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Amyloid SAA is an apoprotein of mouse plasma high density lipoprotein
Abstract
Mouse plasma contains a protein antigenically related to mouse protein AA, the principal protein derived from tissue deposits of amyloid substance in mice. In this work the plasma antigen, SAA, was found mainly as a high molecular weight form (2 x 10(5)) residing for the most part in the density interval 1.063-1.21 g/cm3 (high density lipoprotein, HDL); the largest amount of SAA, absolute and relative to total protein, was found in the density interval 1.125-12.1 g/cm3 (HDL3). When apoproteins of the mouse HDL obtained by delipidation of the lipoprotein particles were chromatographed in acid/urea, the antigenic activity appeared in the 10,000- to 15,000-dalton portion of the apoprotein complex. In these characteristics mouse SAA closely resembles human SAA and the behavior of the protein related to amyloid protein AA indicates that is one of the apoproteins of the HDL complex in both species. Therefore we suggest that it be named apoSAA.
... Although proteolysis of SAA yields the N-terminal fragment(s) found in the microfibrils in secondary amyloid deposits the responsible protease(s) has(ve) not yet been unequivocally identified. SAA is poorly soluble in aqueous solutions; in blood it is partitioned into high density lipoproteins (HDL (Benditt and Eriksen 1977;Benditt et al. 1979)). It functions as an apolipoprotein in this regard, possibly changing the character of the HDL particles (see below and (Benditt et al. 1979;Hoffman and Benditt 1982;Cabana et al. 1989;Kisilevsky and Subrahmanyan 1992)). ...
... SAA is poorly soluble in aqueous solutions; in blood it is partitioned into high density lipoproteins (HDL (Benditt and Eriksen 1977;Benditt et al. 1979)). It functions as an apolipoprotein in this regard, possibly changing the character of the HDL particles (see below and (Benditt et al. 1979;Hoffman and Benditt 1982;Cabana et al. 1989;Kisilevsky and Subrahmanyan 1992)). Aside from its apolipoprotein character, no function was initially identified for SAA although its APR association implied that it might be related to primordial host defense or inflammation, similar to the situation for CRP. ...
... Early studies showed SAA association with lipids, particularly HDL (Benditt and Eriksen 1977;Benditt et al. 1979). Predicted amphipathic features for SAA monomers implicated regions for lipid binding (Meeker and Sack 1998;Lu et al. 2014;Frame and Gursky 2016). ...
Serum amyloid A (SAA) proteins were isolated and named over 50 years ago. They are small (104 amino acids) and have a striking relationship to the acute phase response with serum levels rising as much as 1000-fold in 24 hours. SAA proteins are encoded in a family of closely-related genes and have been remarkably conserved throughout vertebrate evolution. Amino-terminal fragments of SAA can form highly organized, insoluble fibrils that accumulate in "secondary" amyloid disease. Despite their evolutionary preservation and dynamic synthesis pattern SAA proteins have lacked well-defined physiologic roles. However, considering an array of many, often unrelated, reports now permits a more coordinated perspective. Protein studies have elucidated basic SAA structure and fibril formation. Appreciating SAA's lipophilicity helps relate it to lipid transport and metabolism as well as atherosclerosis. SAA's function as a cytokine-like protein has become recognized in cell-cell communication as well as feedback in inflammatory, immunologic, neoplastic and protective pathways. SAA likely has a critical role in control and possibly propagation of the primordial acute phase response. Appreciating the many cellular and molecular interactions for SAA suggests possibilities for improved understanding of pathophysiology as well as treatment and disease prevention.
... Several SAA subtypes are present across diverse animal species (22), including invertebrates (23), suggesting important conserved functions. Since SAA is poorly soluble in aqueous solutions, it circulates associated with lipoproteins, in particular high density lipoprotein (HDL), and is considered an apolipoprotein (24,25). Functions of particular SAA subtypes include roles in host defense (26)(27)(28)(29)(30), chemoattraction (31)(32)(33)(34), lipid metabolism (35)(36)(37), and inflammation (38). ...
... HDL-bound SAA also may play a role in atherogenesis. When SAA is secreted by the liver as part of the acute or chronic inflammatory response, it circulates in plasma bound to HDL, although it can associate with less dense lipoproteins under certain circumstances (24,25,37,143). HDL particles that carry SAA, so-called "inflammatory HDL", is less atheroprotective than normal HDL, with reduced inhibition of inflammation in cells due to its being trapped by cell surface proteoglycans (191), versican in the case of adipocytes and biglycan produced by macrophages (118). Trapping of SAA-containing HDL at the cell surface prevents it from adequately promoting reverse cholesterol transport (192). ...
Serum amyloid A (SAA) subtypes 1–3 are well-described acute phase reactants that are elevated in acute inflammatory conditions such as infection, tissue injury, and trauma, while SAA4 is constitutively expressed. SAA subtypes also have been implicated as playing roles in chronic metabolic diseases including obesity, diabetes, and cardiovascular disease, and possibly in autoimmune diseases such as systemic lupus erythematosis, rheumatoid arthritis, and inflammatory bowel disease. Distinctions between the expression kinetics of SAA in acute inflammatory responses and chronic disease states suggest the potential for differentiating SAA functions. Although circulating SAA levels can rise up to 1,000-fold during an acute inflammatory event, elevations are more modest (∼5-fold) in chronic metabolic conditions. The majority of acute-phase SAA derives from the liver, while in chronic inflammatory conditions SAA also derives from adipose tissue, the intestine, and elsewhere. In this review, roles for SAA subtypes in chronic metabolic disease states are contrasted to current knowledge about acute phase SAA. Investigations show distinct differences between SAA expression and function in human and animal models of metabolic disease, as well as sexual dimorphism of SAA subtype responses.
... On acute infection, SAA proteins are produced by hepatocytes and other cells and are secreted into the circulation. Although the majority of circulating SAAs are associated with HDL (13,21), a fraction of SAAs circulate freely in the mouse bloodstream after acute infection (22). ...
... We then fractionated serum from Salmonella typhimurium-infected wild-type and Saa −/− mice by size-exclusion chromatography and analyzed the fractions for SAA content ( Fig. 1 A and B). In the infected wild-type mice, the majority of serum SAA was associated with the HDL peak, as expected ( Fig. 1B) (21,22). However, SAAs also eluted beyond the HDL peak, suggesting that a fraction of SAA circulates freely and is not associated with HDL. ...
Serum amyloid A (SAA) proteins are strongly induced in the liver by systemic infection and in the intestine by bacterial colonization. In infected mice, SAA proteins circulate in association with the vitamin A derivative retinol, suggesting that SAAs transport retinol during infection. Here we illuminate a structural basis for the retinol–SAA interaction. In the bloodstream of infected mice, most SAA is complexed with high-density lipoprotein (HDL). However, we found that the majority of the circulating retinol was associated with the small fraction of SAA proteins that circulate without binding to HDL, thus identifying free SAA as the predominant retinol-binding form in vivo. We then determined the crystal structure of retinol-bound mouse SAA3 at a resolution of 2.2 Å. Retinol-bound SAA3 formed a novel asymmetric trimeric assembly that was generated by the hydrophobic packing of the conserved amphipathic helices α1 and α3. This hydrophobic packing created a retinol-binding pocket in the center of the trimer, which was confirmed by mutagenesis studies. Together, these findings illuminate the molecular basis for retinol transport by SAA proteins during infection.
... SAA contains an N-terminal α-helical domain (amino acid 1-28) capable of binding highdensity lipoproteins (HDL) [25,26], the smallest lipoproteins that carry cholesterol, triglycerides, and phospholipids within the water-based blood stream. The capture of SAA by HDL results in the displacement of apolipoproteins (Apo-AI) and formation of larger HDL particles (up to 200 kDa) [27,28]. At physiologically relevant concentrations (>100 μg/ml), HDL almost completely blocks the chemoattractant activities of SAA [20], suggesting HDL as a natural inhibitor of SAA in the circulation. ...
... It has been shown that HDL, at physiologically relevant concentrations (>100 μg/ml), can capture SAA [27,28] and attenuate its chemoattractant activities [20]. To test whether HDL similarly affects SAA-induced sPLA 2 secretion, macrophages were stimulated with SAA in the presence of HDL at various concentrations. ...
Human serum amyloid A (SAA) has been demonstrated as a chemoattractant and proinflammatory mediator of lethal systemic inflammatory diseases. In the circulation, it can be sequestered by a high-density lipoprotein, HDL, which carries cholesterol, triglycerides, phospholipids and apolipoproteins (Apo-AI). The capture of SAA by HDL results in the displacement of Apo-AI, and the consequent inhibition of SAA’s chemoattractant activities. It was previously unknown whether HDL similarly inhibits SAA-induced sPLA2 expression, as well as the resultant HMGB1 release, nitric oxide (NO) production and autophagy activation. Here we provided compelling evidence that human SAA effectively upregulated the expression and secretion of both sPLA2-IIE and sPLA2-V in murine macrophages, which were attenuated by HDL in a dose-dependent fashion. Similarly, HDL dose-dependently suppressed SAA-induced HMGB1 release, NO production, and autophagy activation. In both RAW 264.7 cells and primary macrophages, HDL inhibited SAA-induced secretion of several cytokines (e.g., IL-6) and chemokines (e.g., MCP-1 and RANTES) that were likely dependent on functional TLR4 signaling. Collectively, these findings suggest that HDL counter-regulates SAA-induced upregulation and secretion of sPLA2-IIE/V in addition to other TLR4-dependent cytokines and chemokines in macrophage cultures.
... To assess inflammatory activity, expression of the genes for two wellknown diagnostic markers of IBD, S100a9 (calprotectin) and serum amyloid A3 (Saa3), in the colon was determined (22,23). The mRNA levels of both S100a9 and Saa3 were found to be upregulated after treatment with DSS in mice fed the standard diet (1,350.2-fold ...
... The presence of active gut inflammation in IBD is associated with an acute-phase reaction and migration of leukocytes to the gut, which is translated into enhanced expression of acute-phase proteins like Saa3 (23). The level of Saa3 mRNA was significantly increased in both DSS-treated groups but was greatly reduced in animals fed the bacterial meal. ...
Dietary inclusion of a bacterial meal has recently been shown to efficiently abolish soybean meal-induced enteritis in Atlantic
salmon. The objective of this study was to investigate whether inclusion of this bacterial meal in the diet could abrogate
disease development in a murine model of epithelial injury and colitis and thus possibly have therapeutic potential in human
inflammatory bowel disease. C57BL/6N mice were fed ad libitum a control diet or an experimental diet containing 254 g/kg of body weight BioProtein, a bacterial meal consisting of Methylococcus capsulatus (Bath), together with the heterogenic bacteria Ralstonia sp., Brevibacillus agri, and Aneurinibacillus sp. At day 8, colitis was induced by 3.5% dextran sulfate sodium (DSS) ad libitum in the drinking water for 6 days. Symptoms of DSS treatment were less profound after prophylactic treatment with the diet
containing the BioProtein. Colitis-associated parameters such as reduced body weight, colon shortening, and epithelial damage
also showed significant improvement. Levels of acute-phase reactants, proteins whose plasma concentrations increase in response
to inflammation, and neutrophil infiltration were reduced. On the other, increased epithelial cell proliferation and enhanced
mucin 2 (Muc2) transcription indicated improved integrity of the colonic epithelial layer. BioProtein mainly consists of Methylococcus capsulatus (Bath) (88%). The results that we obtained when using a bacterial meal consisting of M. capsulatus (Bath) were similar to those obtained when using BioProtein in the DSS model. Our results show that a bacterial meal of the
noncommensal bacterium M. capsulatus (Bath) has the potential to attenuate DSS-induced colitis in mice by enhancing colonic barrier function, as judged by increased
epithelial proliferation and increased Muc2 transcription.
... One such APP known to be synthesized primarily by the liver is serum amyloid A (SAA). SAA belongs to a family of evolutionary conserved proteins which is involved in inflammation (3,4). The SAA isoforms are expressed at different levels in response to inflammatory stimuli. ...
It has been established that the acute phase protein, Serum amyloid A (SAA), which is usually synthesized by the liver, is also synthesized by cancer cells and cancer-associated cells in the tumor microenvironment. SAA also activates modulators of autophagy, such as the PI3K/Akt and MAPK signaling pathways. However, the role of SAA in autophagy in breast cancer still remains to be elucidated. The aim of this study was to investigate the role of SAA in the regulation of signaling pathways and autophagy in in vitro and in vivo models of breast cancer. The MDA-MB-231 and MCF7 cell lines were transiently transfected to overexpress SAA1. A tumor-bearing SAA1/2 knockout mouse model was also utilized in this study. SAA1 overexpression activated ERK signaling in the MDA-MB-231 cells, downregulated the PI3K pathway protein, PKB/Akt, in the MCF7 cell line, while SAA1/2 knockout also inhibited Akt. Furthermore, SAA1 overexpression in vitro downregulated autophagy, while the expression of SQSTM1/p62 was increased in the MCF7 cells, and SAA1/2 knockout induced autophagy in vivo. SAA overexpression in the MDA-MB-231 and MCF7 cells resulted in an increase in cell viability and increased the expression of the proliferation marker, MCM2, in the MCF7 cells. Furthermore, knockout of SAA1/2 resulted in an altered inflammatory profile, evident in the decrease of plasma IL-1β, IL-6 and IL-10, while increasing the plasma levels of MCP-1 and TNF-α. Lastly, SAA1/2 knockout promoted resistance to apoptosis and necrosis through the regulation of autophagy. SAA thus regulates autophagy in breast cancer cells to promote tumorigenesis.
... Early analysis of human serum revealed the majority of SAA to float at the same density as HDL 3 [79]. In line with this, murine SAA showed 75 % reactivity within high-density moieties following density gradient centrifugation [80]. As such, HDL particles are the prime carrier of SAA during the APR. ...
... SAA levels range between 1 to >3,000 mg/L, with higher levels occurring when production is upregulated to potentially a 1000-fold during acute inflammation [34,35]. SAA is synthesised by hepatocytes under regulatory control of proinflammatory cytokines such as tumour necrosis factor (TNF), interleukin 1 (IL-1), interleukin 6 (IL-6) and transcription factors such as SAA activating factor and lipopolysaccharide [36][37][38]. ...
BACKGROUND: Amyloidosis is a disorder arising from the variable physiological effects of dysregulated, extracellular protein deposition. There are >30 different subtypes, all possessing the same histological characteristics and the two major organs affected are the kidneys and the heart. AIMS AND HYPOTHESIS: To evaluate current UK histological practices leading to a misdiagnosis of amyloidosis; To establish proteomics as a new diagnostic technique for identifying amyloid, in the UK; To investigate the usefulness of a relatively new biomarker i.e. Retinol Binding Protein (RBP), across amyloid subtypes and correlate values with biopsy findings, which has not previously been done; To identify the cause of death in patients with Stage III/ IV cardiac amyloidosis using, for the first time, Implantable Loop Recorders; To present the first comprehensive review of Light Chain Deposition Disease highlighting the relationship between haematological response and overall prognosis. RESULTS: In 65% of cases where renal amyloidosis was misdiagnosed as minimal change disease, Congo red staining was not undertaken and in 35% of cases neither Congo red staining, with cross-polarised light visualisation, nor electron microscopy was undertaken. Proteomics has now been established as a specific and sensitive technique by which to diagnose amyloid and the subtype, demonstrated by distinguishing Fibrinogen Aα-Chain (AFib) renal biopsies from other subtypes. Urinary RBP/Creatinine (RCR) correlated with the: degree of tubular atrophy, number of light chains, eGFR, presence of glycosuria and degree of tubular phosphate reabsorption. RCR values were especially high in AFib and AA amyloidosis. Pulseless Electrical Activity was identified as the terminal rhythm in patients with Stage III/IV cardiac amyloidosis and this was preceded by a high degree AV block. Deep clonal responses to chemotherapy are associated with improved renal and overall outcomes in LCDD and should be pursued even in advanced chronic kidney disease.
... A major player in this process is serum amyloid A (SAA, ~12 kDa), an ancient acute-phase response protein whose plasma levels increase transiently up to 1,000-fold and reach 1 mg/ml in inflammation and injury, making SAA a major plasma protein (13,14). SAA circulates mainly on HDL surface where it can displace other HDL-associated proteins, such as PON1 and other lipases, as well as a fraction of apoA-I (15). ...
Oxidative stress and inflammation, which involves a dramatic increase in serum amyloid A (SAA) levels, are critical in the development of atherosclerosis. Most SAA circulates on plasma HDL particles, altering their cardioprotective properties. SAA-enriched HDL have diminished anti-oxidant effects on LDL, which may contribute to atherogenesis. We determined combined effects of SAA enrichment and oxidation on biochemical changes in HDL. Normal human HDL were incubated with SAA, oxidized by various factors (Cu2+, myeloperoxidase, H2O2, OCl-), and analyzed for lipid and protein modifications and biophysical remodeling. Three novel findings are reported: addition of SAA reduces oxidation of HDL and LDL lipids; oxidation of SAA-containing HDL in the presence of OCl- generates an SAA-apoA-I heterodimer that resists the release from HDL; and mild oxidation promotes spontaneous release of proteins (SAA and apoA-I) from SAA-enriched HDL. We show that the anti-oxidant effects of SAA extend to various oxidants and are mediated mainly by the unbound protein. We propose that free SAA sequesters lipid hydroperoxides and delays lipoprotein oxidation, though much less efficiently than other anti-oxidant proteins such as apoA-I that SAA displaces from HDL. These findings prompt us to reconsider the role of SAA in lipid oxidation in vivo.
... B . Pepys, manuscript in preparation) involved absorption of a fixed dose of rabbit anti-AA antibodies (produced by immunization with acute-phase mouse HDL [16]) by the tissue AA . Residual anti-AA activity was then measured in a microtiter plate solid-phase system with degraded AA amyloid (17) coated on the wells and ' 2 'I-labeled immunopurified goat anti-rabbit IgG antibody for detection of bound anti-AA . ...
Highly specific, high-resolution scintigraphic images of amyloid-laden organs in mice with experimentally induced amyloid A protein (AA) amyloidosis were obtained after intravenous injection of 123I-labeled serum amyloid P component (SAP). Interestingly, a much higher proportion (up to 40%) of the injected dose of heterologous human SAP localized to amyloid and was retained there than was the case with isologous mouse SAP, indicating that human SAP binds more avidly to mouse AA fibrils than does mouse SAP. Specificity of SAP localization was established by the failure of the related proteins, human C-reactive protein and Limulus C-reactive protein, to deposit significantly in amyloid and by the absence of human SAP deposition in nonamyloidotic organs. However, only partial correlations were observed between the quantity of SAP localized and two independent estimates, histology and RIA for AA of the amount of amyloid in particular organs. It is not clear which of the three methods used reflects better the extent or clinical significance of the amyloid deposits but in vivo localization of radiolabeled SAP, detectable and quantifiable by gamma camera imaging, is apparently extremely sensitive. These findings establish the use of labeled SAP as a noninvasive in vivo diagnostic probe in experimental amyloidosis, potentially capable of revealing the natural history of the condition, and suggest that it may also be applicable generally as a specific targeting agent for diagnostic and even therapeutic purposes in clinical amyloidosis.
... Serum amyloid A (SAA) is a very important protein that is markedly up-regulated in acute phase inflammatory responses. Four SAA isoforms have been identified at the protein level in the mouse, and were shown to be approximately 10 kDa in size after the signal peptide was removed [1][2][3][4]. Mouse SAA1 and SAA2 are well-known to be expressed in hepatocytes, whereas mSAA3 is expressed extrahepatically in cells including adipocytes, epithelial cells, and endothelial cells [5][6][7]. While these three isoforms are inducible isoforms, mSAA4 is constitutively expressed in hepatocytes. ...
Serum amyloid A3 (SAA3) possesses characteristics distinct from the other serum amyloid A isoforms, SAA1, SAA2, and SAA4. High density lipoprotein contains the latter three isoforms, but not SAA3. The expression of mouse SAA3 (mSAA3) is known to be up-regulated extrahepatically in inflammatory responses, and acts as an endogenous ligand for the toll-like receptor 4/MD-2 complex. We previously reported that mSAA3 plays an important role in facilitating tumor metastasis by attracting circulating tumor cells and enhancing hyperpermeability in the lungs. On the other hand, human SAA3 (hSAA3) has long been regarded as a pseudogene, which is in contrast to the abundant expression levels of the other isoforms. Although the nucleotide sequence of hSAA3 is very similar to that of the other SAAs, a single oligonucleotide insertion in exon 2 causes a frame-shift to generate a unique amino acid sequence. In the present study, we identified that hSAA3 was transcribed in the hSAA2-SAA3 fusion transcripts of several human cell lines. In the fusion transcript, hSAA2 exon 3 was connected to hSAA3 exon 1 or hSAA3 exon 2, located approximately 130kb downstream from hSAA2 exon 3 in the genome, which suggested that it is produced by alternative splicing. Furthermore, we succeeded in detecting and isolating hSAA3 protein for the first time by an immunoprecipitation-enzyme linked immune assay system using monoclonal and polyclonal antibodies that recognize the hSAA3 unique amino acid sequence. We also demonstrated that hSAA3 bound oxidized low density lipoprotein receptor (oxLDL receptor, LOX-1) and elevated the phosphorylation of ERK, the intracellular MAP-kinase signaling protein.
... There are also indications that SAA influences HDL function. In mice, approximately 90% of SAA in plasma is found in the HDL fraction 142 . SAA is associated with HDL also in humans 143 . ...
... Similarly, in transgenic mice, plasma hSAA levels were strongly correlated with total fat mass in the HF fed animals. Furthermore, in humans and mice, A-SAA in the circulation is associated with HDL 66,67 . To test if this was true also in the transgenic animals, pooled plasma samples were subjected to fast protein liquid chromatography (FPLC) separation. ...
... For example, it has been documented that a constellation of systemic reactions termed Acute Phase Response (APR) is induced in metabolic disorders of type 2 diabetes and related to obesity. APR is a systemic response to inflammatory processes accompanied by increased circulation of acute phase proteins such as Ceruloplasmin, C-reactive protein (CRP), and serum amyloid A protein (SAA) [28,29]. Although the process remains unclear, a number of theories have been proposed to explain the underlying mechanisms involved in the activation of APR, including insulin resistance, obesity, and other diabetic complications [30,31]. ...
... rheumatoid arthritis, familial Mediterranean fever, and tuberculosis [1], with renal failure as main clinical outcome. The main amyloid constituent in this form of amyloid disease is N-terminal fragments [2,3] of the acute phase reactant, serum amyloid A (SAA) [4]. SAA is produced by hepatocytes in response to inflammatory cytokines (TNF-a, IL-1 and IL-6) and circulates in plasma associated with high-density lipoproteins (HDL) [5,6]. ...
AA amyloidosis is a systemic disease that develops secondary to chronic inflammatory diseases Macrophages are often found in the vicinity of amyloid deposits and considered to play a role in both formation and degradation of amyloid fibrils. In spleen reside at least three types of macrophages, red pulp macrophages (RPM), marginal zone macrophages (MZM), metallophilic marginal zone macrophages (MMZM). MMZM and MZM are located in the marginal zone and express a unique collection of scavenger receptors that are involved in the uptake of blood-born particles. The murine AA amyloid model that resembles the human form of the disease has been used to study amyloid effects on different macrophage populations. Amyloid was induced by intravenous injection of amyloid enhancing factor and subcutaneous injections of silver nitrate and macrophages were identified with specific antibodies. We show that MZMs are highly sensitive to amyloid and decrease in number progressively with increasing amyloid load. Total area of MMZMs is unaffected by amyloid but cells are activated and migrate into the white pulp. In a group of mice spleen macrophages were depleted by an intravenous injection of clodronate filled liposomes. Subsequent injections of AEF and silver nitrate showed a sustained amyloid development. RPMs that constitute the majority of macrophages in spleen, appear insensitive to amyloid and do not participate in amyloid formation.
... 3Rs | disease model A myloid A (AA) amyloidosis is a rare but important complication of otherwise treatable chronic inflammatory conditions (1) in which the amyloid deposits are derived from serum amyloid A protein (SAA) (2), an apolipoprotein of HDL. SAA is a nonspecific acute phase protein, with a dynamic range of 1 to >3,000 mg/L in humans, synthesized by hepatocytes under the control of proinflammatory cytokines (3). The deposition of AAtype amyloid in the viscera, connective tissue, and blood vessel walls damages tissue structure and function, with the kidney as the major target organ (4). ...
Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid.
... Soon after its discovery, SAA was shown to be an acutephase protein produced by the liver within hours of tissue injury regardless of cause. Its plasma concentration can increase a 1000-fold within 24 h [18,19]. In plasma, SAA is associated with HDL [20,21] and, during severe inflammation, can contribute 80% of its apo-protein composition [22]. ...
Background
The development of osteoporosis is associated with several risk factors, such as genetic polymorphisms and enviromental factors. This study assessed the correlation between SAA1 gene rs12218 polymorphism and HDL-C lelvels and osteoporosis in a population of Chinese women.
Methods
A total of 387 postmenopausal female patients who were diagnosed with osteoporosis (case group) based on bone mineral density measurements via dual-energy x-ray absorptiometry and 307 females with no osteoporosis (control group) were included in this study. Correlations between SAA1 gene rs12218 polymorphism and osteoporosis and HDL-C level were investigated through the identification of SAA1 gene rs12218 polymorphism genotypes using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
Results
The TT genotype of rs12218 was more frequently in osteoporosis patients than in control subjects (P <0.001). And the rs12218 was found to be associated with plasma TG, HDL-C, LDL-C, and BMD levels in osteoporosis patients (P<0.05).
Conclusions
The present results indicate that both osteoporosis and lipids levels are associated with the TT genotype of rs12218 in the human SAA1 gene.
Expression of mouse serum amyloid A (SAA1, -2, and -3) mRNAs can be induced up to 1,000-fold in the liver in response to acute inflammation. This large increase is primarily the result of a 200-fold increase in the rates of SAA gene transcription. To analyze the cis-acting regulatory element(s) responsible for regulating transcription, we fused 306 base pairs of the mouse SAA3 promoter to a reporter gene, the chloramphenicol acetyltransferase (CAT) gene, and transfected this chimeric DNA into cultured cells. In transient expression assays, this 5' sequence was sufficient to confer cell-specific expression: CAT activity was readily detectable when the construct was transfected into liver-derived cells but was not detectable in nonliver cells. Furthermore, when liver cells transfected with this construct were treated with conditioned media prepared from activated mixed lymphocyte cultures or with recombinant interleukin-1, a 10- to 15-fold increase in CAT activity was detected. Deletion analyses showed two regions of interest: a proximal region that enhanced CAT expression in a cell-specific manner and a distal region that conferred responsiveness to both conditioned media and recombinant interleukin-1. This distal responsive element had properties of an inducible transcriptional enhancer, and deletion of the proximal cell-specific region rendered the distal element responsive to stimulation by conditioned media in nonliver cells.
Amyloid-A mediated (AA) amyloidosis is the pathogenic byproduct of body’s prolonged exposure to inflammatory conditions. It is described by the aggregation of mutated/misfolded serum amyloid A1 (SAA1) protein in various tissues and organs. Genetic polymorphism G90D is suspected to cause AA amyloidosis, although the causal mechanism remains cryptic. Recent experimental findings insinuate that heparan sulphate (HS), a glycosaminoglycans, exhibits binding with SAA1 to promote its aggregation. To foster the enhanced binding of HS, we computationally determined the pernicious modifications in G90D mutant SAA1 protein. Also, we examined the influence of HS on the dynamic conformation of mutant SAA1 that could potentially succor amyloidosis. Accordingly, the protein-ligand binding studies indicate that upon SNP G90D, SAA1 protein exhibited an augmented association with HS. Further, the simulation of HS bound mutant SAA1 complex delineates an increase in RMSD, Rg, and RMSF. Also, both RMSD and Rg evinced a fluctuating trajectory. Further, the complex showed increase of beta turn in its secondary structural composition. Additionally, the free energy landscape of mutant SAA1-HS complex posits the occurrence of multiple global minima conformers as opposed to the presence of a single global energy minima conformation in native SAA1 protein. In conclusion, the aforementioned conformational ramifications induced by HS on SAA1 could potentially be the proteopathic incendiary behind AA amyloidosis; this incendiary will need to be considered in future studies for developing effective therapeutics against AA amyloidosis.
Communicated by Ramaswamy H. Sarma
Background
Serum amyloid A protein (SAA) is known as an inflammatory factor and an apolipoprotein that can replace apolipoprotein A-I/II components as the major apolipoprotein of high-density lipoprotein (HDL), which is related to atherosclerosis. The present study is aimed to evaluate whether the SAA gene polymorphism is involved in ischemic stroke in northern Chinese Han population.
Methods
In a case-control study, the participants included 396 patients (239 males, 157 females) with ischemic stroke and 360 healthy subjects (211 males, 149 females). The rs12218 polymorphism of the SAA gene was analyzed by polymerase chain reaction and restriction fragment length polymorphism, while the rs2468844 polymorphism of the SAA gene was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Results
The frequencies of the CC genotype and the C allele of rs12218 were higher in participants with ischemic stroke than in the control group (P = 0.020 in males, P = 0.001 in large-artery atherosclerosis group, LAA). The frequencies of the AG genotype and the G allele of rs2468844 were higher in participants with ischemic stroke than in the control group (P = 0.040 in males, P = 0.011 in large-artery atherosclerosis group). Multiple logistic regression analysis revealed the significance of the rs12218 in males and in large-artery atherosclerosis group after adjustment for confounding factors.
Conclusion
The rs12218 polymorphism of the SAA gene was associated with ischemic stroke in males and in patients with large-artery atherosclerosis group in northern Chinese Han population.
Accumulating evidence suggests that O3 exposure may contribute to CNS dysfunction. Here, we posit that inflammatory and acute-phase proteins in the circulation increase after O3 exposure and systemically convey signals of O3 exposure to the CNS. To model acute O3 exposure, female Balb/c mice were exposed to 3 ppm O3 or forced air for 2 h and were studied after 6 or 24 h. Of 23 cytokines and chemokines, only KC/CXCL1 was increased in blood 6 h after O3 exposure. The acute-phase protein serum amyloid A (A-SAA) was significantly increased by 24 h, whereas C-reactive protein was unchanged. A-SAA in blood correlated with total leukocytes, Mϕs, and neutrophils in bronchoalveolar lavage from O3-exposed mice. A-SAA mRNA and protein were increased in the liver. We found that both isoforms of A-SAA completely crossed the intact blood-brain barrier, although the rate of SAA2.1 influx was approximately 5 times faster than that of SAA1.1. Finally, A-SAA protein, but not mRNA, was increased in the CNS 24 h post-O3 exposure. Our findings suggest that A-SAA is functionally linked to pulmonary inflammation in our O3 exposure model and that A-SAA could be an important systemic signal of O3 exposure to the CNS.-Erickson, M. A., Jude, J., Zhao, H., Rhea, E. M., Salameh, T. S., Jester, W., Pu, S., Harrowitz, J., Nguyen, N., Banks, W. A., Panettieri Jr., R. A., Jordan-Sciutto, K. L. Serum amyloid A: an ozone-induced circulating factor with potentially important functions in the lung-brain axis.
Reactive systemic amyloidosis is a serious clinical disorder characterized by the extracellular accumulation of fibrils of protein AA (1,2). Recent studies of experimentally induced systemic amyloidosis in mice have shown that despite equal expression of the major murine gene products under experimental conditions, only one isotype is deposited as AA fibrils in vivo (3,4). This may also be the situation in the genetic amyloidosis associated with familial Mediterranean fever (FMF) in humans (5). Important questions thus arise as to how the chemistry and metabolism of different AA proteins might be related to particular differences in their sequences.
Amyloidosis can be induced in mice by casein, endotoxin, or other substances. Results of biochemical studies on the amyloid protein in experimentally induced amyloidosis (1–2) are similar to the findings of secondary amyloidosis in humans. There have also been numerous reports on spontaneous amyloidosis in mice (3–6). However, information about the biochemical features of amyloid proteins in spontaneous amyloidosis has been sparse until recently.
A new amyloid fibril protein was isolated from the livers of Senescence Accelerated Mice (SAM-P), a strain characterized by accelerated senescence and a high incidence of age-associated systemic amyloidosis. This 5,200 dalton amyloid protein “ASSAM” differs in amino acid composition from the amyloid protein of murine secondary amyloidosis. Sequence analysis revealed a blocked N-terminus. Double immunodiffusion tests showed no relationship between ASSAM and murine protein AA or immunoglobulin components.
Sera obtained from normal mice contained a substance that reacted with anti-ASSAM antiserum. This physiological substance, termed “SASSAM” (Serum ASSAM-related antigenic substance) migrated to the albumin/prealbumin region and stained positively with both protein and lipid stains. Fractionation of lipoprotein from normal mouse serum disclosed that SASSAM was present mainly in high density lipoprotein (HDL). ASSAM immunoreactivity appeared in the low molecular weight proteins of apo HDL separated through Sephadex G-200. Purified apoprotein of SASSAM with crossreactivity to anti-ASSAM antiserum was obtained with ion-exchange chromatography of low molecular weight proteins of apo HDL. This apoprotein termed “apo SASSAM” has about the same molecular weight (5,200) and amino acid composition as ASSAM. Electrophoretic analysis revealed that apo SASSAM corresponds presumably to apo A-II, a major apolipoprotein of HDL.
We investigated the effects of colchicine on the acute phase serum amyloid A protein (SAA) response and splenic amyloid A protein (AA) deposition in CBA/J mice undergoing chronic inflammatory stimulation with silver nitrate (AgNO3), and on accelerated amyloid deposition induced by amyloid-enhancing factor (AEF). Colchicine (10 μg daily) significantly lowered splenic AA levels after 25 days of inflammation, as determined by radioimmunoassay. Pretreatment (3 days) with colchicine decreased SAA levels 24 h after AgNO3. It was (unexpectedly) observed that brief pretreatment (12 h) with colchicine augmented the acute phase SAA response to AgNO3 at 24 h. Colchicine stimulated production of both the SAA inducer and lymphocyte-activating factor (LAF) activities of interleukin 1 (IL 1) by macrophages. Decreased SAA levels did not appear to be the mechanism by which colchicine inhibited amyloidosis, since SAA levels fell both in colchicine-treated and control mice after 25 days of inflammation. Colchicine only partially lowered AA deposition after injection of AEF. This effect could be explained by decreased acute phase SAA levels. It is postulated that colchicine inhibits amyloidosis in the pre-deposition period by altering the production of factors (e.g., AEF) required in the deposition phase.
Serum amyloid A protein (SAA) was isolated from mink, horse, and human serum by ultracentrifugation and gel filtration and characterized by two-dimensional gel electrophoresis, Western blotting followed by autoradiography and N-terminal amino acid analysis. SAA was found in similar quantities in the high density lipoprotein (HDL) fraction of serum from a patient suffering from systemic juvenile rheumatoid arthritis (JRA) and mink stimulated with lipopolysaccharide (LPS), and in somewhat smaller quantities in serum from horses stimulated with Escherichia coli cultures. Only very small quantities were present in normal human controls and not detectable in normal mink and horse. Striking similarities were found between human and mink SAA with respect to molecular weight, isoelectric point and degree of heterogeneity, while the molecular weight of horse SAA seemed to be somewhat lower, and no obvious heterogeneity could be demonstrated in this protein using two-dimensional gel electrophoresis. Immunologic cross-reactivity between SAA from the three species was not found. In contrast to human and horse HDL, mink HDL was found not to contain apoA-II and only minute amounts of apoC proteins. Normal horse HDL also contained additional apoproteins not present in HDL from the other species. N-terminal amino acids analysis of SAA from mink and horse demonstrated the same similarity with the corresponding AA protein as previously reported for human SAA/AA.
We developed a recombinant DNA system to overexpress a fusion protein between the small, minimally soluble acute phase serum protein, serum amyloid A (SAA), and the bacterial enzyme staphylococcal nuclease (SN), This fusion protein is very soluble and is immunoreactive to polyclonal anti-SAA antibodies, Tryptophan fluorescence shows smooth denaturation curves for the fusion protein in guanidinium HCl or potassium thiocyanate, Fluorescence also indicates that only a single tryptophan residue (of the four present) is accessible to iodide quenching and, presumably, is exposed on the surface of the fusion protein, Circular dichroism (CD) shows a significant signal indicating a-helix, which can be attributed to the SAA portion of the molecule; these are the first CD spectral data available for SAA. pH titration shows persistence of helix domains for the fusion protein at pH 3.0, in contrast to the denaturation of SN under the same conditions, (The entire fusion protein shows a random coil pattern below pH 3.0.) By exploiting the structural and solubility properties of SN, this fusion protein has provided the first structural data about SAA-the precursor of the amyloid deposits in secondary amyloidosis, This fusion protein should be useful for further physical and physiologic studies of SAA. (C) 1998 Wiley-Liss, Inc.
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.
Amyloidosis was probably first described by Nicolaus Fontanus, however, he certainly did not use this term. In his book edited in 1639 and entitled “Responsinum et curationum medicinalium liber unus” he presented an autopsy record of a young man who had jaundice, nasal bleeding and protrusion of the belly due to ascites. Macroscopic study of the organs revealed hepatomegaly — with liver abscess and splenomegaly — with “white stones” in the spleen. This phenomenon termed “sago spleen” is still used.
The changes in plasma protein levels that constitute the acute phase response are the result of selectively altered hepatic protein synthesis. Macrophage-derived interleukin-1 (IL-1) affects gene expression, possibly through a dual signal system, decreasing mRNA for negative acute phase reactants like albumin and increasing mRNA for positive acute phase reactants (1). In the mouse, mRNA for serum amyloid A increases up to 500 fold to constitute up to 2.5% of total hepatic protein synthesis (2).
The acute phase response is a multi-system adaptation induced by autologous cell death or by products of exogenous invasion (Kushner 1982). These influence macrophages to secrete various monokines that, among other functions, affect hepatic gene expression for many proteins (Ramadori et al. 1985). This response consists of a variety of biochemical, cellular, hormonal and metabolic changes with characteristic increases in many plasma proteins. Serum amyloid A protein (apo-SAA) is particularly notable in that it is normally present in trace amounts in human serum, but the concentration increases up to 100-fold within 48 hours of initiation of the acute phase. (Eriksen and Benditt 1984; McAdam et al. 1978; de Beer et al. 1982). Segrest et al. (1976) first noted that a 45-residue fragment of the apo-SAA cleavage product, amyloid A protein (AA), formed stable complexes with phospholipid. This finding preceded the discovery, by Benditt and Eriksen (1977), that apo-SAA is transported in human plasma mainly with high density lipoproteins (HDL) and is thus classified as an apolipoprotein. Although HDL (expressed as HDL cholesterol) is a significant negative acute phase reactant, it can play a role in inflammation. HDL has been reported to function as a vehicle to transfer damaged cellular constituents to the liver and to bind bacterial lipopolysaccharides (Ulevitch et al. 1981) and neutrophil elastase (Jacob et al. 1981). The function of apo-SAA rich HDL in response to injury is, however, unknown. The phylogenetic conservation of the association of apo-SAA with HDL suggests that it plays an important role.
Kinetic models have been established for the study of plasma proteins in which tracer quantities of radiolabelled proteins are administered intravascularly to animals and disappearance of radioactivity from the plasma compartment is monitored. Two conditions must be met. First, it is essential that the tracer be distributed and metabolized identically to the tracee. Secondly, the system must be in dymamic equilibrium, i.e., plasma concentrations must not fluctuate during the course of the study. If these criteria are met, one can plot a disappearance curve of radioactivity remaining in the plasma versus time on a log-linear scale. Slopes and intercepts extrapolated from the curve are then used to calculate half-life and fractional catabolic rate. If one also knows the plasma concentration of the protein of interest, it is possible to calculate synthesis rate. The total plasma mass of the protein can be determined from the initial specific activity (usually measured 5–10 minutes after infusion of tracer) and the dose of tracer (1).
Amyloid protein AA is the presumptive fragment of an acute phase serum apolipoprotein, apoSAA. Two major murine apoSAA isotypes (apoSAA1 and apoSAA2) have been identified. The NH2-terminal amino acid sequences of purified murine apoSAA1 and apoSAA2 have been examined and compared with that of murine amyloid protein AA. Our results indicate that apoSAA1 and apoSAA2 are separate gene products and that amyloid protein AA has NH2-terminal amino acid sequence identity with only one of these isotypes, namely apoSAA2.
Serum amyloid A (SAA) is an acute-phase protein that circulates mainly on plasma high-density lipoprotein (HDL). SAA interactions with its functional ligands and its pathogenic deposition in reactive amyloidosis depend, in part, on the structural disorder of this protein and its propensity to oligomerize. In vivo, SAA can displace substantial fraction of the major HDL protein, apoA-I, and thereby influence the structural remodeling and functions of acute-phase HDL in ways that are incompletely understood. We use murine SAA1.1 to report the first structural stability study of human plasma HDL that has been enriched with SAA. Calorimetric and spectroscopic analyses of these and other SAA-lipid systems reveal two surprising findings. First, progressive displacement of the exchangeable fraction of apoA-I by SAA has little effect on the structural stability of HDL and its fusion and release of core lipids. Consequently, the major determinant for HDL stability is the non-exchangeable apoA-I. A structural model explaining this observation is proposed, which is consistent with functional studies of acute-phase HDL. Second, we report an alpha-helix folding/unfolding transition in SAA in the presence of lipid at near-physiologic temperatures. This new transition may have potentially important implications for normal functions of SAA and its pathogenic misfolding.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Lipoproteins are plurimolecular, typically quasi-spherical, pseudomicellar com-plexes composed of polar and non-polar lipids solubilized by proteins with spe-cialized structure and function, the apolipoproteins. Lipids and apolipoproteins thus constitute major building blocks of lipoproteins. The principal lipid-binding structural motifs of apolipoproteins include amphipathic alpha-helixes and beta-sheets. Major lipoproteins in human plasma are, in the order of increasing den-sity and decreasing size, chylomicrons, very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoprotein (LDL), lipopro-tein (a) [Lp(a)] and high-density lipoprotein (HDL). Whereas chylomicrons, VLDL, IDL, LDL and Lp(a) are commonly regarded as large, light, lipid-rich particles, HDL represents a small, dense, protein-rich lipoprotein with a mean size of 8–10 nm and density of 1.063-1.21 g/ml (Fig. 1.1).
While oxidative modification of LDL, a crucial step in atherogenesis, occurs in the arterial subintimal space and oxidized LDL (oxLDL) is observed chiefly in arterial lesions, oxLDL has also been shown to exist in the circulation, where it can become a surrogate biomarker of cardiovascular disease. The serum amyloid A-LDL (SAA-LDL) complex is currently considered to be a novel oxLDL marker in a broad sense. There have been several reports using SAA-LDL measurements in human subjects with lipid disorders, the metabolic syndrome and coronary artery disease. These reports have shown that there are higher circulating SAA-LDL levels in the aforementioned disease status and that a reduction in the levels of SAA-LDL can be achieved by intervention treatments including lifestyle modifications and drugs such as highly purified eicosapentaenoic acid. Data also suggest a prognostic value of SAA-LDL on cardiac events in patients with coronary artery disease. This article summarizes the current clinical data that indicate the usefulness of SAA-LDL measurements for understanding the pathophysiology of and assessing the risk of cardiovascular disease development.
The serum amyloid A (SAA) superfamily comprises a number of differentially expressed genes with a high degree of homology in mammalian species. SAA4, an apolipoprotein constitutively expressed only in humans and mice, is associated almost entirely with lipoproteins of the high-density range. The presence of SAA4 mRNA and protein in macrophage-derived foam cells of coronary and carotid arteries suggested a specific role of human SAA4 during inflammation including atherosclerosis. Here we underline the importance of ribosome binding site (rbs)-like sequences (also known as Shine-Dalgarno sequences) in the SAA4 cDNA for expression of recombinant SAA4 protein in Escherichia coli. In contrast to rbs sequences coded by the expression vectors, rbs-like sequences in the cDNA of target protein(s) are known to interfere with protein translation via binding to the small 16S ribosome subunit, yielding low or even no expression. Here we show that PCR mutations of two rbs-like sequences in the human SAA4 cDNA promote expression of considerable amounts of recombinant SAA4 in E.coli.
Abstract Amyloid A (AA) amyloidosis is a fatal disease caused by extracellular deposition of fibrils derived from serum AA (SAA). AA amyloid fibril formation has previously been modeled in macrophage cultures using highly amyloidogenic mouse SAA1.1, but attempts to do the same with human SAA invariably failed. Our objective was to define conditions that support human SAA-derived amyloid formation in peripheral blood mononuclear cell (PBMC) cultures. Two conditions were found to be critical - omission of fetal calf serum and use of StemPro34, a lipid-enriched medium formulated for hematopoietic progenitor cells. Cultures maintained in serum-free StemPro34 and provided with recombinant human SAA1 in the complete absence of amyloid-enhancing factor exhibited amyloid deposition within 7 d. Amyloid co-localized with cell clusters that characteristically included cells of fibrocytic/dendritic morphology as well as macrophages. These cells formed networks that appeared to serve as scaffolding within and upon which amyloid accumulated. Cells in amyloid-forming cultures demonstrated increased adherence, survival and expression of extracellular matrix components. Of the three human SAA1 isoforms, SAA1.3 showed the most extensive amyloid deposition, consistent with it being the most prevalent isoform in Japanese patients with AA amyloidosis. Attesting to the reproducibility and general applicability of this model, amyloid formation has been documented in cultures established from eight PBMC donors.
Serum amyloid A (apoSAA) is a polymorphic protein encoded by a family of SAA genes in which new members continue to be identi3ed. Those isoforms or allelic forms that are precursors for the amyloidfibril protein A(AA) in reactive (secondary) amyloidosis, are among the acute phase or regulated apoSAA apoproteins complexed with high densig lipoprotein (HDL); other isoforms are expressed constitutively. The chieffunction of apoSAA, which is a well conserved protein found in birds and mammals, has probably not yet been disclosed, but is thought to be part of the host response to infections and tissue damage. Structural and functional aspects of apoSAA. and its role in AA amyloidosis are complex due to the presence of multiple isoforms. Most species appear to possess two main acute phase apoSAA isoforms of hepatic origin in their serum (apoSAA1 and apoSAA2 and a third form that has predominant extrahepatic expression (apoSAA3) A single constitutive isoform, apoSAA5 has been identijied in human and apoSAA, has been described in BALB/c mice. Thus, apoSAA proteins can be regarded or subclassified as either acute phase reactants (regulated apoSAA also termed A-SAA) or constitutive proteins (C-SAA).
Purified spleen macrophages (SM) and liver Kupffer cells (KC) were incubated with HDL3−SAA for up to 72 h. The uptake of serum amyloid A (SAA) by cells was determined by comparing the amount of SAA, as measured by ELISA, remaining in the supernatant at different time points of incubation. The uptake of SAA by SM significantly increased during the 72 h incubation period. SM obtained from animals undergoing an amyloidosis induction protocol had a similar uptake of SAA when compared to SM obtained from normal animals, thus suggesting that the uptake (and/ or catabolism) of HDL−−SAA by SM during induction of amyloidosis remains stable. No significant increase in the uptake of HDL3−SAA, however, was seen in KC over the 72 h incubation period. The addition of serum amyloid P protein (SAP) to the culture abrogated the uptake of SAA by SM but did not modify the uptake seen with KC. Amyloid P protein (AP) may contribute to the pathogenesis of the disease by inhibiting the cellular uptake of the precursor in the affected organs.
Lipoprotein metabolism was assessed in hamsters following subcutaneous injection of AgNO3. Apolipoprotein serum amyloid A (apoSAA) peaked at 36 h, followed by elevations in plasma cholesterol at 56 h and triglycerides at 96 h. There was a striking increase in LDL and a de-crease in HDL. Migration of all acute phase (AP) lipoproteins was retarded compared to controls and SDS-PAGE electrophoretic analysis was consistent with agarose gel profiles that revealed increased apoB-rich VLDL, IDL and LDL and decreased apoAl-rich HDL2 fractions. Cholesterol transported by LDL of AgNO3 treated hamsters was double that of controls while the pool of HDL-cholesterol was only two-thirds that of controls. Fasting triglyceride and cholesterol secretion rates were depressed sharply at 24 h. After E. coli lipopolysaccharide (LPS) injection, apoSAA-HDL particles bound avidly to cultured peritoneal macrophages (møs) but in vitro exposure of tissue møs to LPS did not alter the binding characteristics of either control-or apoSAA-HDL, Finally, 125I radiolabelled apoSAAHDL and apoAl-HDL decreased during 2–4 h exposure to møs but only apoAl remained associated with cells. Collectively, these data support the hypothesis that apoSAA may commandeer HDL during the AP response in order to deliver phospholipids and cholesterol to cells involved in tissue repair at sites of inflammation.
Heterogeneity in Physicochemical PropertiesHeterogeneity in Chemical CompositionRelationships Between HDL Subfractions Separated by Different Methods
References
The administration of dimethylnitrosamine (DMN) into rabbit induced liver fibrosis/cirrhosis and finally caused a lethal hepatic failure. Blood collected from the rabbit was centrifuged and the supernatant was analyzed by two-dimensional gel electrophoresis (2-DE) for the study of proteome in serum. Compared with 2-DE gel of serum from healthy rabbit, a significant reduction in the number of protein spots having molecular weights (MWs) below 21 kDa was observed in the gels of the serum from the rabbit treated with DMN, while the secretion of albumin was kept at a high level. Separated spots in the two-dimensional gel were cut, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. Serum amyloid A-3 protein precursor (SAA3) and other serum amyloid A (SAA) protein precursors were identified by matching the peptide masses with those in database. In the SAA family of acute-phase/inflammatory response proteins, SAA3 is mainly synthesized in the liver. The SAA3 secreted level in the serum decreased with time after DMN administration as the result of hepatic dysfunctions.
Serum amyloid A (SAA) has been implicated by three independent studies to increase in concentration with ageing. The present study measured SAA concentration in 395 samples from 302 healthy individuals ranging in age from 21 to 100 years. The average SAA concentration was 20 microgram/ml, with only five serum samples falling below 5 microgram/ml. SAA concentrations are expressed in terms of cross-reactivity of purified, denatured SAA with anti-AA antibodies, rather than the purified, denatured amyloid fibril protein AA from tissues, which has been used in the past. No age-related increase in SAA concentration was observed in the present study. The average SAA concentration in these normal, healthy individuals was almost a hundred-fold less than values measured in acute phase human serum in a separate study with the same reagents.
Evidence is presented that the major extractable protein of the amyloid substance induced in mice by the injection of Candida albicans is homologous in the NH 2 terminal region with nonimmunoglobulin amyloid proteins previously found in tissues of men, monkeys, and ducks and recently assigned the name protein AA. Further, a protein extracted from livers of mice with casein induced amyloidosis although it was not subjected to sequence analysis, was found to possess characteristic properties of protein AA. For the major protein extracted from amyloid deposits induced in mice by injection of either Candida albicans cells or sodium caseinate had chromatographic and electrophoretic properties and an amino acid composition characteristic of the AA class of amyloid proteins. The homology of the mouse protein with protein AA from man and monkey was established by determination of the sequence of the first 28 amino acid residues.
The serum precursor SAA of the secondary amyloid protein AA has been detected by solid-phase radioimmunoassay as a normal serum alpha-globulin of mol wt 160,000, which dissociates to a more stable 12,500 dalton moiety on treatment with formic acid. In 12 strains of mice, including T-cell-deficient nude mice, treated with the amyloid-inducing agents lipopolysaccharide (LPS) or casein, SAA behaved as an acute-phase reactant. SAA concentration rose to about 750 mug/ml by 24 h and returned to less than 1 mug/ml by 48 h. Since the amyloid-resistant colchicine-treated mice and AJ mice had a normal SAA response to LPS, it appears that their resistance to amyloid induction is due to the nature of their SAA processing rather than decreased SAA production. C3H/HeJ mice, which have defective B-lymphocyte responses to LPS, required extremely high dosages of LPS to cause SAA elevation, although their SAA response to casein was normal. This suggests that SAA is an acute-phase protein produced as a result of B-lymphocyte stimulation. Preliminary evidence suggests that at the height of an acute SAA response, liver homogenates are particularly rich in protein AA cross-reacting material.
SAA is a normal acute-phase serum protein and is thought to be the precursor of amyloid protein AA which is deposited as insoluble beta-pleated sheet fibrils in secondary amyloidosis. Native SAA has a molecular weight of 160,000 and has not been isolated; it has been most frequently purified as a species (designated SAAL) of 12,500 mol. wt. by gel filtration in dissociating solutions. The conformational properties of SAA proteins in patients with and without amyloidosis have been compared in an effort to determine the factors involved in the induction of the beta-pleated sheet conformation in the amyloid SAA protein prior to fibril deposition. Amyloid and nonamyloid SAA proteins are similar in that they readily undergo conformational changes which result in the formation of heterogenous mol. wt. SAA species and in an increased exposure of antigenic determinants which cross-react with AA fibril proteins. Amyloid and nonamyloid SAA are different, however, in that amyloid SAA is more resistant to dissociation to SAAL. Amyloid SAAL, while similar to nonamyloid SAAL in immunoreactivity, shows a greater tendency toward aggregation. The relative resistance of both amyloid SAA and SAAL to complete dissociation may play an important role in amyloid fibril formation from these precursors.
A sensitive and specific double antibody radioimmunoassay for the major apolipoprotein (apo A-I) of human serum high density lipoprotein (HDL) was developed. Initial studies indicated that direct measurements of apo A-I concentration in whole untreated sera or isolated high density lipoprotein fractions yielded variable results, which were lower than those obtained in the corresponding samples which had been subjected to delipidation. Subsequently, it was observed that heating diluted sera or HDL for 3 hr at 52 °C prior to assay resulted in maximal increases in apo A-I immunoreactivity to levels comparable to those found in the delipidated specimens. This simple procedure permitted multiple sera to be assayed efficiently with full recovery of apo A-I.
Serum apo A-I in healthy normolipemic males was 130 ± 3 mg/dl (range 95–165), while the values in females were significantly higher 154 ± 6 mg/dl (range 107–199) (P < 0.005). The apo A-I levels correlated with the total serum cholesterol (males r = 0.46, P < 0.005; females r = 0.58, P < 0.001).
Preliminary results with sera from patients with abetalipoproteinemia, hypercholeskrdemia and Tangier disease indicated that alterations in the low density lipoprotein concentration and changes in the physical chemical properties of the high density lipoproteins did not affect the optimal conditions for mnwuring serum apo A-I. The apo A-I concentrations were abnormally low in each of the above disorders.
Antisera have been prepared against the major nonimmunoglobulin component of secondary and familial Mediterranean fever (FMF) associated amyloid which has been called A component or acid soluble fraction (ASF). The antisera were shown to be monospecific for ASF by precipitation of (125)I-labeled antigen and gave a reaction of identity with four different ASF preparations. The antisera were able to detect a circulating component in human serum that migrated in the alpha1-globulin region. This circulating component gave a line of identity with degraded ASF by double immunodiffusion. 57 normal sera and 89 sera from patients with diseases known to be frequently associated with amyloidosis were tested by immunodiffusion for the circulating ASF component. 7% of normal sera and 50-80% of the pathologic sera had elevated amounts of this component. Absorption studies showed that all normal sera probably have small amounts of this component while cord sera do not have detectable amounts. This component was partially purified and was shown to be slightly larger than albumin. The relation of the circulating component to the acid soluble fraction of amyloid is discussed.
Amyloidosis was produced experimentally in guinea pigs by multiple casein injections. Amyloid fibrils were isolated and fractionated and a protein obtained that had an amino acid composition comparable with A protein, a unique nonimmunoglobulin constituent of secondary amyloid deposits. N-terminal sequence analysis demonstrated a sequence homologous with that of A proteins from human and monkey preparations but preceded by a 5-residue peptide which had an N-terminal histidine. A definite species specificity in A protein from human and guinea pig was identified on immunologic analysis.
A group of human amyloid fibril proteins having chemical homology and antigenic similarities to immunoglobulin light polypeptide chains has been previously described. The amino acid sequence of a different type of human amyloid fibril protein is reported. This protein lacks valine, proline, threonine, and half-cystine and its sequence is not homologous with any presently known immunoglobulin sequence. This sequence defines a new class of amyloid fibril proteins of as yet unknown origin.
Antisera have been prepared against the major nonimmunoglobulin component of secondary and familial Mediterranean fever (FMF) associated amyloid which has been called A component or acid soluble fraction (ASF). The antisera were shown to be monospecific for ASF by precipitation of 125I-labeled antigen and gave a reaction of identity with four different ASF preparations. The antisera were able to detect a circulating component in human serum that migrated in the α1-globulin region. This circulating component gave a line of identity with degraded ASF by double immunodiffusion. 57 normal sera and 89 sera from patients with diseases known to be frequently associated with amyloidosis were tested by immunodiffusion for the circulating ASF component. 7% of normal sera and 50–80% of the pathologic sera had elevated amounts of this component. Absorption studies showed that all normal sera probably have small amounts of this component while cord sera do not have detectable amounts. This component was partially purified and was shown to be slightly larger than albumin. The relation of the circulating component to the acid soluble fraction of amyloid is discussed.
With direct immunoprecipitation or gel filtration under dissociating conditions, amyloid-related serum protein SAA has been isolated as a low molecular weight protein from the serum of two patients with rheumatoid arthritis but without known amyloidosis. The isolated protein SAA showed antigenic identity and an amino acid composition that was similar, but not identical, with isolated fibril protein AA. Molecular weight estimations suggest that protein SAA is approximately 50% larger than protein AA and has a molecular weight of 14,000-15,000 daltons. Preliminary results indicate that protein SAA from a patient with amyloidosis has a similar small molecular weight subunit.
The amyloid-related serum protein SAA has been isolated by gel filtration in 10% formic acid from three animal species: mink, mouse, and rabbit. Sera used in the isolation procedure were obtained from animals in which high concentrations of SAA had been induced by treatment with LPS.
The isolated SAA proteins had a subunit size similar to that of human SAA, with m.w. values ranging from 10,000 to 11,700 (estimated by gel filtration in 6 M guanidine-HCl) or 12,400 to 15,000 (estimated by SDS-PAGE). The m.w. studies and amino acid sequence data indicated that SAA and the amyloid fibril protein AA in the mouse, and probably also the mink, are related in the same way as in man, the two proteins having common NH2-terminal amino acid sequences and SAA being extended by 20 to 40 residues at the COOH-terminal end of the molecule.
A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d equal to 1.085-1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in the incubation mixture, and accurate with a variability of only 3-5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.
Temperature-dependent conformational changes of the principal apoprotein of human plasma high density lipoprotein (HDL), apoA-I, have been studied in the isolated apoprotein, in complexes of apoprotein with phospholipid, and in intact HDL. Differential scanning calorimetry shows that in solution apoA-I undergoes a reversible, two-state thermal denaturation (midpoint temperature 54 degrees). The enthalpy (2.4 cal/g)(10.0 J/g) and specific heat change (0.08 cal/degrees C per g)(0.33 J/degrees C per g) associated with the denaturation were used to calculate the free energy difference (deltaG) between native and unfolded apoA-I at 37 degrees. DeltaG (2.4 kcal/mol)(10.0 kJ/mol) is less than that of other globular proteins (typically 8-14 kcal/mol)(33-59 kJ/mol), indicating that at 37 degrees native apoA-I has a loosely folded conformation. Turbidity studies show that apoA-I is able to solubilize phospholipid in its native but not in its denatured form. Mixtures of apo-HDL (the total apoprotein of HDL) or apoA-I with dimyristoyl lecithin show a thermal transition at about 85 degrees not present in the lecithin or the apoprotein alone, which indicates that the native conformation of the apoprotein is stabilized by phospholipid. Scanning calorimetry of intact HDL shows a high-temperature endotherm associated with disruption of the HDL particle, suggesting that in HDL the conformation of apoA-I is also stabilized by interaction with lipid. The loosely folded conformation of native, uncomplexed apoA-I may be especially adapted to the binding of lipid, since this process may involve both hydrophobic sites on the surface of the protein and concealed apolar amino acid residues that are exposed by a cooperative, low energy unfolding process.
1.1. Serum lipoproteins of rat, mouse, gerbil, rabbit, pig and man have been studied by agarose gel electrophoresis, immunoelectrophoresis and crossed immunoelectrophoresis. Lipoproteins (HDL, VLDL and LDL) from the serum of rat, mouse, gerbil and pig were isolated by a procedure used earlier for the isolation of human serum lipoproteins, and they were characterized by electrophoretical methods.2.2. Electrophoretical and immunoelectrophoretical analyses show a pattern of three to four stainable fractions in the prealbumin-α and β regions of rat, mouse, rabbit, pig and man. In the gerbil an additional lipoprotein fraction was found in the β2-γ region. Immunoelectrophoresis revealed that this fraction was not identical to the immunoglobulin precipitates.3.3. The results, especially those from electrophoretical studies on isolated lipoproteins, demonstrate the problems in obtaining homologous lipoprotein patterns between animal species and thus the difficulties involved in adopting a nomenclature and classification applicable to a greater number of animal species. It is evident that these problems must be considered each time a new species is investigated or when a new method for lipoprotein assay is used.
Human plasma low density lipoprotein displays a reversible thermal transition between 20 and 40 degrees C, due to a phase transition of its core cholesterol ester from a smectic to a more liquid-like state. To determine if the cholesterol of high density lipoprotein (HDL) displays similar thermal behavior, the human lipoprotein and its extracted lipid have been examined by differential scanning calorimetry, low angle X-ray scattering and polarizing microscopy. Neither HDL2**(d 1.063--1.125--1.21 g/ml) nor HDL3(d1.125--1.21g/ml) show thermal transitions between O and 60 degrees C. By contrast cholesterol ester isolated from HDL and mixtures of cholesterol oleate and linoleate show reversible liquid crystalline transitions between 20 and 40 degreesC. X-ray scattering studies of HDL2 and HDL3 performed at 10 degreesC show no scattering fringes attributable to a smectic phase of cholesterol ester. When HDL is heated to temperatures above 60 degreesC a broad, double-peaked endotherm is observed. The first component (peak temperature=71 degreesC) corresponds to a selective release of apoprotein A-1 from the lipoprotein, and the second component (peak temperature=90 degreesC) to a more generalized disruption of lipoprotein structure with release of cholesterol ester and apoprotein A-2. Following the thermal disruption of HDL, reversible liquid crystalline transitions of cholesterol ester can be seen by differential scanning calorimetry and polarizing microscopy, showing the presence of large domains of cholesterol ester. The absence of cholesterol ester transitions in intact HDL may indicate an interaction of cholesterol ester molecules with the protein-phospholipid surface of HDL that prevents the formation of an organized lipid phase. The high temperature behavior of HDL indicates that apoprotein A-1 is less important than apoprotein A-2 in maintaining the HDL apolar lipids in the form of a stable miroemulsion.
Human serums contain a protein antigenically related to protein AA, the principal protein of a major class of amyloid substance. The serum antigen, SAA, occurs mainly in a high molecular weight form, 1 to 2 X 10(5). This work shows that the bulk of the SAA sediments at density 1.12 g/cm3 and floats at density 1.21 g/cm3, as does the high density lipoprotein HDL3. SAA is associated with the apolipoproteins ApoA-I and ApoA-II. The total cholesterol:total protein ratio of the fraction with density 1.12-1.21 g/cm3 is 0.2:1, consistent with that of SAA constituent of the HDL3 fraction into low molecular weight species of the order of 13,000. The quantity of SAA may vary from 0.1% or more of the total protein of HDL3.
Study of the binding, internalization, and degradation of 125l-labeled low density lipoprotein (LDL) in human fibroblasts has disclosed a new mutant allele at the LDL receptor locus. This mutant allele, designated Rb+,io, specifies a receptor molecule that is able to bind 125l-LDL but is unable to facilitate the internalization of the receptor-bound lipoprotein. This allele has been identified through analysis of fibroblasts from four heterozygous relatives of patient J.D., the previously described subject with the clinical syndrome of homozygous familial hypercholesterolemia whose fibroblasts bind but do not internalize 125l-LDL (Brown and Goldstein, 1976c). The current pedigree data reveal that J.D. is a genetic compound. From his father, he has inherited the Rb+,io allele. From his mother, J.D. has inherited the Rbo allele, a previously characterized mutant allele that specifies a receptor that is unable to bind LDL and hence is biochemically silent in J.D. Thus the only detectable receptors in the J.D. cells are the products of the Rb+,io allele that can bind, but not internalize, 125l-LDL. The demonstration that the LDL-binding and internalization defects do not complement one another in J.D. supports the conclusion that the mutations causing these two defects are allelic. This in turn suggests that the LDL receptor contains two functional sites on the same polypeptide chain-one that binds LDL and one that is necessary to mediate the internalization of the receptorbound lipoprotein.
Plasma lipoproteins are the macromolecular complexes with reproducible lipid-protein ratios and stability in aqueous solutions. This chapter describes the classification of the plasma lipoproteins, molecular properties of apolipoproteins, molecular organization of lipoprotein particles, and lipoprotein metabolism. The chapter focuses critically on the recent advances in the field of lipoprotein structure and metabolism. Significant advances about the knowledge of the primary structure and molecular properties of several of the apolipoproteins have been observed. The realization that a number of apolipoproteins undergo self-association provides important, as well as fascinating, insight into the molecular properties of this unique group of proteins. Studies on the synthesis, catabolism, and metabolic interrelationships of plasma lipoproteins provide a number of insights into lipoprotein metabolism, including the precursor-product relationship of very low density lipoproteins (VLDL)-low density lipoproteins (LDL), the peripheral metabolism of lipoprotein particles, as well as the metabolic and molecular defect(s) in patients with disorders of lipid metabolism. Current information on plasma lipoproteins, despite rapid advances in the field, must be regarded as preliminary. However, the availability of current techniques and knowledge of lipoprotein structure and metabolism will undoubtedly provide the necessary background required for the major progress in the knowledge of plasma lipoproteins.
Two apolipoproteins that are minor components normally of human plasma lipoproteins were discovered. They comprise up to 25% or more of the apolipoproteins in high density lipoproteins of some individuals with lipoprotein abnormalities associated with certain metabolic diseases and in individuals treated with amphotericin B for coccidiomycosis infection. The new apolipoproteins are distinctly different in amino acid composition, isoelectric point, and electrophoretic mobility from other known apolipoproteins. The new apolipoproteins are similar in amino acid composition. Both are unusually low in threonine, poor in valine and leucine, and rich in aspartic acid, glycine, alanine, and phenylalanine. They are relatively rich in arginine, although not as rich as the arginine-rich apolipoprotein, and have higher isoelectric points than other apolipoproteins except apoC-I. The more abundant of the two new apolipoproteins, of pI approximately 6.5, exists mainly as a dimeric protein of about 40 000 daltons and is dissociated by mercaptoethanol in the presence of sodium dodecyl sulfate. The less abundant one, of pI approximately 6.0, appears to be about 10 000 daltons. The threonine-poor apolipoproteins are associated mainly with plasma high density lipoproteins, particularly the denser HDLâ subfraction, but small amounts were found in the very low and intermediate density lipoproteins. The HDLâ with high content of threonine-poor apolipoproteins were indistinguishable from the normal HDLâ by agarose or large pore polyacrylamide gel electrophoresis or by analytical ultracentrifugation. The lipid moiety of the abnormal HDL was richer in triglycerides and similar in phospholipid content to that of normal HDL.
In the present study evidence is presented that SAA in serum complexes to a carrier protein with a molecular weight of 100,000-200,000 daltons, with mobility in the alpha-region on electrophoresis, and with a rather low normal serum concentration. The carrier protein is apparently not albumin. SAA isolated from the carrier protein has a molecular weight of 14,000 daltons and does not complex to any considerable extent with itself under neutral conditions.
Amino acid sequence studies of the amino terminal 25 residues of amyloid A (AA) protein and the serum precursor (SAA) induced with casein or LPS indicate differences in the sequence at position 6 and significant heterogeneity at several other positions in SAA. These findings suggest that SAA is a polymorphic serum protein and raise the possibility that only certain forms of SAA are processed to the tissue amyloid fibril.
The murine serum protein SAA, has been found to have a structure similar to human SAA, the precursor of human secondary amyloid fibril protein AA. SAA is detected by its cross-reaction in radioimmunoassay with antibodies raised to denatured amyloid fibrils of protein AA isolated from tissues of mice with amyloidosis. Murine SAA exists in the native state as a 160,000 molecular weight species, and can be isolated as a 12,500 molecular weight moiety, SAAL, by gel filtration in 10% formic acid. The quaternary structure of SAA is such that its AA determinants are relatively inaccessible for immunoreaction. Unfolding of these determinants can occur spontaneously; however, it is promoted by dissociation of SAA to SAAL.
Polypeptide segments, composed of alpha helixes with specific surface topography termed amphipathic helixes, have been proposed as the basic lipid-associating domains of apolipoproteins from the plasma lipoproteins. A computer search for proteins having sequences that could form amphipathic helixes indicated that amyloid A, a pathologically occurring protein usually associated with "secondary" amyloidosis, also contained amphipathic helixes. In studies reported here, amyloid A is shown to associate spontaneously with phospholipid vesicles with the following results: (a) the formation of a protein-lipid complex isolated by equilibrium density gradient ultracentrifugation, (b) a 100% increase in alpha helicity as measured by circular dichroism, (c) a 9-nm shift in the fluorescence maximum due to the single tryptophan residue located in the amphipathic region, indicating the tryptophan is moving from a polar to a nonpolar environment, and (d) the formation of stacked disk-like protein-lipid complexes as visualized by negative stain electron microscopy. The temperature dependence of the circular dichroic spectrum of the amyloid A-phospholipid complex suggests that the complex is formed by insertion of protein between the fatty acyl chains of the lipid. These findings suggest that the amphipathic helix is an important structural unit in lipid-associating proteins and that this unit can be recognized on the basis of its amino acid sequence. In addition, these studies have implications for the origin and function of amyloid A protein.
Experimental amyloidosis was induced in mink by repeated injections with endotoxin. Amyloid fibrils extracted from liver and spleen were fractionated by gel filtration after treatment with guanidine-hydrochloride and a reducing agent, dithiothreitol. An elution profile very similar to that of human amyloid fibrils, having protein AA as a major component, was obtained. The mink amyloid protein eluted at a position similar to that of human protein AA was by amino acid composition and partial sequence studies shown to be very similar to the latter protein and was called mink protein AA. In addition, a protein AA-related component (protein SAA) was found in increased amounts in serum of amyloidotic mink, providing further evidence of the homology with human amyloids. Experimental amyloidosis in mink represents a suitable model for studying amyloid proteins and related serum components.
A 12,000 dalton serum amyloid A protein (SAA) has been isolated by chromatography on Sephadex in 10% formic acid. It is similar immunologically to the previously characterized 8500 dalton tissue amyloid A (AA) protein. The results of amino acid analyses, peptide maps, and the identity of the first 11 residues of the SAA and AA proteins support the idea that AA represents the amino terminal fragment of SAA and is derived from it by proteolysis.
Physical factors leading to the separation of oligopeptides in the molecular weight range of 1,200 to 10,000 daltons by analytical-scale electrophoresis in polyacrylamide gel with sodium dodecyl sulfate are described. Increased acrylamide concentration, cross-linkage, and inclusion of 8 M urea to decrease gel porosity, increased gel length, and buffer ions of low mobility are factors which yield improved separation of such peptides. Electrophoretic mobilities of eleven peptides were linearly related to the logarithm of their molecular weights with a standard deviation of 18% in a system of improved resolution. The intrinsic charge and conformation of peptides were found to be relatively more important determinants of electrophoretic mobilities than for proteins larger than 10,000 daltons. Such determinants were relatively more important with four of the eleven peptides examined, leading to deviations from the log-linear slope greater than 18%. Because of the importance of intrinsic charge and conformation, the system, although allowing a first approximation in molecular weight determination, may also be applicable to peptide “mapping,” particularly for “insoluble” peptide mixtures with prominent hydrophobic association, such as encountered in cellular membranes, viruses, and proteolytic digests.
IT has been shown by Glenner et al.1 that certain amyloid fibrils, chiefly derived from primary amyloidosis or amyloidosis associated with myelomatosis, consist of immunoglobulin chains or their fragments. The principal component of these fibrils seems to be derived from the variable part of monoclonal kappa or lambda light chains (Bence-Jones proteins). In addition, a ``non-immunoglobulin'' amyloid fibril protein with a molecular weight of 85,000, named by us protein AS (amyloid subunit), which comprises an essential part of some secondary amyloid fibrils, has been detected by Benditt et al.2 and by other groups3-5. The protein AS is found in secondary amyloid, chiefly associated with chronic inflammations, and has been suggested to be a fragment of a hitherto unknown larger protein4. Except for the detection of some light chain material connected with amyloid fibrils of the ``non-immunoglobulin'' type6,7, no chemical link between the primary (``light chain'') and secondary (``non-immunoglobulin'') classes of amyloid substances has been established. On the contrary, the question has been raised whether there are two completely different chemical classes of amyloid8.
The apolipoproteins from rat plasma high density lipoproteins were separated into three fractions by Sephadex gel chromatography.
Five low molecular weight proteins were purified from the last Sephadex fraction by ion exchange chromatography. All five
proteins have apparent homologues among the human apolipoproteins. The A-II apoprotein has COOH-terminal alanine, a blocked
NH2 terminus and contains no histidine, tryptophan or cysteine. Unlike human A-II, it appears to occur in monomeric form. The
C-I apoprotein has COOH-terminal alanine, NH2-terminal aspartic acid, contains 16% lysine, and lacks valine, tyrosine, and cysteine. The C-II apoprotein has NH2-terminal threonine, COOH-terminal glutamic acid and contains no cysteine. Two forms of the C-III apoprotein were found with
COOH-terminal proline and NH2-terminal aspartic acid. One form, C-III-0, contains no carbohydrate, while the other, C-III-3, contains 3 moles of sialic
acid and 1 mole of galactosamine per mole of protein.
The results of antigenic and chemical studies on subcomponents of amyloid fibrils secondary to chronic inflammatory diseases are reported. Treatment of amyloid with either guanidine and dithiothreitol or sodium hydroxide followed by gel filtration on Sephadex G-100 revealed two major protein components, one eluted in the void volume (m. w. >200, 000), the other having an m. w. of 5, 000. Results from amino acid composition and N-terminal sequences and also antigenic analysis suggest that the components are two different proteins not related to immunoglobulins.
An enzymatic method is described for determination of total serum cholesterol by use of a single aqueous reagent. The method requires no prior treatment of sample and the calibration curve is linear to 600 mg/dl. Cholesterol esters are hydrolized to free cholesterol by cholesterol ester hydrolase (EC 3.1.1.13). The free cholesterol produced is oxidized by cholesterol oxidase to cholest 4 en 3 one with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4 aminoantipyrine and phenol in the presence of peroxidase to yield a chromogen with maximum absorption at 500 nm. The method is reproducible, and the results correlate well with those obtained by automated Liebermann Burchard procedures and the method of Abell et al. The present method affords better specificity than those previously reported and has excellent precision.
The complete amino acid sequence of a protein, acid soluble fraction, (ASF) which constitutes up to 50% of amyloid fibrils from a patient with familial Mediterranean fever has been obtained. Partial amino acid sequences of three other proteins from patients with secondary amyloidosis were identical in the regions studied except for an alanine-valine interchange in one. The ASF contains no cysteine, does not resemble any known immunoglobulin, and has not been detected as yet in myeloma-associated amyloid.
Concentrates of amyloid substance derived from organs of 10 human patients representing a variety of clinical entities were characterized according to their amino acid compositions, their electrophoretic constituents mobile in urea-starch gel at pH 3 and their stability with respect to the binding of Congo red in the pH interval 9-12.5. The analyses revealed the existence of two major classes of amyloid substance. Material from one class, embracing those cases that had a chronic inflammatory disease as the principal associated process (type A amyloidosis), had a similar amino acid composition and was distinguished by the presence in the amyloid substance of a large amount of an electrophoretically well-defined group of low-molecular-weight proteins with an unusual amino acid composition (amyloid protein A); type A amyloid substance lost the typical binding of Congo red dye at pH 11.5 or lower. Concentrates of amyloid substance in the other class (type B amyloidosis), represented by a case of multiple myeloma, 3 other cases of neoplastic disease and a case of primary cardiac amyloidosis, were distinguished by the presence of an electrophoretically more heterogeneous group of protein constituents with different mobilities and with apparently higher molecular weights than that of protein A, by an amino acid composition that is clearly different from that of the first class, and by retention of the typical Congo red-binding property at pH 12 or higher. The major constituent, protein A, of amyloid substances of the first class is clearly different from ordinary fragments of immunoglobulins in size, electrophoretic mobility, and amino acid composition.
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