to 48 h after trauma, inflammation or infection. Individuals with chronically increased SAA levels may develop AA amyloidosis. SAA has been divided into two groups according to the encoding genes and the source of protein production. These two groups are acute phase SAA (A-SAA) and constitutive SAA (C-SAA). Although the liver is the primary site of the synthesis of A-SAA and C-SAA, extrahepatic production of both SAAs has been observed in animal models and cell culture experiments of several mammalian species and chicken. The functions of A-SAA are thought to involve lipid metabolism, lipid transport, chemotaxis and regulation of the inflammatory process. There is growing evidence that extrahepatic A-SAA formation may play a crucial role in amyloidogenesis and enhances amyloid formation at the site of SAA production.     

Human serum amyloid A (SAA): biosynthesis and postsynthetic processing of preSAA and structural variants defined by complementary DNA

To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence. The SAA-specific cDNA clone (pA1) contains the nucleotide sequence coding for the mature SAA and 10 amino acids of the 18-residue signal peptide. It also includes a 70 nucleotide long 3'-untranslated region and approximately 120 bases of the poly(A) tail. The derived amino acid sequence of pA1 is identical with the alpha form of apoSAA1. A fragment of pA1 containing the conserved (residues 33-38) region of SAA also hybridized with RNA from human acute phase liver and acute phase stimulated, but not unstimulated, mouse and rabbit liver. In contrast, a fragment corresponding to the variable region hybridized to a much greater extent with human than with rabbit or murine RNA. Human acute phase liver SAA mRNA (approximately 600 nucleotides in length) directs synthesis of preSAA (Mr 14 000) in a cell-free translating system. In a Xenopus oocyte translation system preSAA is synthesized and processed to the mature Mr 12 000 product. The complete 18 amino acid signal peptide sequence of preSAA was derived from sequencing cDNA synthesized by "primer extension" from the region of SAA mRNA corresponding to the amino terminus of the mature product. Two other SAA-specific cDNA clones (pA6 and pA10) differed from pA1 in that they lack the internal PstI restriction enzyme site spanning residues 54-56 of pA1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-18 induces serum amyloid A (SAA) protein production from rheumatoid synovial fibroblasts

Interleukin-18 (IL-18) is a novel proinflammatory cytokine that was recently found in synovial fluids and synovial tissues from patients with rheumatoid arthritis (RA). To investigate the role of IL-18 in rheumatoid synovitis, the levels of IL-18 and serum amyloid A (SAA) were measured in synovial fluids from 24 patients with rheumatoid arthritis (RA) and 13 patients with osteoarthritis (OA). The levels of IL-18 and SAA in the synovial fluids were elevated in RA patients. In contrast, the levels of IL-18 in synovial fluids from OA patients were significantly lower compared to those of RA patients. SAA was not detected in synovial fluids from OA patients. The expression of SAA mRNA in rheumatoid synovial cells was also examined. SAA4 mRNA, which was constitutively expressed by rheumatoid synovial cells, was not affected by IL-18 stimulation. Although acute phase SAA (A-SAA, SAA1 + 2) mRNA was not detected in unstimulated synovial cells, its expression was induced by IL-18 stimulation. By immunoblot, we demonstrated that IL-18 induced the SAA protein synthesis from rheumatoid synovial cells in a dose-dependent manner. These results indicate a novel role for IL-18 in rheumatoid inflammation through the synovial SAA production.     

Serum amyloid A-luciferase transgenic mice: response to sepsis, acute arthritis, and contact hypersensitivity and the effects of proteasome inhibition

Acute phase serum amyloid A proteins (A-SAAs) are multifunctional apolipoproteins produced in large amounts during the acute phase of an inflammation and also during the development of chronic inflammatory diseases. In this study we present a Saa1-luc transgenic mouse model in which SAA1 gene expression can be monitored by measuring luciferase activity using a noninvasive imaging system. When challenged with LPS, TNF-alpha, or IL-1beta, in vivo imaging of Saa1-luc mice showed a 1000- to 3000-fold induction of luciferase activity in the hepatic region that peaked 4-7 h after treatment. The induction of liver luciferase expression was consistent with an increase in SAA1 mRNA in the liver and a dramatic elevation of the serum SAA1 concentration. Ex vivo analyses revealed luciferase induction in many tissues, ranging from several-fold (brain) to >5000-fold (liver) after LPS or TNF-alpha treatment. Pretreatment of mice with the proteasome inhibitor bortezomib significantly suppressed LPS-induced SAA1 expression. These results suggested that proteasome inhibition, perhaps through the NF-kappaB signaling pathway, may regulate SAA1 expression. During the development of acute arthritis triggered by intra-articular administration of zymosan, SAA1 expression was induced both locally at the knee joint and systemically in the liver, and the induction was significantly suppressed by bortezomib. Induction of SAA1 expression was also demonstrated during contact hypersensitivity induced by topical application of oxazolone. These results suggest that both local and systemic induction of A-SAA occur during inflammation and may contribute to the pathogenesis of chronic inflammatory diseases associated with amyloid deposition.     

Expression and sequence analyses of serum amyloid A in the Syrian hamster

Reactive amyloidosis occurs during chronic inflammation and involves deposition of amyloid A (AA) fibrils in many organs. Amyloid A is derived by proteolysis from serum amyloid A component (SAA), a major acute-phase reactant in many species. Since spontaneous amyloidosis occurs commonly in Syrian hamsters, we have studied the structure and expression of SAA genes during inflammation in these animals. Two cDNA clones and one genomic clone were sequenced, suggesting that Syrian hamster SAA is encoded by at least two genes. Hepatic mRNA analyses showed that SAA was inducible in many hamster organs during acute inflammation. These studies also demonstrated that SAA mRNA for one isotype is maximally expressed at a site of local tissue damage.     

Alpha 1-antitrypsin and serum albumin mRNA accumulation in normal, acute phase and ZZ human liver

Alpha 1-Antitrypsin and albumin mRNA levels of 4 human livers were assessed using a newly sequenced cDNA clone of the carboxyterminal third of alpha 1-antitrypsin and a previously cloned albumin cDNA sequence. The relative concentration of alpha 1-antitrypsin mRNA was the same in poly(A)-containing RNA isolated from acute phase (MM) and alpha1-antitrypsin deficient (ZZ) individuals. In the acute phase liver relative to the normal (MM) liver, total RNA extracts showed a marked decrease in albumin mRNA concentration but no increase in alpha 1-antitrypsin mRNA. The ZZ liver showed decreased total and poly(A)-containing RNA content but the same proportion of alpha 1-antitrypsin to albumin mRNA as in the normal (MM) liver. This supports other evidence that ZZ alpha 1-antitrypsin deficiency is due to a defect in polypeptide processing (secretion) rather than a deficiency in mRNA accumulation.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.     

Expression of serum amyloid A, in normal, dysplastic, and neoplastic human colonic mucosa: implication for a role in colonic tumorigenesis.

Serum amyloid A (SAA) is an acute phase reactant, whose level in the blood is elevated in response to trauma, infection, inflammation, and neoplasia. Elevated levels of SAA in the serum of cancer patients were suggested to be of liver origin rather than a tumor cell product. The role of SAA in human malignancies has not been elucidated. We investigated the expression of SAA at various stages of human colon carcinoma progression. Nonradioactive in situ hybridization applied on paraffin tissue sections from 26 colon cancer patients revealed barely detected SAA mRNA expression in normal looking colonic epithelium. Expression was increased gradually as epithelial cells progressed through dysplasia to neoplasia. Deeply invading colon carcinoma cells showed the highest levels of SAA. Expression was also found in colon carcinoma metastases. Cells of lymphoid follicles of the intestinal wall, inflammatory cells, ganglion cells, and endothelial cells, also expressed SAA mRNA. Immunohistochemical staining revealed SAA protein expression that colocalized with SAA mRNA expression. RT-PCR analysis confirmed the expression of the SAA1 and SAA4 genes in colon carcinomas, expression that was barely detectable in normal colon tissues. These findings indicate local and differential expression of SAA in human colon cancer tissues and suggest its role in colonic tumorigenesis.     

Mink serum amyloid A protein. Expression and primary structure based on cDNA sequences

The nucleotide sequences of two mink serum amyloid A (SAA) cDNA clones have been analyzed, one (SAA1) 776 base pairs long and the other (SAA2) 552 base pairs long. Significant differences were discovered when derived amino acid sequences were compared with data for apoSAA isolated from high density lipoprotein. Previous studies of mink protein SAA and amyloid protein A (AA) suggest that only one SAA isotype is amyloidogenic. The cDNA clone for SAA2 defines the "amyloid prone" isotype while SAA1 is found only in serum. Mink SAA1 has alanine in position 10, isoleucine in positions 24, 67, and 71, lysine in position 27, and proline in position 105. Residue 10 in mink SAA2 is valine while arginine and asparagine are at positions 24 and 27, respectively, all characteristics of protein AA isolated from mink amyloid fibrils. Mink SAA2 also has valine in position 67, phenylalanine in position 71, and amino acid 105 is serine. It remains unknown why these six amino acid substitutions render SAA2 more amyloidogenic than SAA1. Eighteen hours after lipopolysaccharide stimulation, mink SAA mRNA is abundant in liver with relatively minor accumulations in brain and lung. Genes encoding both SAA isotypes are expressed in all three organs while no SAA mRNA was detectable in amyloid prone organs, including spleen and intestine, indicating that deposition of AA from locally synthesized SAA is unlikely. A third mRNA species (2.2 kilobases) was identified and hybridizes with cDNA probes for mink SAA1 and SAA2. In addition to a major primary translation product (molecular mass 14,400 Da) an additional product with molecular mass 28,000 Da was immunoprecipitable.     

Mouse C-reactive protein. Generation of cDNA clones, structural analysis, and induction of mRNA during inflammation.

A full-length C-reactive protein (CRP) cDNA clone has been isolated from a CBA/J-strain-mouse acute-phase liver library. The 1614-nucleotide cDNA specifies mRNA 5' and 3' untranslated regions of 81 and 858 bases respectively that flank 675 bases encoding mouse pre-CRP. The derived amino acid sequence predicts a 19-residue leader peptide followed by a 206-residue mature mouse CRP that shows considerable sequence identity with both human and rabbit CRP. Northern-blot analysis of mouse liver CRP mRNA concentrations after inflammatory stimuli and comparison with hepatic induction of mRNA for the major mouse acute-phase protein serum amyloid P component established that CRP, a major acute-phase reactant in human and rabbit, is a minor acute-phase reactant in mouse. The size and organization of the mouse CRP mRNA 5' and 3' untranslated regions are significantly different from those of human and rabbit CRP mRNA and may have implications for its anomalous minimal induction during acute inflammation.     

Expression of serum amyloid A in chondrocytes and myoblasts differentiation and inflammation: possible role in cholesterol homeostasis.

Serum amyloid A (SAA) is synthesized by the liver during the acute phase. Local expression of SAA mRNA has been reported also in non-liver cells, a potential local source of SAA protein not related to the systemic acute phase response. SAA function has not been established yet. In the present study, we identified SAA as a protein expressed by chondrocytes and myoblasts in response to inflammatory stimula. In both cell systems, SAA mRNA and protein expression is strongly stimulated by bacterial lipopolysaccharide treatment. SAA mRNA expression is also enhanced during terminal differentiation of cells of the chondrogenic and myogenic lineage; mRNA is barely detectable in prechondrogenic cells and is highly expressed in differentiated hyperthrophic chondrocytes. An increased level of SAA mRNA was also observed in vivo when we compared mRNA extracted from tibiae of 10 day embryos, still fully cartilaginous, with tibiae from 18 day embryos, a stage when the endochondral ossification process has already started. p38 activation, a well-known event of the chondrogenesis signaling cascade, controls expression of SAA in cartilage following inflammatory stimuli. SAA secreted by stimulated chondrocytes is associated with cholesterol. Cholesterol is synthesized by the same chondrocytes and is also increased in inflammatory conditions. A role of SAA in cholesterol homeostasis in chondrocytes is proposed.     

Sequence and acute phase regulation of rat alpha 1-inhibitor III messenger RNA

cDNA clones coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from an acute phase rat liver library. The isolates could be divided into four groups with characteristic BamHI restriction fragment patterns. The identity of the prototype clone pRLA1I3/2J was established by comparison with the published amino acid sequence of the purified protein. It codes for a 1477-amino acid precursor polypeptide with a 24-residue signal peptide. The mature protein shares 58% overall sequence identity with rat alpha 2-macroglobulin and contains a typical internal thiolester sequence. Twenty-two of its twenty-three cysteinyl residues are conserved with alpha 2-macroglobulin implying similar tertiary structure. However, the prototype alpha 1-inhibitor III sequence differed significantly from the rat and human alpha 2-macroglobulin sequences in its bait region suggesting alpha 1-inhibitor III possesses proteinase inhibitory specificities different from those of alpha 2-macroglobulin. The variant alpha 1-inhibitor III clone pRLA1I3/2J from a second cDNA group also differed from the prototype in the bait region coding sequence, although both specify similar signal peptides and NH2 termini. The observation of variant cDNA classes suggests that acute phase rat livers produce a heterogeneous mixture of alpha 1-inhibitor III mRNA molecules. Evidence was obtained for the presence of at least four different alpha 1-inhibitor III-related genes in the rat genome. During the first 24 h of an acute phase response the abundance of hepatic alpha 1-inhibitor III mRNA was decreased 3-4-fold. This decrease was of the same order of magnitude as the reported reduction of the corresponding plasma protein concentration, suggesting that in the early phase of the acute inflammatory response the plasma concentration of this protein is mainly controlled through the abundance of its hepatic mRNA.     

Isolation and characterization of a partial cDNA clone for heparin cofactor II1

A human fetal liver cDNA library constructed in lambda gt11 was screened with affinity-purified rabbit antibodies raised against heparin cofactor II. One positive clone was plaque purified and the cDNA insert was completely sequenced. The clone encodes the C-terminal 167 amino acid residues of heparin cofactor II as well as the entire 3'-untranslated region of the message. Proline and leucine were identified in the P2 and P1 positions of the protease cleavage site, providing a possible explanation for the ability of heparin cofactor II to inhibit both thrombin and chymotrypsin-like proteases. The coding sequence is identical to that of the recently published human leuserpin 2 (Ragg (1986) Nucl. Acids Res. 14, 1073).     

The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin

The complete amino acid sequence of human insulin-like growth factor I (IGF-I), a polypeptide isolated from serum, has been determined. IGF-I is a single chain polypeptide of 70 amino acid residues cross-linked by three disulfide bridges. The calculated molecular weight is 7649. IGF-I displays obvious homology to proinsulin: positions 1 to 29 are homologous to insulin B chain and positions 42 to 62 to insulin A chain. A shortened "connecting" peptide with 12 residues (positions 30 to 41) compared to 30 to 35 in proinsulins shows no homology to proinsulin C peptide. An octapeptide sequence at the COOH-terminal end is also a feature not found in proinsulins. The number of differences in amino acid positions between IGF-I and insulins suggests that duplication of the gene of the common ancestor of proinsulin and IGF occurred before the time of appearance of the vertebrates. Of the 19 residues known to be invariant in all insulins so far sequenced, only glutamine A5 and asparagine A21 are replaced in IGF-I by glutamic acid and alanine, respectively. The fact that all half-cystine and glycine residues and most nonpolar core residues of the insulin monomer are conserved is compatible with a three-dimensional structure of IGF-I similar to that of insulin.