DWN12088, A Prolyl-tRNA Synthetase Inhibitor, Alleviates Hepatic Injury in Nonalcoholic Steatohepatitis.

BACKGRUOUND: Nonalcoholic steatohepatitis (NASH) is a liver disease caused by obesity that leads to hepatic lipoapoptosis, resulting in fibrosis and cirrhosis. However, the mechanism underlying NASH is largely unknown, and there is currently no effective therapeutic agent against it. DWN12088, an agent used for treating idiopathic pulmonary fibrosis, is a selective prolyl-tRNA synthetase (PRS) inhibitor that suppresses the synthesis of collagen. However, the mechanism underlying the hepatoprotective effect of DWN12088 is not clear. Therefore, we investigated the role of DWN12088 in NASH progression. METHODS: Mice were fed a chow diet or methionine-choline deficient (MCD)-diet, which was administered with DWN12088 or saline by oral gavage for 6 weeks. The effects of DWN12088 on NASH were evaluated by pathophysiological examinations, such as real-time quantitative reverse transcription polymerase chain reaction, immunoblotting, biochemical analysis, and immunohistochemistry. Molecular and cellular mechanisms of hepatic injury were assessed by in vitro cell culture. RESULTS: DWN12088 attenuated palmitic acid (PA)-induced lipid accumulation and lipoapoptosis by downregulating the Rho-kinase (ROCK)/AMP-activated protein kinase (AMPK)/sterol regulatory element-binding protein-1c (SREBP-1c) and protein kinase R-like endoplasmic reticulum kinase (PERK)/α subunit of eukaryotic initiation factor 2 (eIF2α)/activating transcription factor 4 (ATF4)/C/EBP-homologous protein (CHOP) signaling cascades. PA increased but DWN12088 inhibited the phosphorylation of nuclear factor-κB (NF-κB) p65 (Ser536, Ser276) and the expression of proinflammatory genes. Moreover, the DWN12088 inhibited transforming growth factor ß (TGFß)-induced pro-fibrotic gene expression by suppressing TGFß receptor 1 (TGFßR1)/Smad2/3 and TGFßR1/glutamyl-prolyl-tRNA synthetase (EPRS)/signal transducer and activator of transcription 6 (STAT6) axis signaling. In the case of MCD-diet-induced NASH, DWN12088 reduced hepatic steatosis, inflammation, and lipoapoptosis and prevented the progression of fibrosis. CONCLUSION: Our findings provide new insights about DWN12088, namely that it plays an important role in the overall improvement of NASH. Hence, DWN12088 shows great potential to be developed as a new integrated therapeutic agent for NASH.

Emerging Roles of Vascular Cell Adhesion Molecule-1 (VCAM-1) in Immunological Disorders and Cancer.

Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that triggers the expression of inflammatory molecules, including other cytokines and cell adhesion molecules. TNFα induces the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 was originally identified as a cell adhesion molecule that helps regulate inflammation-associated vascular adhesion and the transendothelial migration of leukocytes, such as macrophages and T cells. Recent evidence suggests that VCAM-1 is closely associated with the progression of various immunological disorders, including rheumatoid arthritis, asthma, transplant rejection, and cancer. This review covers the role and relevance of VCAM-1 in inflammation, and also highlights the emerging potential of VCAM-1 as a novel therapeutic target in immunological disorders and cancer.

A Review of Anti-Angiogenic Targets for Monoclonal Antibody Cancer Therapy.

Tumor angiogenesis is a key event that governs tumor progression and metastasis. It is controlled by the complicated and coordinated actions of pro-angiogenic factors and their receptors that become upregulated during tumorigenesis. Over the past several decades, vascular endothelial growth factor (VEGF) signaling has been identified as a central axis in tumor angiogenesis. The remarkable advent of recombinant antibody technology has led to the development of bevacizumab, a humanized antibody that targets VEGF and is a leading clinical therapy to suppress tumor angiogenesis. However, despite the clinical efficacy of bevacizumab, its significant side effects and drug resistance have raised concerns necessitating the identification of novel drug targets and development of novel therapeutics to combat tumor angiogenesis. This review will highlight the role and relevance of VEGF and other potential therapeutic targets and their receptors in angiogenesis. Simultaneously, we will also cover the current status of monoclonal antibodies being developed to target these candidates for cancer therapy.

Systematic investigation of protein kinase A substrate proteins using on-chip protein kinase kinetic profiling.

Protein kinases play a critical role in a wide variety of cellular processes through post-translational protein phosphorylation and identification of their substrate proteins is important for understanding the enzymes' mechanism of action and elucidating their functions in physiological and disease processes. However, there have been few systematic investigations of protein kinase substrates due to a lack of high-throughput kinetic assays. Thus, we designed an on-chip protein kinase assay for profiling kinase kinetic parameters by introducing the phosphorylation rate (Vp) under physiological conditions, instead of the maximal velocity (Vmax), in a high-throughput manner. We applied this method to the kinetic analysis of protein kinase A (PKA) for 28 nuclear, cytosolic, plasma membrane, and extracellular target proteins to determine the substrate affinity (Km), Vp (FIATP10/s), and substrate preference (Vp/Km). We then constructed a map of PKA's kinetic parameters with respect to the 28 proteins based on subcellular localization. Thus, the on-chip protein kinase kinetic profiling is an ideal approach for systematically investigating substrate proteins of protein kinases and fully characterizing the enzymes' physiological functions.

Plasmodium vivax GPI-anchored micronemal antigen (PvGAMA) binds human erythrocytes independent of Duffy antigen status.

Plasmodium vivax, a major agent of malaria in both temperate and tropical climates, has been thought to be unable to infect humans lacking the Duffy (Fy) blood group antigen because this receptor is critical for erythrocyte invasion. Recent surveys in various endemic regions, however, have reported P. vivax infections in Duffy-negative individuals, suggesting that the parasite may utilize alternative receptor-ligand pairs to complete the erythrocyte invasion. Here, we identified and characterized a novel parasite ligand, Plasmodium vivax GPI-anchored micronemal antigen (PvGAMA), that bound human erythrocytes regardless of Duffy antigen status. PvGAMA was localized at the microneme in the mature schizont-stage parasites. The antibodies against PvGAMA fragments inhibited PvGAMA binding to erythrocytes in a dose-dependent manner. The erythrocyte-specific binding activities of PvGAMA were significantly reduced by chymotrypsin treatment. Thus, PvGAMA may be an adhesion molecule for the invasion of Duffy-positive and -negative human erythrocytes.

Identification of transglutaminase 2 kinase substrates using a novel on-chip activity assay.

Transglutaminase 2 (TG2) is an enzyme that plays a critical role in a wide variety of cellular processes through its multifunctional activities. TG2 kinase has emerged as an important regulator of apoptosis, as well as of chromatin structure and function. However, systematic investigation of TG2 kinase substrates is limited due to a lack of a suitable TG2 kinase activity assays. Thus, we developed a novel on-chip TG2 kinase activity assay for quantitative determination of TG2 kinase activity and for screening TG2 kinase substrate proteins in a high-throughput manner. Quantitative TG2 kinase activity was determined by selective detection of substrate protein phosphorylation on the surface of well-type amine arrays. The limit of detection (LOD) of this assay was 4.34µg/ml. We successfully applied this new activity assay to the kinetic analysis of 27 TG2-related proteins for TG2 kinase activity in a high-throughput manner and determined Michaelis-Menten constants (Km) of these proteins. We used the Km values and cellular locations of the TG2-related proteins to construct a substrate affinity map for TG2 kinase. Therefore, this on-chip TG2 kinase activity assay has a strong potential for the systematic investigation of substrate proteins and will be helpful for studying new physiological functions.

Sensitive array-based assay for determination of serological protein kinase A autoantibody levels based on its antigen protein activation.

OBJECTIVES: We investigated the effect of cPKAα conformational states during protein immobilization on an array platform for cPKA autoantibody assays for sensitive and high-throughput profiling of protein kinase A (PKA) autoantibody levels in human sera. DESIGN AND METHODS: We prepared activated human cPKAα protein arrays by addition of cofactors including ATP, MgCl2, and Triton X-100 to incubation buffer. Anti-human cPKAα antibody or PKA autoantibody levels in human sera were analyzed using activated human cPKAα protein arrays. RESULTS: Activation of cPKAα with ATP, Mg(2+), and Triton X-100 enhanced the sensitivity of the assay by increasing the signal/noise ratio and lowering the limit of detection. cPKAα activation also enhanced the sensitivity of cPKA autoantibody detection in human sera. We successfully applied this assay to determine cPKA autoantibody levels in human sera from normal individuals (n=30) and hepatic cancer patients (n=30). CONCLUSIONS: Our results demonstrate that cPKAα activation enhanced the sensitivity of array-based PKA autoantibody assays, and that this assay is suitable for high-throughput analyses of cPKA autoantibodies in human sera.

A peptide array-based serological protein kinase A activity assay and its application in cancer diagnosis.

Protein kinase A (PKA) plays a crucial role in several biological processes; however, there is no assay with sufficient sensitivity and specificity to determine serological PKA (sPKA) activity. Here we present an on-chip activity assay that employs cysteine-modified kemptide arrays to determine specific sPKA activity in human sera that eliminates the potential contributions of other kinases with a protein kinase peptide inhibitor. The sensitivity of the on-chip sPKA activity assay was greatly enhanced by Triton X-100, with a 0.01 U mL(-1) detection limit. sPKA activity was determined by subtracting nonspecific sPK activity from total sPK activity. Our assay provided greater sensitivity and specificity and more accurate area under the curve values for gastric cancer compared to the total sPK activity assay. sPKA activities in human sera from patients with hepatic (n = 30), gastric (n = 30), lung (n = 30), and colorectal (n = 30) cancers were significantly higher than those in controls (n = 30, p < 10(-4)), but no significant difference in sPKA activities between normal and inflammation groups was observed. These results demonstrate that the on-chip assay accurately measures sPKA activity in human sera and that the sPKA activity may be a potential biomarker for cancer diagnosis.

Characterization of Plasmodium vivax Early Transcribed Membrane Protein 11.2 and Exported Protein 1.

In Plasmodium, the membrane of intracellular parasites is initially formed during invasion as an invagination of the red blood cell surface, which forms a barrier between the parasite and infected red blood cells in asexual blood stage parasites. The membrane proteins of intracellular parasites of Plasmodium species have been identified such as early-transcribed membrane proteins (ETRAMPs) and exported proteins (EXPs). However, there is little or no information regarding the intracellular parasite membrane in Plasmodium vivax. In the present study, recombinant PvETRAMP11.2 (PVX_003565) and PvEXP1 (PVX_091700) were expressed and evaluated antigenicity tests using sera from P. vivax-infected patients. A large proportion of infected individuals presented with IgG antibody responses against PvETRAMP11.2 (76.8%) and PvEXP1 (69.6%). Both of the recombinant proteins elicited high antibody titers capable of recognizing parasites of vivax malaria patients. PvETRAMP11.2 partially co-localized with PvEXP1 on the intracellular membranes of immature schizont. Moreover, they were also detected at the apical organelles of newly formed merozoites of mature schizont. We first proposed that these proteins might be synthesized in the preceding schizont stage, localized on the parasite membranes and apical organelles of infected erythrocytes, and induced high IgG antibody responses in patients.

Antigenicity and immunogenicity of PvRALP1, a novel Plasmodium vivax rhoptry neck protein.

BACKGROUND: Proteins secreted from the rhoptry in Plasmodium merozoites are associated with the formation of tight junctions and parasitophorous vacuoles during invasion of erythrocytes and are sorted within the rhoptry neck or bulb. Very little information has been obtained to date about Plasmodium vivax rhoptry-associated leucine (Leu) zipper-like protein 1 (PvRALP1; PVX_096245), a putative rhoptry protein. PvRALP1 contains a signal peptide, a glycine (Gly)/glutamate (Glu)-rich domain, and a Leu-rich domain, all of which are conserved in other Plasmodium species. METHODS: Recombinant PvRALP1s were expressed as full-length protein without the signal peptide (PvRALP1-Ecto) and as truncated protein consisting of the Gly/Glu- and Leu-rich domains (PvRALP1-Tr) using the wheat germ cell-free expression system. The immunoreactivity to these two fragments of recombinant PvRALP1 protein in serum samples from P. vivax-infected patients and immunized mice, including analysis of immunoglobulin G (IgG) subclasses, was evaluated by enzyme-linked immunosorbent assay or protein microarray technology. The subcellular localization of PvRALP1 in blood stage parasites was also determined. RESULTS: Recombinant PvRALP1-Ecto and PvRALP1-Tr proteins were successfully expressed, and in serum samples from P. vivax patients from the Republic of Korea, the observed immunoreactivities to these proteins had 58.9% and 55.4% sensitivity and 95.0% and 92.5% specificity, respectively. The response to PvRALP1 in humans was predominantly cytophilic antibodies (IgG1 and IgG3), but a balanced Th1/Th2 response was observed in mice. Unexpectedly, there was no significant inverse correlation between levels of parasitaemia and levels of antibody against either PvRALP1-Ecto (R2=0.11) or PvRALP1-Tr (R2=0.14) antigens. PvRALP1 was localized in the rhoptry neck of merozoites, and this was the first demonstration of the localization of this protein in P. vivax. CONCLUSIONS: This study analysed the antigenicity and immunogenicity of PvRALP1 and suggested that PvRALP1 may be immunogenic in humans during parasite infection and might play an important role during invasion of P. vivax parasites.

Profiling the humoral immune responses to Plasmodium vivax infection and identification of candidate immunogenic rhoptry-associated membrane antigen (RAMA).

Completion of sequencing of the Plasmodium vivax genome and transcriptome offers the chance to identify antigens among >5000 candidate proteins. To identify those P. vivax proteins that are immunogenic, a total of 152 candidate proteins (160 fragments) were expressed using a wheat germ cell-free system. The results of Western blot analysis showed that 92.5% (148/160) of the targets were expressed, and 96.6% (143/148) were in a soluble form with 67.7% of solubility rate. The proteins were screened by protein arrays with sera from 22 vivax malaria patients and 10 healthy individuals to confirm their immune profile, and 44 (27.5%, 44/160) highly reactive P. vivax antigens were identified. Overall, 5 candidates (rhoptry-associated membrane antigen [RAMA], Pv-fam-a and -b, EXP-1 and hypothetical protein PVX_084775) showed a positive reaction with >80% of patient sera, and 21 candidates with 50% to 80%. More than 23% of the highly immunoreactive proteins were hypothetical proteins, described for the first time in this study. One of the top immunogenic proteins, RAMA, was characterized and confirmed to be a serological marker of recent exposure to P. vivax infection. These novel immunoproteomes should greatly facilitate the identification of promising novel malaria antigens and may warrant further study. BIOLOGICAL SIGNIFICANCE: The establishment of high-throughput cloning and expression systems has permitted the construction of protein arrays for proteome-wide study of Plasmodium vivax. In this study, high-throughput screening assays have been applied to investigate blood stage-specific immune proteomes from P. vivax. We identified 44 antigenic proteins from the 152 putative candidates, more than 23% of which were hypothetical proteins described for the first time in this study. In addition, PvRAMA was characterized further and confirmed to be a serological marker of exposure to infections. The expression of one-third of the selected antigenic genes were shifted between P. vivax and Plasmodium falciparum, suggesting that these genes may represent important factors associated with P. vivax selectivity for young erythrocytes and/or with immune evasion. These novel immune proteomes of the P. vivax blood stage provide a baseline for further prospective serological marker studies in malaria. These methods could be used to determine immunodominant candidate antigens from the P. vivax genome.

Comparative characterization of direct and indirect substrate probes for on-chip transamidating activity assay of transglutaminases.

The development of molecular probes is a prerequisite for activity-based protein profiling. This strategy helps in characterizing the catalytic activity and function of proteins, and how these proteins and protein complexes control biological processes of interest. These probes are composed of a reactive functional group and a reporter tag. The reactive group of these substrate probes has been considered to be important to their design, while the significance of the reporter tag is relatively underestimated. In this study we compare TAMRA-cadaverine and biotin-cadaverine, two substrate probes that have different reporter tags but an identical reactive functional group. We assess the on-chip transamidating activity of two transglutaminases; transglutaminase 2 and blood coagulation factor XIII. Activity assays were more easily executed when using the direct probe TAMRA-cadaverine. However the indirect probe, biotin-cadaverine, provided a wider dynamic range, higher signal-to-noise ratio, and lower limit of detection compared to TAMRA-cadaverine. Additionally, we successfully used the on-chip activity assay using the indirect probe to determine TG2 and FXIII activities in Hela cell lysates and human plasma samples, respectively. These results demonstrate that the reporter tag of the substrate probe is critical for protocol execution, sensitivity, and dynamic range of enzyme activity assays. Furthermore, this study provides a helpful guide for development of new probes, which is necessary for the identification of potential biomarkers and therapeutic targets for treating enzyme-related diseases.

Serological responses to a soluble recombinant chimeric Plasmodium vivax circumsporozoite protein in VK210 and VK247 population.

BACKGROUND: Circumsporozoite protein (CSP) is essential for sporozoite formation and sporozoite invasion into human hepatocyte. Previously, a recombinant P. vivax CSP based on chimeric repeats (rPvCSP-c) representing two major alleles VK210 and VK247 within central region has been designed. Naturally acquired humoral immune responses study show that antigenicity of rPvCSP-c was much higher than that of native strain. However, the serologic reactivity of rPvCSP-c was still unclear in detail. METHODS: In present study, recognition of rPvCSP-c in vivax malaria typed VK210 and VK247 alleles was assessed. VK210 typed and VK247 typed sera from adult residents reacted specifically with rPvCSP-c using protein array and immunoblot assay. Additionally, anti-rPvCSP-c serum recognized the fixed VK210 and VK247 sporozoites by immunofluorescence assay. Furthermore, statistic analysis was performed for correlational detection. RESULTS: The rPvCSP-c reacted with both VK210 typed and VK247 typed P. vivax infected patient sera and anti-rPvCSP-c immune serum also reacted with VK210 and VK247 sporozoite parasites of P. vivax specifically. There was a positive correlation between increased antibody level, age of patients and also associated with pvcsp repeat number, although the level of responses did vary considerably in their reactivity to the rPvCSP-c from negative to very high level within each age group. CONCLUSIONS: These data confirmed the serologic reactivity of the novel rPvCSP-c in exposed both VK210 and VK247 populations. These results strongly suggested that this recombinant CSP was biologically active and potently immunogenic across major strains and raised the prospect that this protein could be used as serologic marker.

The Plasmodium vivax merozoite surface protein 1 paralog is a novel erythrocyte-binding ligand of P. vivax.

Merozoite surface protein 1 of Plasmodium vivax (PvMSP1), a glycosylphosphatidylinositol-anchored protein (GPI-AP), is a malaria vaccine candidate for P. vivax. The paralog of PvMSP1, named P. vivax merozoite surface protein 1 paralog (PvMSP1P; PlasmoDB PVX_099975), was recently identified and predicted as a GPI-AP. The similarities in genetic structural characteristics between PvMSP1 and PvMSP1P (e.g., size of open reading frames, two epidermal growth factor-like domains, and GPI anchor motif in the C terminus) led us to study this protein. In the present study, different regions of the PvMSP1P protein, demarcated based on the processed forms of PvMSP1, were expressed successfully as recombinant proteins [i.e., 83 (A, B, and C), 30, 38, 42, 33, and 19 fragments]. We studied the naturally acquired immune response against each fragment of recombinant PvMSP1P and the potential ability of each fragment to bind erythrocytes. The N-terminal fragment (83A) and two C-terminal fragments (33 and 19) reacted strongly with sera from P. vivax-infected patients, with 50 to 68% sensitivity and 95 to 96% specificity, respectively. Due to colocalization of PvMSP1P with PvMSP1, we supposed that PvMSP1P plays a similar role as PvMSP1 during erythrocyte invasion. An in vitro cytoadherence assay showed that PvMSP1P, especially the 19-kDa C-terminal region, could bind to erythrocytes. We also found that human sera from populations naturally exposed to vivax malaria and antisera obtained by immunization using the recombinant molecule PvMSP1P-19 inhibited in vitro binding of human erythrocytes to PvMSP1P-19. These results provide further evidence that the PvMSP1P might be an essential parasite adhesion molecule in the P. vivax merozoite and is a potential vaccine candidate against P. vivax.

Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays.

Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

Integrative proteomic profiling of protein activity and interactions using protein arrays.

Proteomic studies based on abundance, activity, or interactions have been used to investigate protein functions in normal and pathological processes, but their combinatory approach has not been attempted. We present an integrative proteomic profiling method to measure protein activity and interaction using fluorescence-based protein arrays. We used an on-chip assay to simultaneously monitor the transamidating activity and binding affinity of transglutaminase 2 (TG2) for 16 TG2-related proteins. The results of this assay were compared with confidential scores provided by the STRING database to analyze the functional interactions of TG2 with these proteins. We further created a quantitative activity-interaction map of TG2 with these 16 proteins, categorizing them into seven groups based upon TG2 activity and interaction. This integrative proteomic profiling method can be applied to quantitative validation of previously known protein interactions, and in understanding the functions and regulation of target proteins in biological processes of interest.

Monitoring of proteolytic enzyme activity using phase transition-based peptide arrays.

We have developed an assay using peptide arrays based on phase transition from the glass substrate to the liquid for monitoring quantitative protease activity in real-time. Peptide arrays were fabricated using a bifunctional cross-linker, N-[γ-maleimidobutyryloxy] sulfosuccinimide ester, and a substrate peptide containing two functional groups, cysteine and tetramethyl-6-carboxyrhodamine (TAMRA) on the C- and N-terminus, respectively. The phase transition-based peptide arrays were characterized by analyzing the substrate peptide cleaved from the solid substrate by matrix metalloproteinase-3 (MMP-3). We successfully used this assay to determine the quantitative proteolytic activity of MMP-3 in a dose-dependent manner. In addition, parameters including Michaelis constant (K(m)), maximum rate of enzymatic reaction (V(max)), and half maximal inhibitory concentration (IC(50)) were determined by analyzing the concentrations of substrate peptide cleaved by MMP-3. Therefore, this new assay has potential for the quantitative analysis of enzyme kinetics of protease and informs research developments in drug discovery utilizing kinetic studies.

Normalization using a tagged-internal standard assay for analysis of antibody arrays and the evaluation of serological biomarkers for liver disease.

For minimizing systemic experimental variation in the analysis of antibody array data, we developed a novel median-centered/IgM-tagged-internal standard (TIS) assay normalization using median-centering and TIS assay-based determination of serum IgM concentrations. We evaluated five normalization methods by analyzing correlation coefficients and coefficients of variation for six serum proteins using human serum samples from normal controls (n=25) and patients with liver cirrhosis (n=25) or hepatocellular carcinoma (HCC; n=29). Median-centered normalization improved correlation coefficients, while IgM-based normalizations improved coefficients of variation. The TIS assay was more efficient, economical, and reproducible for determining IgM concentrations than enzyme-linked immunosorbent assay. Additionally, we normalized antibody array data for six serum proteins using the median-centered/IgM-TIS assay, and evaluated serum biomarkers through distribution analysis of normalized fluorescence intensities and receiver operating characteristic analyses for the diagnosis of liver cirrhosis and HCC. Apolipoprotein A-1 and a combination of alpha-fetoprotein and C-reactive protein were determined to be potential serological biomarkers for liver cirrhosis and HCC, respectively. Thus, median-centered/IgM-TIS assay normalization is a useful approach for analyzing antibody array data and evaluating serological biomarkers for the diagnosis of liver disease or cancers.

Fucosylated glycoproteomic approach to identify a complement component 9 associated with squamous cell lung cancer (SQLC).

Human lung cancer is a major cause of cancer mortality worldwide. Understanding the pathophysiological features and the development of novel biomarkers for diagnosis as well as treatment are major tasks. In the present study, sera from ten SQLC patients and healthy control (HEC) were collected and pooled, respectively. The pooled sera were depleted via an immunoaffinity method and further subjected to fucosylation enrichment. Enriched fucosylated glycoproteins were resolved by SDS-PAGE and subsequently analyzed by LC-ESI-MS/MS. From comparative proteomic analysis, we selected the C9 protein. C9 protein levels were validated by Western blot, protein arrays and the fucosylation levels of C9 by hybrid lectin ELISA (HLE) in the sera of 120 HEC and 118 SQLC patients. The C9 protein level was 6.4-fold higher in SQLC patients compared to HEC, as determined by Western blot analysis. The results were concurrently confirmed by a protein array that showed a C9 level significantly higher in SQLC patients, as compared to HEC, with a sensitivity of 53% and a specificity of 89%. C9 fucosylation levels were significantly higher in SQLC patients compared to HEC (p<0.05) when tested by HLE. These findings suggest that C9 and fucosylated form could serve as a useful marker for SQLC.

Rapid determination of blood coagulation factor XIII activity using protein arrays for serodiagnosis of human plasma.

We developed a novel on-chip assay using protein arrays for quantitative and rapid analysis of blood coagulation factor XIII (FXIII) activity in human plasma. FXIII is activated by concerted action of thrombin and Ca(2+) and plays essential roles in hemostasis, angiogenesis, and wound healing. We fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane layer of well-type arrays and determined FXIII activity by analyzing biotinylated fibrinogen with Cy3-conjugated streptavidin. We determined optimal concentrations of Ca(2+), thrombin, and 5-(biotinamido)pentylamine (BAPA) for the on-chip activity assay, and the detection limit was 0.01 Lowey U/mL (9.9 pM). Using the on-chip activity assay, hepatocellular carcinoma patients (n = 24), but not hepatitis (n = 24) or liver cirrhosis patients (n = 41), had significantly lower FXIII activities (p < 0.001) than normal individuals (n = 41), indicating that FXIII activity is a possible diagnostic marker for hepatocellular carcinoma. In addition, we have successfully used this activity assay to reveal individual variations (37-57%, n = 65) in the inhibition rate of FXIII activity by isoniazid, the first-line antituberculosis agent. Thus, our optimized on-chip FXIII activity assay provides a quantitative and high-throughput approach to investigating the role(s) of FXIII in human diseases. Moreover, it has a strong potential to be applied toward FXIII-related personalized medicines.