Serum amyloid A: a marker of adiposity-induced low-grade inflammation but not of metabolic status

Objective: Adipocytes secrete a series of acute phase proteins including serum amyloid A (SAA); the link with metabolic status is unknown. We studied the variations of expression of the SAA gene in adipose and liver tissues and of SAA serum levels, as well as their relationships with metabolic features during weight loss. Research Methods and Procedures: Plasmatic variations of SAA, inflammatory markers (high sensitivity C‐reactive protein, interleukin‐6, fibrinogen, and orosomucoid), and adipokines (adiponectin, leptin) were studied in 60 morbidly obese patients before and after gastric surgery. For 10 subjects, SAA mRNA expression was measured at baseline in subcutaneous white adipose tissue (scWAT) and visceral white adipose tissue (vWAT) and in the liver. The evolution of SAA mRNA expression was also studied after surgery in scWAT. Results: SAA serum concentration displayed a significant reduction 3 months after surgery and remained stable beyond 6 months. mRNA expression of inducible SAA isoforms (SAA 1 and 2) in scWAT was higher than in vWAT (p = 0.01) and the liver (p < 0.01) and correlated significantly with BMI, SAA, and high sensitivity C‐reactive protein serum concentrations but not with metabolic markers (glucose, insulin, lipid parameters, adiponectin). SAA serum level and its variation during weight loss significantly correlated with adiposity markers (BMI and adipocyte volume, p < 0.01) and inflammatory markers but not with variations of metabolic parameters. The variations of SAA expression in scWAT after surgery correlated with changes in BMI and SAA protein serum levels (p < 0.05). Discussion: SAA can be considered as a marker of adiposity‐induced low‐grade inflammation but not of the metabolic status of obese subjects.     

Silent mutations in secondary Shine-Dalgarno sequences in the cDNA of human serum amyloid A4 promotes expression of recombinant protein in Escherichia coli.

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.     

Exploring the expression bar code of SAA variants for gastric cancer detection

We reported an integrated platform to explore serum protein variant pattern in cancer and its utility as a new class of biomarker panel for diagnosis. On the model study of serum amyloid A (SAA), we employed nanoprobe‐based affinity mass spectrometry for enrichment, identification and quantitation of SAA variants from serum of 105 gastric cancer patients in comparison with 54 gastritis patients, 54 controls, and 120 patients from other cancer. The result revealed surprisingly heterogeneous and most comprehensive SAA bar code to date, which comprises 24 SAA variants including SAA1‐ and SAA2‐encoded products, polymorphic isoforms, N‐terminal–truncated forms, and three novel SAA oxidized isotypes, in which the variant‐specific peptide sequence were also confirmed by LC‐MS/MS. A diagnostic model was developed for dimension reduction and computational classification of the 24 SAA‐variant bar code, providing good discrimination (AUC = 0.85 ± 3.2E?3) for differentiating gastric cancer group from gastritis and normal groups (sensitivity, 0.76; specificity, 0.81) and was validated with external validation cohort (sensitivity, 0.71; specificity, 0.74). Our platform not only shed light on the occurrence and modification extent of under‐represented serum protein variants in cancer, but also suggested a new concept of diagnostic platform by serum protein variant profile.     

Large-scale isotype-specific quantification of Serum amyloid A 1/2 by multiple reaction monitoring in crude sera

Quantification is an essential step in biomarker development. Multiple reaction monitoring (MRM) is a new modified mass spectrometry-based quantification technology that does not require antibody development. Serum amyloid A (SAA) is a positive acute-phase protein identified as a lung cancer biomarker in our previous study. Acute SAA exists in two isoforms with highly similar (92%) amino acid sequences. Until now, studies of SAA have been unable to distinguish between SAA1 and SAA2. To overcome the unavailability of a SAA2-specific antibody, we developed MRM methodology for the verification of SAA1 and SAA2 in clinical crude serum samples from 99 healthy controls and 100 lung adenocarcinoma patients. Differential measurement of SAA1 and SAA2 was made possible for the first time with the developed isotype-specific MRM method. Most healthy control samples had small or no MS/MS peaks of the targeted peptides otherwise, higher peak areas with 10- to 34-fold increase over controls were detected in lung cancer samples. In addition, our SAA1 MRM data demonstrated good agreement with the SAA1 enzyme-linked immunosorbent assay (ELISA) data. Finally, successful quantification of SAA2 in crude serum by MRM, for the first time, shows that SAA2 can be a good biomarker for the detection of lung cancers.     

Structural studies of the C‐terminal 19‐peptide of serum amyloid A and its Pro→Ala variants interacting with human cystatin C

Serum amyloid A (SAA) is a multifunctional acute‐phase protein whose concentration in serum increases markedly following a number of chronic inflammatory and neoplastic diseases. Prolonged high SAA level may give rise to reactive systemic amyloid A (AA) amyloidosis, where the N‐terminal segment of SAA is deposited as amyloid fibrils. Besides, recently, well‐documented association of SAA with high‐density lipoprotein or glycosaminoglycans, in particular heparin/heparin sulfate (HS), and specific interaction between SAA and human cystatin C (hCC), the ubiquitous inhibitor of cysteine proteases, was proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, a hCC binding site in the SAA sequence was determined as SAA(86–104). The role of this SAA C‐terminal fragment as a ligand‐binding locus is still not clear. It was postulated important in native SAA folding and in pathogenesis of AA amyloidosis. In the search of conformational details of this SAA fragment, we did its structure and affinity studies, including its selected double/triple Pro→Ala variants. Our results clearly show that the SAA(86–104) 19‐peptide has rather unordered structure with bends in its C‐terminal part, which is consistent with the previous results relating to the whole protein. The results of affinity chromatography, fluorescent ELISA‐like test, CD and NMR studies point to an importance of proline residues on structure of SAA(86–104). Conformational details of SAA fragment, responsible for hCC binding, may help to understand the objective of hCC–SAA complex formation and its importance for pathogenesis of reactive amyloid A amyloidosis. Copyright © 2015 John Wiley & Sons, Ltd.     

Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Circular-dichroism studies on two murine serum amyloid A proteins.

C.d. studies have shown that mouse SAA2 (serum amyloid A2) protein has about one-half of the alpha-helix content of the SAA1 (serum amyloid A1) analogue (15 as against 32%), although secondary-structure prediction analyses based on sequence data do not suggest such a large difference between the forms. The decreased helical content may be a reflection or indication of a stronger propensity to aggregation of the SAA2 form compared with SAA1. The main elements of secondary structure in both proteins are beta-sheets/turns. Interactions with Ca2+ are accompanied by small losses in alpha-helix content, whereas binding to chondroitin-6-sulphate in the presence of millimolar Ca2+ also decreases the amount of secondary structure. However, SAA2 binding to heparan sulphate increases its beta-sheet structure, whereas with SAA1 secondary structure is not apparently altered by its interaction with heparan sulphate. Computer-generated surface profiles show slight differences in accessibility, hydrophilicity and flexibility between the proteins. Understanding these differences may help to explain why SAA2 is found in amyloid fibrils whereas SAA1 is not. In particular, a stronger tendency to aggregation might be the reason why SAA2 is deposited exclusively in these fibrils.     

Cellular mechanism of fibril formation from serum amyloid A1 protein

Serum amyloid A1 (SAA1) is an apolipoprotein that binds to the high‐density lipoprotein (HDL) fraction of the serum and constitutes the fibril precursor protein in systemic AA amyloidosis. We here show that HDL binding blocks fibril formation from soluble SAA1 protein, whereas internalization into mononuclear phagocytes leads to the formation of amyloid. SAA1 aggregation in the cell model disturbs the integrity of vesicular membranes and leads to lysosomal leakage and apoptotic death. The formed amyloid becomes deposited outside the cell where it can seed the fibrillation of extracellular SAA1. Our data imply that cells are transiently required in the amyloidogenic cascade and promote the initial nucleation of the deposits. This mechanism reconciles previous evidence for the extracellular location of deposits and amyloid precursor protein with observations the cells are crucial for the formation of amyloid.     

Interaction of serum amyloid A with human cystatin C—identification of binding sites

Serum amyloid A (SAA) is a multifunctional acute‐phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N‐terminal 76‐amino acid residue‐long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAA–hCC complex, using a combination of selective proteolytic excision and high‐resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86–104), whereas the binding site in hCC sequence was identified as hCC(96–102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAA–hCC complex. Copyright © 2012 John Wiley & Sons, Ltd.     

Secretome proteins as candidate biomarkers for aggressive thyroid carcinomas

Using proteomics in tandem with bioinformatics, the secretomes of nonaggressive and aggressive thyroid carcinoma (TC) cell lines were analyzed to detect potential biomarkers for tumor aggressiveness. A panel of nine proteins, activated leukocyte cell adhesion molecule (ALCAM/CD166), tyrosine‐protein kinase receptor (AXL), amyloid beta A4 protein, amyloid‐like protein 2, heterogeneous nuclear ribonucleoprotein K, phosphoglycerate kinase 1, pyruvate kinase isozyme M2, phosphatase 2A inhibitor (SET), and protein kinase C inhibitor protein 1 (14–3‐3 zeta) was chosen to confirm their expression in TC patients’ sera and tissues. Increased presurgical circulating levels of ALCAM were associated with aggressive tumors (p = 0.04) and presence of lymph node metastasis (p = 0.018). Increased serum AXL levels were associated with extrathyroidal extension (p = 0.027). Furthermore, differential expression of amyloid beta A4 protein, AXL, heterogeneous nuclear ribonucleoprotein K, phosphoglycerate kinase 1, pyruvate kinase muscle isozyme M2, and SET was observed in TC tissues compared to benign nodules. Decreased nuclear expression of AXL can detect malignancy with 90% specificity and 100% sensitivity (AUC = 0.995, p < 0.001). In conclusion, some of these proteins show potential for future development as serum and/or tissue‐based biomarkers for TC and warrant further investigation in a large cohort of patients.     

Preliminary proteome analysis of rabbit serum with hepatic failure

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.     

Amyloid-related serum protein SAA from three animal species: comparison with human SAA.

The amyloid-relates serum protein SAA has been isolated by gel filtration in 10% formic acid from three animal species: mink, mouse, 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-HC1) 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.     

Serum amyloid A is found on ApoB‐containing lipoproteins in obese humans with diabetes

Objective:

In murine models of obesity/diabetes, there is an increase in plasma serum amyloid A (SAA) levels along with redistribution of SAA from high‐density lipoprotein (HDL) to apolipoprotein B (apoB)‐containing lipoprotein particles, namely, low‐density lipoprotein and very low‐density lipoprotein. The goal of this study was to determine if obesity is associated with similar SAA lipoprotein redistribution in humans.

Design and Methods:

Three groups of obese individuals were recruited from a weight loss clinic: healthy obese (n = 14), metabolic syndrome (MetS) obese (n = 8), and obese with type 2 diabetes (n = 6). Plasma was separated into lipoprotein fractions by fast protein liquid chromatography, and SAA was measured in lipid fractions using enzyme‐linked immunosorbent assay and Western blotting.

Results:

Only the obese diabetic group had SAA detectable in apoB‐containing lipoproteins, and SAA reverted back to HDL with active weight loss.

Conclusions:

In human subjects, SAA is found in apoB‐containing lipoprotein particles only in obese subjects with type 2 diabetes, but not in healthy obese or obese subjects with MetS.     

Ail provides multiple mechanisms of serum resistance to Yersinia pestis

Ail, a multifunctional outer membrane protein of Yersinia pestis, confers cell binding, Yop delivery and serum resistance activities. Resistance to complement proteins in serum is critical for the survival of Y. pestis during the septicemic stage of plague infections. Bacteria employ a variety of tactics to evade the complement system, including recruitment of complement regulatory factors, such as factor H, C4b‐binding protein (C4BP) and vitronectin (Vn). Y. pestis Ail interacts with the regulatory factors Vn and C4BP, and Ail homologs from Y. enterocolitica and Y. pseudotuberculosis recruit factor H. Using co‐sedimentation assays, we demonstrate that two surface‐exposed amino acids, F80 and F130, are required for the interaction of Y. pestis Ail with Vn, factor H and C4BP. However, although Ail‐F80A/F130A fails to interact with these complement regulatory proteins, it still confers 10,000‐fold more serum resistance than a Δail strain and prevents C9 polymerization, potentially by directly interfering with MAC assembly. Using site‐directed mutagenesis, we further defined this additional mechanism of complement evasion conferred by Ail. Finally, we find that at Y. pestis concentrations reflective of early‐stage septicemic plague, Ail weakly recruits Vn and fails to recruit factor H, suggesting that this alternative mechanism of serum resistance may be essential during plague infection.     

NMR screening of new carbocyanine dyes as ligands for affinity chromatography

Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N‐carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α‐chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un‐substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α‐chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α‐chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD‐NMR technique was successfully used to screen cyanine–protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography. Copyright © 2014 John Wiley & Sons, Ltd.     

Inflammation protein SAA2.2 spontaneously forms marginally stable amyloid fibrils at physiological temperature

For nearly four decades, the formation of amyloid fibrils by the inflammation-related protein serum amyloid A (SAA) has been pathologically linked to the disease amyloid A (AA) amyloidosis. However, here we show that the nonpathogenic murine SAA2.2 spontaneously forms marginally stable amyloid fibrils at 37 °C that exhibit cross-beta structure, binding to thioflavin T, and fibrillation by a nucleation-dependent seeding mechanism. In contrast to the high stability of most known amyloid fibrils to thermal and chemical denaturation, experiments monitored by glutaraldehyde cross-linking/SDS-PAGE, thioflavin T fluorescence, and light scattering (OD(600)) showed that the mature amyloid fibrils of SAA2.2 dissociate upon incubation in >1.0 M urea or >45 °C. When considering the nonpathogenic nature of SAA2.2 and its ~1000-fold increased concentration in plasma during an inflammatory response, its extreme in vitro amyloidogenicity under physiological-like conditions suggest that SAA amyloid might play a functional role during inflammation. Of general significance, the combination of methods used here is convenient for exploring the stability of amyloid fibrils that are sensitive to urea and temperature. Furthermore, our studies imply that analogous to globular proteins, which can possess structures ranging from intrinsically disordered to extremely stable, amyloid fibrils formed in vivo might have a broader range of stabilities than previously appreciated with profound functional and pathological implications.     

Binding forces between a novel Schiff base palladium(II) complex and two carrier proteins: human serum albumi and β-lactoglobulin

Ligand binding studies on carrier proteins are crucial in determining the pharmacological properties of drug candidates. Here, a new palladium(II) complex was synthesized and characterized. The in vitro binding studies of this complex with two carrier proteins, human serum albumin (HSA), and β-lactoglobulin (βLG) were investigated by employing biophysical techniques as well as computational modeling. The experimental results showed that the Pd(II) complex interacted with two carrier proteins with moderate binding affinity (K_b ≈ .5 × 10⁴ M^?1 for HSA and .2 × 10³ M^?1 for βLG). Binding of Pd(II) complex to HSA and βLG caused strong fluorescence quenching of both proteins through static quenching mechanism. In two studied systems hydrogen bonds and van der Waals forces were the major stabilizing forces in the drug-protein complex formation. UV–Visible and FT-IR measurements indicated that the binding of above complex to HSA and βLG may induce conformational and micro-environmental changes of two proteins. Protein–ligand docking analysis confirmed that the Pd(II) complex binds to residues located in the subdomain IIA of HSA and site A of βLG. All these experimental and computational results suggest that βLG and HSA might act as carrier protein for Pd(II) complex to deliver it to the target molecules.     

Independent association of serum amyloid P component, protein S, and complement C4b with complement C4b-binding protein and subsequent association of the complex with membranes

C4b-binding protein (C4BP) is a large complex assembly of eight subunits that functions as an inhibitor of the complement cascade. A portion of the C4BP in serum exists as a complex with protein S. This study demonstrated that another protein, serum amyloid P component (SAP), also formed a calcium-dependent complex with C4BP. The C4BP.SAP complex was detected by several methods including light scattering intensity, gel filtration, and sucrose density gradient ultracentrifugation. This complex was of high affinity relative to serum levels of these proteins so that no dissociation was detected at 3% of serum protein concentrations. The C4BP.SAP complex was also detected in normal serum and the results suggested that there was virtually no free SAP or uncomplexed C4BP in normal serum. In addition to its complex with C4BP, SAP underwent other calcium-dependent associations such as binding to phospholipid vesicles and self-aggregation. Self-aggregation was highly cooperative with kinetics corresponding to a reaction that was 6th-order with respect to calcium and required about 1.5 mM calcium. In contrast, formation of the SAP.C4BP complex and interaction of SAP with membranes required only about 0.4 and 1.0 mM calcium, respectively. Thus, selection of the correct conditions allowed study of the SAP.C4BP interaction without interference from self-aggregation. All three of these interactions of SAP were mutually exclusive and the SAP. C4BP interaction appeared to be favored over self-aggregation or binding of SAP to phospholipids. It seems likely that the biologically dominant interaction for SAP is with C4BP. The SAP.C4BP complex interacted with protein S and these binding sites appeared to be entirely independent. Furthermore, SAP had little or no effect on the ability of C4BP to bind C4b. Finally, the entire complex of proteins (C4BP, SAP, protein S, and C4b) could associate with membranes in the presence of calcium. Membrane binding occurred through the protein S component. This rather complicated assemblage of proteins probably functions in a regulatory role for the complement cascade or other biological systems. It is possible that elevated levels of SAP or nonequivalent levels of SAP and C4BP could contribute to certain pathological conditions.     

The inverse relation of HDL anti‐oxidative functionality with serum amyloid a is lost in metabolic syndrome subjects

Objective:

Anti‐oxidative properties of high density lipoproteins (HDL) are relevant for atheroprotection. HDL carry serum amyloid A (SAA), which may impair HDL functionality. We questioned whether HDL anti‐oxidative capacity is determined by SAA.

Design and Methods:

Relationships of HDL anti‐oxidative capacity (% inhibition of low density lipoprotein oxidation in vitro) with SAA were determined in 54 non‐diabetic subjects without metabolic syndrome (MetS) and 68 subjects with MetS (including 51 subjects with Type 2 diabetes mellitus).

Results:

SAA levels were higher in MetS subjects, coinciding higher high sensitive C‐reactive protein (hs‐CRP) and lower HDL cholesterol and apolipoprotein (apo) A‐I levels (P<0.001 for all). HDL anti‐oxidative capacity was not different between subjects with and without MetS (P=0.76), but the HDL anti‐oxidation index (HDL anti‐oxidative capacity multiplied by individual HDL cholesterol concentrations), as a measure of global anti‐oxidative functionality of HDL, was lower in Mets subjects (P<0.001). HDL anti‐oxidative capacity was correlated inversely with SAA levels in subjects without MetS (r=‐0.286, P=0.036). Notably, this relationship was independent of HDL cholesterol or apoA‐I (P<0.05 for both). In contrast, no relation of HDL anti‐oxidative capacity with SAA was observed in MetS subjects (r=0.032, P=0.80). The relationship of SAA with HDL anti‐oxidative capacity was different in subjects with MetS compared to subjects without MetS (P=0.039 for the interaction between the presence of MetS and SAA on HDL anti‐oxidative capacity) taking age and diabetes status into account.

Conclusion:

Higher SAA levels may impair HDL anti‐oxidative functionality. The relationship of this physiologically relevant HDL functionality measure with circulating SAA levels is apparently disturbed in metabolic syndrome.