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.     

Selected expression profiling of full-length proteins and their variants in human plasma

With increased interest in clinical proteomics—the comparative investigation of differential protein expression patterns for use in the diagnostic and prognostic assessment of disease states—the demand for techniques that can readily identify changes in select proteome components is greater than ever before. This article describes a targeted proteomics approach to recover and quantify C-reactive protein (CRP) directly from human plasma. CRP, a putative biomarker for cardiac health, was isolated from microliter volumes of human plasma by using novel proteomics tools that combine micro-scale affinity capture with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) detection. Native CRP was analyzed along with serum amyloid P component (SAP) and retinol binding protein (RBP), that were intentionally targeted to generate a selected protein expression profile. A number of qualitative changes were readily observed within these profiles, including micro heterogeneity in the SAP glycan, C-terminally truncated versions of RBP, and detection of a novel truncated variant of CRP. After quantitative validation of increasing plasma CRP concentrations, the approach was applied to the analysis of eight plasma samples obtained from individuals with known medical histories. The result of the analyses are eight protein profiles, revealing increasing CRP levels that can be associated with individuals ailing from post-surgery inflammation, chronic rheumatoid arthritis, and recent acute myocardial infarction. The technique described in this article lays the foundation for selected protein profiling for use in biomarker discovery, as well as in clinical and diagnostic applications.     

Identification of a plasma proteomic signature to distinguish pediatric osteosarcoma from benign osteochondroma

Osteosarcoma (OS) is the most common malignant bone tumor in children. To identify a plasma proteomic signature that can detect OS, we used SELDI MS to perform proteomic profiling on plasma specimens from 29 OS and 20 age-matched osteochondroma (OC) patients. Nineteen statistically significant ion peaks that were differentially expressed in OS when compared with OC patients were identified (p < 0.001 and false discovery rate < 10%). Using the proteomic profiles, we constructed a multivariate 3-nearest neighbors classifier to distinguish OS from OC patients with a sensitivity of 97% and a specificity of 80% based on external leave-one-out crossvalidation. Permutation test showed that the classification result was statistically significant (p < 0.00005). One of the proteins (m/z 11 704) in the proteomic signature was identified as serum amyloid protein A (SAA) by PMF. The higher plasma level of SAA in OS patients was further validated by Western blotting when compared to that of osteochrondroma patients and normal subjects as reference. The classifier based on this plasma proteomic signature may be useful to differentiate malignant bone cancer from benign bone tumors and for early detection of OS in high-risk individuals.     

Identification of tumor-associated plasma biomarkers using proteomic techniques: from mouse to human

In an effort to identify tumor-associated proteins from plasma of tumor-bearing mice that may be used as diagnostic biomarkers, we developed a strategy that combines a tumor xenotransplantation model in nude mice with comparative proteomic technology. Five human cancer cell lines (SC-M1, HONE-1, CC-M1, OECM1, GBM 8401) derived from stomach, nasopharyngeal, colon, oral and brain cancers were subcutaneously inoculated into nude mice and compared to control nude mice injected with phosphate-buffered saline. One month later, plasma from mice inoculated with cancer cells was collected for proteomic analysis using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Comparison of plasma 2-DE maps from tumor-bearing mice with those produced from control mice revealed the overexpression of several mouse acute phase proteins (APPs) such as haptoglobin. Another APP, serum amyloid A (SAA), was found only in mice bearing tumors induced by the stomach cancer cell line SC-M1, which has not previously been demonstrated in xenotransplatation experiment. Furthermore, by using immunohistochemistry, SAA and haptoglobin were found to originate from the mouse hosts and not from the human cancer cell line donors. The protein alterations were further confirmed on patients with stomach cancers where up-regulated levels of SAA were also observed. These results indicate that APPs may be used as nonspecific tumor-associated serum markers. SAA in particular may serve as a potential marker for detecting stomach cancer. Taken together, the combination of the xenotransplatation model in nude mice and proteomics analysis provided a valuable impact for clinical applications in cancer diagnostics. In addition, our findings demonstrate that a panel of APPs might serve as screening biomarkers for early cancer detection.     

Structural basis of ligand specificity in the human pentraxins, C-reactive protein and serum amyloid P component

The normal physiological roles of the phylogenetically conserved human plasma proteins C-reactive protein (CRP) and serum amyloid P component (SAP) are not known. Novel drugs targeting their ligand specificities are in clinical development as both proteins have significant pathophysiological effects, SAP in promoting amyloidosis and CRP in exacerbating ischemic injury. Both proteins bind to phosphoethanolamine and we show here that, under physiological conditions, phosphoethanolamine is bound with higher affinity by human SAP than by human CRP. An explanation is provided by X-ray crystal structures that show SAP residue Tyr74 allowing additional hydrophobic protein-ligand interactions compared with the equivalent Thr76 of CRP. Docking simulations show many more low energy positions for phosphoethanolamine bound by CRP than by SAP and are consistent with the crystallographic and functional binding results. These fundamental observations on structure-activity relationships will aid the design of improved pentraxin targeting drugs.     

Complete cDNA sequence of SAP-like pentraxin from Limulus polyphemus: implications for pentraxin evolution

The serum amyloid P component (SAP)-like pentraxin Limulus polyphemus SAP is a recently discovered, distinct pentraxin species, of known structure, which does not bind phosphocholine and whose N-terminal sequence has been shown to differ markedly from the highly conserved N terminus of all other known horseshoe crab pentraxins. The complete cDNA sequence of Limulus SAP, and the derived amino acid sequence, the first invertebrate SAP-like pentraxin sequence, have been determined. Two sequences were identified that differed only in the length of the 3' untranslated region. Limulus SAP is synthesised as a precursor protein of 234 amino acid residues, the first 17 residues encoding a signal peptide that is absent from the mature protein. Phylogenetic analysis clusters Limulus SAP pentraxin with the horseshoe crab C-reactive proteins (CRPs) rather than the mammalian SAPs, which are clustered with mammalian CRPs. The deduced amino acid sequence shares 22% identity with both human SAP and CRP, which are 51% identical, and 31-35% with horseshoe crab CRPs. These analyses indicate that gene duplication of CRP (or SAP), followed by sequence divergence and the evolution of CRP and/or SAP function, occurred independently along the chordate and arthropod evolutionary lines rather than in a common ancestor. They further indicate that the CRP/SAP gene duplication event in Limulus occurred before both the emergence of the Limulus CRP variants and the mammalian CRP/SAP gene duplication. Limulus SAP, which does not exhibit the CRP characteristic of calcium-dependent binding to phosphocholine, is established as a pentraxin species distinct from all other known horseshoe crab pentraxins that exist in many variant forms sharing a high level of sequence homology.     

New insights into the role of serum amyloid P component, a novel lipopolysaccharide-binding protein

Serum amyloid P component (SAP) is a highly preserved plasma protein named for its ubiquitous presence in amyloid deposits. Although SAP is described to bind many ligands, no clear biological function has been ascribed to it as yet. This review summarizes the current knowledge about the protein SAP, its ligands and functional properties. Finally, the author focuses on the recent finding of the binding of SAP to lipopolysaccharide (LPS) and Gram-negative bacteria and the possible functional consequences of these interactions.     

快速康复外科理念对结直肠癌根治术患者疗效及机体应激反应的影响

目的:探讨快速康复外科理念应用于结直肠癌根治术患者的疗效及其对机体应激反应的影响。方法:选取2012年1月-2013年6月我院收治的180例结直肠癌患者为研究对象,采用随机数字表法分为对照组与观察组,每组各90例。两组患者均进行结直肠癌根治术治疗,对照组患者围手术期采用传统处理措施,观察组患者围手术期采用快速康复处理措施,对比两组患者术中出血量、手术时间、术后住院时间、住院费用、术后排便时间、首次排气时间,同时比较两组患者手术当日、术后1 d、5 d血清白细胞介素-6(IL-6)、C反应蛋白(CRP)及血清淀粉样蛋白A(SAA)的水平,并观察两组患者并发症发生情况。结果:与对照组相比,观察组患者的术后住院时间、术后排便时间及首次排气时间均缩短,住院费用降低,差异有统计学意义(P0.05),两组患者术中出血量、手术时间对比差异无统计学意义(P0.05)。术前,两组患者的血清IL-6、CRP、SAA水平对比差异无统计学意义(P0.05),术后1 d、5 d,两组患者血清IL-6、CRP、SAA水平均高于术前,术后5 d血清IL-6、CRP、SAA水平低于术后1 d,差异有统计学意义(P0.05),观察组患者术后1 d、5 d血清IL-6、CRP、SAA水平均低于对照组,差异有统计学意义(P0.05)。观察组并发症总发生率为6.67%,与对照组的7.78%比较差异无统计学意义(P0.05)。结论:快速康复外科理念应用于结直肠癌根治术患者能够有效加快患者术后康复,降低术后应激反应,值得临床推广。     

脓毒症患儿血清SAA、PCT、CRP水平与预后的关系及其诊断价值分析

摘要 目的：探讨脓毒症患儿血清淀粉样蛋白A（SAA）、降钙素原（PCT）、C反应蛋白（CRP）与预后的关系,并分析三者对脓毒症的诊断价值。方法：纳入我院于2016年8月～2020年6月期间收治的脓毒症患儿60例开展回顾性研究,作为脓毒症组,选取同期于我院进行体检的健康儿童40例作为对照组,比较两组血清SAA、PCT、CRP水平。根据脓毒症患儿1个月内的生存、死亡情况,分成生存组（n=42）、死亡组（n=18）,比较两组临床资料及血清SAA、PCT、CRP水平,经COX回归模型分析脓毒症患儿死亡的危险因素。绘制受试者工作特征（ROC）曲线分析血清SAA、PCT、CRP对脓毒症的诊断价值。结果：脓毒症组血清SAA、PCT、CRP水平显著高于对照组（P＜0.05）。死亡组器官障碍数量＞2个、脓毒性休克患儿占比分别为55.56%、44.44%,显著高于生存组的19.05%、9.52%（P＜0.05）;死亡组入院后1 h内使用抗菌治疗患儿占比为38.89%,显著低于生存组的69.05%（P＜0.05）;死亡组血清SAA、PCT、CRP水平高于生存组（P＜0.05）。COX多因素分析结果显示,器官障碍数量＞2、脓毒性休克及血清SAA、PCT、CRP水平升高是脓毒症患儿死亡的危险因素（P＜0.05）,而入院后1 h内使用抗菌治疗是脓毒症患儿死亡风险的保护性因素（P＜0.05）。血清SAA、PCT、CRP单独及三者联合诊断脓毒症的曲线下面积（AUC）分别为0.808、0.780、0.761、0.912。结论：脓毒症患儿血清SAA、PCT、CRP明显升高,三者升高均为脓毒症患儿死亡的危险因素,且对脓毒症具有一定诊断价值。     

胃癌及癌旁组织定量比较蛋白质组学研究

为寻找胃癌特异的肿瘤标记物,用于胃癌临床诊断及药物治疗靶点的选择,本研究采用荧光差异显示凝胶电泳（DIGE）技术分离并筛选 Cy3、Cy5 及 Cy2 荧光素标记的胃癌及对应癌旁组织差异表达蛋白质,用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）或串联质谱技术进行鉴定并分析。结果共筛选出 33 个差异表达蛋白质点,其中 9 个蛋白质点在胃癌组织中上调,24 个蛋白质点下调。对 22 个蛋白质点采用质谱技术成功鉴定,突变结蛋白、锰超氧化物歧化酶、热休克蛋白 60等在胃癌中高表达,热休克蛋白 27、前列腺素 F 合酶、硒结合蛋白 1、锌指蛋白 160、微管蛋白 α6、真核生物翻译延伸因子 1 α1 等在胃癌组织中低表达,并筛选出 5 个未知蛋白。这些差异表达蛋白可望成为胃癌诊断的特异标记物,并与胃癌的发生、发展及预后等有关,为胃癌的诊断、发生机制的研究提供了新的思路。     

Identification and validation of a potential lung cancer serum biomarker detected by matrix-assisted laser desorption/ionization-time of flight spectra analysis

Many abnormalities detected in the thorax by routine conventional imaging studies are benign, yet all require further evaluation because of the concern for cancer. To address this deficiency and develop a serum biomarker for lung cancer, we designed a matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) based platform to display the proteins present in the serum of patients with or without lung cancer, and then challenged the scientific community to analyze these data with the aim of determining specific ion signal differences among the resulting spectra. The most statistically significant ion peak identified by the various analysis algorithms that differentiated the serum of patients with lung cancer from the serum of individuals without lung cancer was found at m/z 11,702. We identified the protein responsible for this ion peak as serum amyloid A (SAA; M(r) = 11,682.7) by partial purification followed by in-gel digestion and peptide mapping. By enzyme-linked immunosorbent assay, we showed SAA to be present at 286 ng/mL in the serum of cancer patients vs. 34.1 ng/mL in the serum of individuals without cancer. These data suggest that the combination of MALDI-TOF MS and computer analysis can be a powerful tool in the search for serum biomarkers of lung cancer and other diseases.     

Serum amyloid A enhances plasminogen activation: implication for a role in colon cancer

We have recently reported that the acute phase protein serum amyloid A (SAA), is locally and differentially expressed in neoplastic tissues of human colon. In the present study, we demonstrate that SAA enhances the plasminogen activation (PA)-activity of HT-29 colon cancer cell line. Cell-associated PA-activity was measured following the plasminogen-dependent ability of the cells to cleave the chromogenic substrate S-2251. The SAA-enhanced PA-activity was inhibited by anti-SAA antibodies. These antibodies also decreased the basal PA-activity of HT-29 cells and neutralized their cytokines (Interleukin-1β + Interleukin-6)—enhanced PA-activity. Using specific chromogenic substrates and the fibrin clot-lysis assay, we found that SAA enhances also the PA-activity mediated by purified urokinase- and tissue-type plasminogen activators. Together, the data indicate that SAA enhances plasminogen activation and suggest its possible role in plasmin(ogen)-mediated colon cancer progression.     

淀粉样蛋白A、D-二聚体、肌酸激酶同工酶联合检测对川崎病患儿冠状动脉损伤的诊断价值分析

摘要 目的：探讨血清淀粉样蛋白A(SAA)、D-二聚体(D-D)、肌酸激酶同工酶(CK-MB)联合检测对川崎病患儿冠状动脉损伤(CAL)的诊断价值。方法：选取2018年9月～2021年5月我院收治的80例川崎病患儿,根据是否合并CAL分为CAL组(n=34)和NCAL组(n=46)。收集患儿基础资料,并检测SAA、D-D、CK-MB水平。多因素Logistic回归分析川崎病患儿CAL影响因素,受试者工作特征（ROC）曲线分析血清SAA、D-D、CK-MB水平对川崎病患儿CAL的诊断价值。结果：与NCAL组比较,CAL组C反应蛋白(CRP)、红细胞沉降率(ESR)、SAA、D-D、CK-MB水平升高(P＜0.05)。多因素Logistic回归分析显示,CRP、ESR、SAA、D-D、CK-MB为川崎病患儿CAL独立影响因素(P＜0.05)。SAA、D-D、CK-MB、三项联合诊断川崎病患儿CAL的曲线下面积（AUC）分别为0.661、0.687、0.746、0.799,联合应用的诊断效能最高。结论：血清SAA、D-D、CK-MB是川崎病患儿CAL独立影响因素,且联合检测以上指标可辅助诊断川崎病患儿CAL。     

Association of serum amyloid A levels with adipocyte size and serum levels of adipokines: Differences between men and women

The aim of this study was to characterize the association between adipocyte enlargement and circulating levels of serum amyloid A (SAA). Furthermore, we wanted to search for possible associations with measures of glycemic control and levels of circulating adipokines and/or inflammatory markers in men and women with a large range in body mass index. The study cohort consisted of 167 subjects, 114 non-diabetic and 53 with Type 2 diabetes. Adipocyte diameter as well as circulating levels of SAA, C-reactive protein (CRP), adiponectin, leptin, interleukin-6, tumor necrosis factor alpha, glucose and insulin were measured. Women had higher serum levels of SAA than men (p = 0.044). SAA levels were weakly but positively correlated with BMI (p = 0.043) and % body fat (p = 0.027) in all subjects as well as subcutaneous adipocyte diameter (p = 0.034) in women. Furthermore, in all subjects we found correlations between SAA levels and levels of CRP (p < 0.001), interleukin-6 (p < 0.001), leptin (p = 0.003), insulin (p = 0.006), HbA1c (p = 0.02) and HOMA-IR (p = 0.002). A majority of the correlations were strongest in women. In conclusion, serum levels of SAA are strongly correlated with serum levels of inflammatory markers as well as measures of glycemic control. There seems to be large sex differences in these associations suggesting that sex-specific factors need to be considered when analyzing SAA levels in relation to metabolic disease.     

Urea-induced denaturation of apolipoprotein serum amyloid A reveals marginal stability of hexamer

Serum Amyloid A (SAA) is an acute phase reactant protein that is predominantly found bound to high-density lipoprotein in plasma. Upon inflammation, the plasma concentration of SAA can increase dramatically, occasionally leading to the development of amyloid A (AA) amyloidosis, which involves the deposition of SAA amyloid fibrils in major organs. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a hexamer containing a central channel. Here we show using various biophysical and biochemical techniques that the SAA2.2 hexamer can be totally dissociated into monomer by approximately 2 M urea, with the concerted loss of its alpha-helical structure. However, limited trypsin proteolysis experiments in urea showed a conserved digestion profile, suggesting the preservation of major backbone topological features in the urea-denatured state of SAA2.2. The marginal stability of hexameric SAA2.2 and the presence of residual structure in the denatured monomeric protein suggest that both forms may interconvert in vivo to exert different functions to meet the various needs during normal physiological conditions and in response to inflammatory stimuli.     

新型冠状病毒肺炎患者血清SAA、ESR、CRP检测结果分析

目的:分析新型冠状病毒肺炎(COVID-19)患者血清淀粉样蛋白A(SAA)、血沉ESR(ESR)、C-反应蛋白CRP(CRP)的临床价值。方法:以我院2020年1月至2020年2月确诊的COVID-19患者51例为实验组:其中重症患者21例,轻症患者30例,选取非感染患者30例为对照组。分析新型冠状病毒肺炎(COVID-19)患者血清SAA、ESR、CRP水平以及临床价值。结果:实验组SAA、ESR、CRP水平高于对照组(P0.05);重症组SAA、ESR、CRP水平高于轻症组(P0.05),由ROC曲线得知SAA、ESR、CRP预测COVID-19的AUC小于三者联合预测的AUC。结论:COVID-19的SAA、ESR、CRP水平会升高,三者联合预测为疾病评估、预测疾病发展的提供依据,值得临床进一步推广应用。     

The interaction of C-reactive protein and serum amyloid P component with nuclear antigens

The pentraxins are a family of proteins characterized by cyclic pentameric structure, calcium-dependent ligand binding and sequence homology. The two main representatives of this family are the serum proteins, C-reactive protein (CRP) and serum amyloid P component (SAP). In man CRP is an acute phase reactant which increases up to 1000 fold during the acute phase response whereas SAP is a constitutive protein expressed at about 30 g/ml. These proteins activate complement through the classical pathway and participate in opsonization of particulate antigens and bacteria. In the past several years it has been determined that both of these pentraxins interact with nuclear antigens including chromatin and small nuclear ribonucleoproteins (snRNPs). Both CRP and SAP have nuclear transport signals which facilitate their entry into the nuclei of intact cells. Furthermore, these pentraxins have been shown to affect the clearance of nuclear antigens in vivo. It is now believed that one of the major functions of the pentraxins could be to interact with the nuclear antigens released from apoptotic or necrotic cells. This interaction could mitigate against deposition of these antigens in tissue and autoimmune reactivity.Abbreviations CRP C-reactive protein - HSA human serum albumin - PC phosphocholine - SAP serum amyloid P component - snRNP small nuclear ribonucleoprotein - SLE systemic lupus erythematosus     

Serum amyloid A promotes ABCA1-dependent and ABCA1-independent lipid efflux from cells

Serum amyloid A (SAA) is an acute phase protein that associates with HDL. In order to examine the role of SAA in reverse-cholesterol transport, lipid efflux was tested to SAA from HeLa cells before and after transfection with the ABCA1 transporter. ABCA1 expression increased efflux of cholesterol and phospholipid to SAA by 3-fold and 2-fold, respectively. In contrast to apoA-I, SAA also removed lipid without ABCA1; cholesterol efflux from control cells to SAA was 10-fold higher than for apoA-I. Furthermore, SAA effluxed cholesterol from Tangier disease fibroblasts and from cells after inhibition of ABCA1 by fixation with paraformaldehyde. In summary, SAA can act as a lipid acceptor for ABCA1, but unlike apoA-I, it can also efflux lipid without ABCA1, by most likely a detergent-like extraction process. These results suggest that SAA may play a unique role as an auxiliary lipid acceptor in the removal of lipid from sites of inflammation.     

Serum amyloid A and inflammasome activation: A link to breast cancer progression?

Breast cancer is the most frequently diagnosed cancer in women globally. Although there have been many significant advances made in the diagnosis and treatment of breast cancer, numerous unresolved challenges remain, which include prevention, early diagnosis, metastasis and recurrence. The role of inflammation in cancer development is well established and is believed to be one of the leading hallmarks of cancer progression. Recently, the role of the inflammasome, a cytosolic multiprotein complex, has received attention in different cancers. By contributing to the activation of inflammatory cytokines the inflammasome intensifies the inflammatory cascade. The inflammasome can be activated through several pathways, which include the binding of pattern associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) to toll-like receptors (TLRs). Serum amyloid A (SAA), a non-specific acute-phase protein, can function as an endogenous DAMP by binding to pattern recognition receptors like TLRs on both breast cancer cells and cancer associated fibroblasts (CAFs). SAA can thus stimulate the production of IL-1β, thereby creating a favourable inflammatory environment to support tumour growth. The aim of this review is to highlight the possible role of SAA as an endogenous DAMP in the tumour microenvironment (TME) thereby promoting breast cancer growth through the activation of the NLRP3 inflammasome.