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Autoimmune diseases such as ankylosing spondylitis (AS) can be driven by emerging neoantigens that disrupt immune tolerance. Here, we developed a workflow to profile posttranslational modifications involved in neoantigen formation. Using mass spectrometry, we identified a panel of cysteine residues differentially modified by carboxyethylation that required 3-hydroxypropionic acid to generate neoantigens in patients with AS. The lysosomal degradation of integrin αIIb [ITGA2B (CD41)] carboxyethylated at Cys96 (ITGA2B-ceC96) generated carboxyethylated peptides that were presented by HLA-DRB1*04 to stimulate CD4 + T cell responses and induce autoantibody production. Immunization of HLA-DR4 transgenic mice with the ITGA2B-ceC96 peptide promoted colitis and vertebral bone erosion. Thus, metabolite-induced cysteine carboxyethylation can give rise to pathogenic neoantigens that lead to autoreactive CD4 + T cell responses and autoantibody production in autoimmune diseases.

sRecent evidence suggests that CD147 serves as a novel receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Blocking CD147 via anti-CD147 antibody could suppress the in vitro SARS-CoV-2 replication. Meplazumab is a humanized anti-CD147 IgG2 monoclonal antibody, which may effectively prevent SARS-CoV-2 infection in coronavirus disease 2019 (COVID-19) patients. Here, we conducted a randomized, double-blinded, placebo-controlled phase 1 trial to evaluate the safety, tolerability, and pharmacokinetics of meplazumab in healthy subjects, and an open-labeled, concurrent controlled add-on exploratory phase 2 study to determine the efficacy in COVID-19 patients. In phase 1 study, 59 subjects were enrolled and assigned to eight cohorts, and no serious treatment-emergent adverse event (TEAE) or TEAE grade ≥3 was observed. The serum and peripheral blood Cmax and area under the curve showed non-linear pharmacokinetic characteristics. No obvious relation between the incidence or titer of positive anti-drug antibody and dosage was observed in each cohort. The biodistribution study indicated that meplazumab reached lung tissue and maintained >14 days stable with the lung tissue/cardiac blood–pool ratio ranging from 0.41 to 0.32. In the exploratory phase 2 study, 17 COVID-19 patients were enrolled, and 11 hospitalized patients were involved as concurrent control. The meplazumab treatment significantly improved the discharged (P = 0.005) and case severity (P = 0.021), and reduced the time to virus negative (P = 0.045) in comparison to the control group. These results show a sound safety and tolerance of meplazumab in healthy volunteers and suggest that meplazumab could accelerate the recovery of patients from COVID-19 pneumonia with a favorable safety profile.

T cell acute lymphoblastic leukemia (T-ALL) is invasive and heterogeneous, and existing therapies are sometimes unsuccessful. Chimeric antigen receptor (CAR) T cell therapy is a breakthrough tumor treatment method, particularly for B cell acute lymphoblastic leukemia. We found that CD147 was highly expressed in tumor T cells of T-ALL patients and T cell lymphoma. Therefore, CD147-CAR T cells that contain a humanized single-chain variable fragment targeting human CD147 and a second-generation CAR frame were constructed for treating T-ALL. CD147-CAR T cells were able to maintain a healthy proliferation rate, preserving a subset of CD62L+/CCR7+ memory T cells. CD147-CAR T cells showed a potent anti-tumor activity against human T-ALL cell line and T-ALL blasts, releasing high level of cytokines in the process. However, CD147-CAR T cells exhibited potential safety toward human normal cells and CD147-deficent cells. NOD/ShiLtJGpt-Prkdc

SARS-CoV-2 mutations contribute to increased viral transmissibility and immune escape, compromising the effectiveness of existing vaccines and neutralizing antibodies. An in-depth investigation on COVID-19 pathogenesis is urgently needed to develop a strategy against SARS-CoV-2 variants. Here, we identified CD147 as a universal receptor for SARS-CoV-2 and its variants. Meanwhile, Meplazeumab, a humanized anti-CD147 antibody, could block cellular entry of SARS-CoV-2 and its variants—alpha, beta, gamma, and delta, with inhibition rates of 68.7, 75.7, 52.1, 52.1, and 62.3% at 60 μg/ml, respectively. Furthermore, humanized CD147 transgenic mice were susceptible to SARS-CoV-2 and its two variants, alpha and beta. When infected, these mice developed exudative alveolar pneumonia, featured by immune responses involving alveoli-infiltrated macrophages, neutrophils, and lymphocytes and activation of IL-17 signaling pathway. Mechanistically, we proposed that severe COVID-19-related cytokine storm is induced by a “spike protein-CD147-CyPA signaling axis”: Infection of SARS-CoV-2 through CD147 initiated the JAK-STAT pathway, which further induced expression of cyclophilin A (CyPA); CyPA reciprocally bound to CD147 and triggered MAPK pathway. Consequently, the MAPK pathway regulated the expression of cytokines and chemokines, which promoted the development of cytokine storm. Importantly, Meplazumab could effectively inhibit viral entry and inflammation caused by SARS-CoV-2 and its variants. Therefore, our findings provided a new perspective for severe COVID-19-related pathogenesis. Furthermore, the validated universal receptor for SARS-CoV-2 and its variants can be targeted for COVID-19 treatment.

Major gaps in understanding the molecular mechanisms of colorectal cancer (CRC) progression and intestinal mucosal repair have hampered therapeutic development for gastrointestinal disorders. Trefoil factor 3 (TFF3) has been reported to be involved in CRC progression and intestinal mucosal repair; however, how TFF3 drives tumors to become more aggressive or metastatic and how TFF3 promotes intestinal mucosal repair are still poorly understood. Here, we found that the upregulated TFF3 in CRC predicted a worse overall survival rate. TFF3 deficiency impaired mucosal restitution and adenocarcinogenesis. CD147, a membrane protein, was identified as a binding partner for TFF3. Via binding to CD147, TFF3 enhanced CD147-CD44s interaction, resulting in signal transducer and activator of transcription 3 (STAT3) activation and prostaglandin G/H synthase 2 (PTGS2) expression, which were indispensable for TFF3-induced migration, proliferation, and invasion. PTGS2-derived PGE2 bound to prostaglandin E2 receptor EP4 subtype (PTGER4) and contributed to TFF3-stimulated CRC progression. Solution NMR studies of the TFF3-CD147 interaction revealed the key residues critical for TFF3 binding and the induction of PTGS2 expression. The ability of TFF3 to enhance mucosal restitution was weakened by a PTGS2 inhibitor. Blockade of TFF3-CD147 signaling using competitive inhibitory antibodies or a PTGS2 inhibitor reduced CRC lung metastasis in mice. Our findings bring strong evidence that CD147 is a novel receptor for TFF3 and PTGS2 signaling is critical for TFF3-induced mucosal restitution and CRC progression, which widens and deepens the understanding of the molecular function of trefoil factors.

Though the great success of paclitaxel, the variable response of patients to the drug limits its clinical utility and the precise mechanisms underlying the variable response to paclitaxel remain largely unknown. This study aims to verify the role and the underlying mechanisms of CD147 in paclitaxel resistance. Immunostaining was used to analyze human non-small-cell lung cancer (NSCLC) and ovarian cancer tissues. RNA-sequencing was used to identify downstream effectors. Annexin V-FITC/propidium iodide and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to detect apoptosis. Co-immunoprecipitation (Co-IP), fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) were performed to determine protein interactions. Fluorescence recovery after photobleaching (FRAP) was performed to measure the speed of microtubule turnover. Xenograft tumor model was established to evaluate sensitivity of cancer cells to paclitaxel in vivo. In vitro and in vivo assays showed that silencing CD147 sensitized the cancer cells to paclitaxel treatment. CD147 protected cancer cells from paclitaxel-induced caspase-3 mediated apoptosis regardless of p53 status. Truncation analysis showed that the intracellular domain of CD147 (CD147ICD) was indispensable for CD147-regulated sensitivity to paclitaxel. Via screening the interacting proteins of CD147ICD, Ran binding protein 1 (RanBP1) was identified to interact with CD147ICD via its C-terminal tail. Furthermore, we showed that RanBP1 mediated CD147-regulated microtubule stability and dynamics as well as response to paclitaxel treatment. These results demonstrated that CD147 regulated paclitaxel response by interacting with the C-terminal tail of RanBP1 and targeting CD147 may be a promising strategy for preventing paclitaxel resistant.

Cluster of differentiation 147 (CD147), a transmembrane protein of the immunoglobulin superfamily, is a potential target of treatment against human non-small cell lung cancer (NSCLC). Although there have been exciting advances in epidermal growth factor receptor (EGFR)-targeted therapy for NSCLC in recent years, additional novel targeted agents are needed to improve the efficiency and to offer more options for patients. Antibody-drug conjugates (ADCs) utilize a chemical linker to conjugate cytotoxic drugs to a monoclonal antibody to maximize the delivery to target cells and minimize the delivery to other normal cells. The aim of this study was to prepare a novel anti-CD147 conjugate and examine the tumoricidal effect on NSCLC in vitro and in vivo. HcHAb18 was conjugated to the drug maytansinoid 1 (DM1) via a non-cleavable thioether linker (SMCC) to prepare HcHAb18-DM1 with an appropriate drug-antibody ratio (DAR). NSCLC cell lines expressing different levels of CD147 were tested in vitro to determine internalization, cell cycle arrest and cytotoxicity. In vivo efficacy and safety of HcHAb18-DM1 were evaluated in NSCLC xenograft mouse models. We found that HcHAb18-DM1 displayed an impressive potency in vitro and in vivo with a favorable safety profile. Upon binding to CD147, HcHAb18 could be internalized and delivered the payload DM1 to disturb mitotic spindle formation by microtubules. Target cells were arrested at G2/M phase and HcHAb18-DM1 exerted antiproliferative activity in vitro. Antigen-antibody binding and target cells with high growth rate were two integral prerequisites for exerting anti-tumor activity of HcHAb18-DM1. Therefore, we suggest HcHAb18-DM1 is a promising CD147-targeted therapeutic for NSCLC.

CD147 is a tumor-associated antigen that plays a key regulatory role in tumor invasion and distant metastasis. However, the exact role of CD147 phosphorylation, which is deregulated during cancer progression, is unknown. Here, the effects of CD147 phosphorylation on the malignant behavior of hepatocellular carcinoma (HCC) cells and its possible underlying mechanisms are explored. An in situ Duolink-proximity ligation assay (PLA) was used to detect CD147 phosphorylation. Tandem mass spectrometry was employed to identify the phosphorylation sites of CD147. The effects of CD147 phosphorylation on the malignant behavior of HCC cells were evaluated using scratch wound healing assays, transwell invasion assays and cell cycle assays. The genes regulated by CD147 phosphorylation were detected by RNA sequencing. We identified phosphorylated serine-246 in the C terminus of CD147 in primary HCC tissues, whereas serine to alanine substitution mutation analysis suggested that CD147 is phosphorylated mainly at serine-252 in HCC-derived Huh-7 cells. Recovery expression of S246A/S252A mutants in CD147 knockout cells revealed significantly increased migration and invasion capacities compared to wildtype CD147 expressing cells. Cyclophilin A (CyPA) treatment decreased the phosphorylation level of CD147, whereas NIMA-related kinase 6 (NEK6) increased the CD147 phosphorylation level. Moreover, the CD147 phosphorylation level was found to be dramatically decreased in HCC tissues in patients with distant metastases, and a low phosphorylation level of CD147 was found to be associated with a high serum AFP level, recurrence and a poor overall survival. From our data we conclude that hypo-phosphorylated CD147 promotes the migration and invasion of HCC cells and correlates with an unfavorable prognosis in HCC patients, indicating that targeting the aberrantly hypo-phosphorylated form of CD147 may be instrumental for the development of novel therapeutic modalities directed against HCC metastasis.

In face of the everlasting battle toward COVID-19 and the rapid evolution of SARS-CoV-2, no specific and effective drugs for treating this disease have been reported until today. Angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, mediates the virus infection by binding to spike protein. Although ACE2 is expressed in the lung, kidney, and intestine, its expressing levels are rather low, especially in the lung. Considering the great infectivity of COVID-19, we speculate that SARS-CoV-2 may depend on other routes to facilitate its infection. Here, we first discover an interaction between host cell receptor CD147 and SARS-CoV-2 spike protein. The loss of CD147 or blocking CD147 in Vero E6 and BEAS-2B cell lines by anti-CD147 antibody, Meplazumab, inhibits SARS-CoV-2 amplification. Expression of human CD147 allows virus entry into non-susceptible BHK-21 cells, which can be neutralized by CD147 extracellular fragment. Viral loads are detectable in the lungs of human CD147 (hCD147) mice infected with SARS-CoV-2, but not in those of virus-infected wild type mice. Interestingly, virions are observed in lymphocytes of lung tissue from a COVID-19 patient. Human T cells with a property of ACE2 natural deficiency can be infected with SARS-CoV-2 pseudovirus in a dose-dependent manner, which is specifically inhibited by Meplazumab. Furthermore, CD147 mediates virus entering host cells by endocytosis. Together, our study reveals a novel virus entry route, CD147-spike protein, which provides an important target for developing specific and effective drug against COVID-19.

Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrate robust, sensitive, and reproducible data generation across eleven international sites on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality., Distributed multi-omic digitization of clinical specimen across multiple sites is a prerequisite for turning molecular precision medicine into reality. Here, the authors show that coordinated proteotype data acquisition is feasible using standardized MS data acquisition and analysis strategies.

SummaryBackgroundSARS-CoV-2 is a novel human coronavirus, there is no specific antiviral drugs. It has been proved that host-cell-expressed CD147 could bind spike protein of SARS-CoV-2 and involve in host cell invasion. Antibody against CD147 could block the infection of SARS-CoV-2. We aimed to assess the efficacy and safety of meplazumab, a humanized anti-CD147 antibody, as add-on therapy in patients with COVID-19 pneumonia.MethodsAll patients received recommended strategy fromChinese Clinical Guidance for COVID-19 Pneumonia Diagnosis and Treatmentsreleased by National Health Commission of China. Eligible patients were add-on administered 10 mg meplazumab intravenously at days 1, 2, and 5. Patients hospitalized in the same period were observed as concurrent control. The endpoints include virological clearance rate, case severity, chest radiographic, and laboratory test. This trial was approved by the Ethics Committee of Institution at the Tangdu hospital, and registered with ClinicalTrials.gov, NCT 04275245.Findings17 patients were enrolled and assigned to meplazumab group between Feb 3, 2020 and Feb 10, 2020. 11 hospitalized patients served as concurrent control. Baseline characteristics were generally balanced across two groups. Compared to control group, meplazumab treatment significantly improved the discharged (p=0.005) and case severity (p=0.021) in patients. The time to virus negative in meplazumab group was reduced than that in control group (median 3, 95%CI[1.5-4.5] vs. 13, [6.5-19.5]; p=0.045, HR=0.374, 95%CI[0.143-0.978]). The percentages of patients recovered to the normal lymphocyte count and CRP concentration were also increased remarkably and rapidly in meplazumab group. No adverse effect was found in meplazumab-treated patients.InterpretationMeplazumab efficiently improved the recovery of patients with SARS-CoV-2 pneumonia with a favorable safety profile. Our results support to carry out a large-scale investigation of meplazumab as a treatment for COVID-19 pneumonia.FundingNational Science and Technology Major Project.

SUMMARYCurrently, COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widely spread around the world; nevertheless, so far there exist no specific antiviral drugs for treatment of the disease, which poses great challenge to control and contain the virus. Here, we reported a research finding that SARS-CoV-2 invaded host cells via a novel route of CD147-spike protein (SP). SP bound to CD147, a receptor on the host cells, thereby mediating the viral invasion. Our further research confirmed this finding. First, in vitro antiviral tests indicated Meplazumab, an anti-CD147 humanized antibody, significantly inhibited the viruses from invading host cells, with an EC50 of 24.86 μg/mL and IC50 of 15.16 μg/mL. Second, we validated the interaction between CD147 and SP, with an affinity constant of 1.85×10−7M. Co-Immunoprecipitation and ELISA also confirmed the binding of the two proteins. Finally, the localization of CD147 and SP was observed in SARS-CoV-2 infected Vero E6 cells by immuno-electron microscope. Therefore, the discovery of the new route CD147-SP for SARS-CoV-2 invading host cells provides a critical target for development of specific antiviral drugs.

Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrated robust, sensitive, and reproducible data generation across eleven sites in nine countries on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality.

Proteomic-based approaches for biomarker discovery are promising strategies used in cancer research. In this study, we performed quantitative proteomic analysis on 16 paired samples of non-small cell lung cancer (NSCLC) and adjacent non-tumor lung tissues using label-free quantitative proteomics and liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS) to identify differentially expressed proteins. A total of 91 proteins were differentially expressed in NSCLC compared with adjacent non-tumor lung tissues among 4047 identified proteins (fold change > 1.5 or < 0.67, P < 0.05). Gene ontology (GO) analysis, Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis and ingenuity pathway analysis (IPA) of 91 dysregulated proteins showed that they were related to the cancer-associated biological processes. We confirmed that the candidate proteins, calreticulin (CALR) and protein disulfide isomerase family A member 3 (PDIA3) were overexpressed in NSCLC by real-time PCR using 20 paired samples and western blot using 5 paired samples. PDIA3 expression was highly associated with CALR expression (Spearman r = 0.345, P = 0.001) and they were co-localized and interacted with each other in A549 and H460 cells. Moreover, survival analysis performed in tissue microarray with 88 samples indicated that low expression of both CALR and PDIA3 in NSCLC was positively associated with poor overall survival. Combination of CALR and PDIA3 might serve as an efficient biomarker and improved the prediction of NSCLC prognosis significantly (P = 0.023). Our results collectively provide a potential biomarker dataset for NSCLC prognosis, especially the prognostic value of combined expression of CALR and PDIA3.

Targeted therapeutics is used as an alternative treatment of non-small cell lung cancer (NSCLC); however, treatment effect is far from being satisfactory, and therefore identification of new targets is needed. We have previously shown that metuzumab inhibit tumor growth in vivo. The present study was performed to investigate the anti-tumor efficacy of metuzumab combined with gemcitabine and cisplatin (GP), paclitaxel and cisplatin (TP) or navelbine and cisplatin (NP) regimens in multiple NSCLC cell lines. Our results demonstrate that, in comparison to single agent metuzumab or GP treated cells, metuzumab combined with GP display inhibitory effects on tumor growth. Furthermore, we found that metuzumab elevated the sensitivity of cell lines to gemcitabine, which was identified by MTT assay. Flow cytometric analysis showed that metuzumab combined with gemcitabine (GEM) treatment led to an obvious G1 arrest and an elevated apoptosis in A549, NCI-H460 and NCI-H520 cells. Western blot analysis also demonstrated a significantly reduced level of cyclin D1, Bcl-2, and an obviously increase level of Bax and full-length caspase-3 in A549, NCI-H460 and NCI-H520 cells treated with metuzumab/gemcitabine combination in comparison with single agent treated cells. In addition, metuzumab/gemcitabine treated A549, NCI-H460 and NCI-H520 cells also demonstrated a significantly increase in deoxycytidine kinase (dCK) protein level compared with single agent metuzumab or gemcitabine treated cells. Xenograft models also demonstrated that this metuzumab/gemcitabine combination led to upregulation of dCK. Taken together, the mechanisms of metuzumab combined with GP repress tumor growth were that the combined treatment significantly inhibited the tumor cell proliferation, apoptosis and cell cycle in vitro and in vivo and at least partially by induction of dCK expression. Our results suggested that metuzumab could significantly enhance chemosensitivity of human NSCLC cells to gemcitabine. Metuzumab/gemcitabine combination treatment may be a potentially useful therapeutic regimen for NSCLC patients.

Lung cancer is the most common malignant tumor and the leading cause of cancer-related deaths worldwide. Because current treatments for advanced non-small cell lung cancer (NSCLC), the most prevalent lung cancer histological subtype, show limited efficacy, screening for tumor-associated biomarkers using bioinformatics reflects the hope to improve early diagnosis and prognosis assessment. In our study, a Gene Expression Omnibus dataset was analyzed to identify genes with prognostic significance in NSCLC. Upon comparison with matched normal tissues, 118 differentially expressed genes (DEGs) were identified in NSCLC, and their functions were explored through bioinformatics analyses. The most significantly upregulated DEGs were TOP2A, SLC2A1, TPX2, and ASPM, all of which were significantly associated with poor overall survival (OS). Further analysis revealed that TOP2A had prognostic significance in early-stage lung cancer patients, and its expression correlated with levels of immune cell infiltration, especially dendritic cells (DCs). Our study provides a dataset of potentially prognostic NSCLC biomarkers, and highlights TOP2A as a valuable survival biomarker to improve prediction of prognosis in NSCLC.

CD147 is a tumor-associated glycoprotein that regulates cell metabolism. However, CD147 methylation and its subsequent role in cancer cell metabolism remain unclear. Here, we detect CD147 di-methylation in 16 non-small-cell lung cancer (NSCLC) tissues using liquid chromatography-tandem mass spectrometry. CD147 is di-methylated to CD147-K234me2 by lysine methyltransferase 5A (KMT5A). The increase in KMT5A expression boosts the levels of CD147-K234me2, further promoting the interaction between CD147 and monocarboxylate transporter 4 (MCT4), which enhances the translocation of MCT4 from the cytoplasm to the membrane. Overexpression of CD147-K234me2 and KMT5A enhances glycolysis and lactate export in NSCLC cells. Clinical analysis shows that high CD147-K234me2 expression is significantly related to cancer progression and overall survival, and has prognostic significance in individuals with NSCLC, especially for those in the early stages. Our findings indicate that CD147-K234me2 plays a critical role in cancer metabolism, and it can be a highly promising therapeutic target for NSCLC.

Although the expression levels of total GalNAc-binding proteins (GNBPs) were up-regulated significantly in human hepatic stellate cells (HSCs) activated with transforming growth factor-β1(TGF-β1), yet little is known about the precise types, distribution and sub-cellular localization of the GNBPs in HSCs. Here, 264 GNBPs from the activated HSCs and 257 GNBPs from the quiescent HSCs were identified and annotated. A total of 46 GNBPs were estimated to be significantly up-regulated and 40 GNBPs were estimated to be significantly down-regulated in the activated HSCs. For example, the GNBPs (i.e. BTF3, COX17, and ATP5A1) responsible for the regulation of protein binding were up-regulated, and those (i.e. FAM114A1, ENO3, and TKT) responsible for the regulation of protein binding were down-regulated in the activated HSCs. The motifs of the isolated GNBPs showed that Proline residue had the maximum preference in consensus sequences. The western blotting showed the expression levels of COX17, and PRMT1 were significantly up-regulated, while, the expression level of CLIC1(B5) was down-regulated in the activated HSCs and liver cirrhosis tissues. Moreover, the GNBPs were sub-localized in the Golgi apparatus of HSCs. In conclusion, the precision alteration of the GNBPs referred to pathological changes in liver fibrosis/cirrhosis may provide useful information to find new molecular mechanism of HSC activation and discover the biomarkers for diagnosis of liver fibrosis/cirrhosis as well as development of new anti-fibrotic strategies.

The interaction of glycan-binding proteins (GBPs) and glycans plays a significant biological role that ranges from cell-cell recognition to cell trafficking, and glycoprotein targeting. The anomalies of GBPs related to the types and/or quantities were not clearly known in cancer incidence. It is imperative to identify and annotate the GBPs related with the canceration. Here the mannose-binding proteins (MBPs) from the clinical sera were isolated and identified by the mannose-magnetic particle conjugates and the high-accuracy MS analysis. Seventy-five MBPs from normal donors' sera and 79 MBPs from hepatocellular carcinoma patients' sera were identified and annotated. By using the stringent criteria of exponentially modified protein abundance index (emPAI) quantification, 12 MBPs were estimated to be significantly upregulated (emPAI ratio > 4) and nine MBPs were estimated to be significantly downregulated (emPAI ratio < 0.25) in the hepatocellular carcinoma sera. Real-time quantitative PCR, Western blotting, and protein microarrays were also used to confirm the altered MBPs expression level and the specific binding between the isolated MBPs and mannose. The sequence recognition motifs and structure preference of the isolated MBPs were characterized. The functional enrichment analysis revealed that over 57% of the isolated MBPs were binding protein and the upregulated MBPs were involved in cell death, tumor progression, and macromolecular complex remodeling.

In our recent study, we found that the expression levels of total xylose-binding proteins (XBPs) were up-regulated significantly in activated hepatic stellate cells (HSCs); however, the denomination, distribution, and function of the XBPs were uncharted. Herein, 70 XBPs from activated HSCs and 64 XBPs from quiescent HSCs were isolated, identified and annotated. A total of 30 XBPs were up-regulated (all fold change ≥ 1.5, p ≤ 0.05) and 14 XBPs were down-regulated (all fold change ≤ 0.67, p ≤ 0.05) in the activated HSCs. The XBPs were localized at the cytoplasm and cytoplasmic membrane in HSCs and cirrhotic liver tissues by cy/histochemistry. The XBPs (i.e. PDIA6 and CFL2) responsible for the regulation of protein binding were up-regulated and those responsible for the regulation of catalytic activity (i.e. TUBB and MX1) were up-regulated in the activated HSCs. 2 candidates (i.e. PDIA6 and APOA1) were then selected for further verification in the sera of patients with HBV-induced chronic hepatitis/cirrhosis using western blotting and serum microarrays. PDIA6 showed a higher discrimination (Area Under Curves, AUCs = 0.8985, p < 0.0001) relative to APOA1 (AUCs = 0.8738, p < 0.0001) in the sera of patients as biomarker candidate. In conclusion, the precision alteration of the XBPs associated with pathological changes in HSCs during liver fibrosis/cirrhosis may provide pivotal information needed to discover potential glycan-binding protein-related biomarkers for diagnosis of liver fibrosis/cirrhosis and for development of new anti-fibrotic strategies.

To determine optical purities of four aromatic 1,2-diol enantiomers synthesized by a Sharpless asymmetric dihydroxylation (AD) reaction of olefins, a simple and reliable separation method was achieved with high resolution (R s > 2.2) by capillary zone electrophoresis (CZE) using hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector and borate combined with methanol additive as background buffer. Furthermore, the developed CZE method was successfully applied to the determination of enantiomeric excess of the tested enantiomers. RSD values of migration time and peak area fell within 1.0 and 3.8%, respectively. This method allowed for the determination of ee (%) values of targeted isomers as high as 99.6%. Impurities of undesired isomers could be detected at levels as low as 0.2% in the presence of the targeted isomers.

Growing evidence indicates that miR-146a is involved in carcinogenesis and tumor progression in several human malignancies. However, the molecular details underlying miR-146a mediated regulation of its target genes and its precise biological function in cancer, especially in hepatocellular carcinoma (HCC) remains unclear. Methods The expression levels of genes including miR-146a, APC, VEGF and HAb18G were examined in HCC cell lines and patient specimens were compared with control levels using quantitative reverse transcription-PCR. The functions of miR-146a and HAb18G in migration/invasion and liver metastasis formation were determined by transwell and spleen injection assays, respectively. miR-146a related genes were determined by PCR array. The potential regulatory targets of miR-146a were determined by bioinformatics and prediction tools, correlation with target protein expression, and luciferase reporter assay. DNA methylation status of miR-146a promoter were performed by PCR analysis of bisulfite-modified genomic DNA. Results We demonstrated that miR-146a expression was markedly downregulated in hepatoma cells and hepatoma tissues compared to immortalized normal liver epithelial cells and normal hepatic tissues. DNA methylation of miR-146a promoter correlated with its downexpression and with liver cancer metastasis. The restoration of miR-146a dramatically suppressed HCC cell invasion and metastasis by repressing VEGF expression through upregulating APC, which inhibits β-catenin accumulation in nucleus, and downregulating NF-κB p65 by targeting HAb18G. In human HCC, miR-146a expression was negative correlated with increased HAb18G, VEGF, NF-κB p65 and beneficial prognosis. Conclusion This study identified a novel target of miR-146a and defined miR-146a as a crucial tumor suppressor in human HCC that acts through multiple pathways and mechanisms to suppress HCC invasion or metastasis. Electronic supplementary material The online version of this article (doi:10.1186/1476-4598-14-5) contains supplementary material, which is available to authorized users.

Hepatoblastoma (HB) is the most common form of liver tumour in infants and children. The cancer cell line HepG2 and normal hepatocellular cell line L02 are valuable cell models and are already widely used in the world while their glycopeoteome profiles are still unknown. This study focuses on N-linked glycoproteomic analysis of these two cell lines using mass spectrometry. Using two complementary approaches, Hydrazide reaction and hydrophilic affinity solid phase extraction methods, almost 400 glycosylation sites were identified from the two cell lines. Functional annotation suggests that N-glycoproteins with molecular binding ability were enriched in HepG2 cells compared to normal liver cells.

Background As a surface glycoprotein, CD147 is capable of stimulating the production of matrix metalloproteinases (MMPs) from neighboring fibroblasts. The aim of the present study is to explore the role of soluble CD147 on MMPs secretion from hepatocellular carcinoma (HCC) cells, and to investigate the diagnostic value of serum soluble CD147 in the HCC detection. Methods We identified the form of soluble CD147 in cell culture supernate of HCC cells and serum of patients with HCC, and explored the role of soluble CD147 on MMPs secretion. Serum CD147 levels were detected by the enzyme-linked immunosorbent assay, and the value of soluble CD147 as a marker in HCC detection was analyzed. Results Full length soluble CD147 was presented in the culture medium of HCC cells and serum of patients with HCC. The extracellular domain of soluble CD147 promoted the expression of CD147 and MMP-2 from HCC cells. Knockdown of CD147 markedly diminished the up-regulation of CD147 and MMP-2 which induced by soluble CD147. Soluble CD147 activated ERK, FAK, and PI3K/Akt pathways, leading to the up-regulation of MMP-2. The level of soluble CD147 in serum of patients with HCC was significantly elevated compared with healthy individuals (P

CD147 is expressed at low levels in normal tissues but frequently highly expressed in a wide range of tumor types such as lung, breast, and liver and therefore it is a potentially unique therapeutic target for these diverse tumor types. We previously generated a murine antibody HAb18 which suppresses matrix met al.loproteinase-2 and matrix metalloproteinase-9 secretion, attenuates cell invasion by blocking the CD147 molecule in tumor cells. Here, we generated a chimeric antibody containing the variable heavy and variable light chains of murine HAb18 and the constant regions of human IgG1γ1 and human κ chain as a potential therapeutic agent (designated cHAb18). Quantitative measurement of cHAb18 antibody affinity for antigen CD147 with surface plasmon resonance showed the equilibrium dissociation constant KD was 2.66 × 10(-10) mol/L, similar to that of KD 2.73 × 10(-10) mol/L for murine HAb18. cHAb18 induced antibody-dependent cell-mediated cytotoxicity in two hepatocellular carcinoma cell lines, SMMC-7721 and Huh-7 cells. It inhibited cancer invasion and migration in hepatocellular carcinoma cells by specifically blocking CD147. Except for the depression of matrix metalloproteinase-2 and matrix metalloproteinase-9 expressions, cHAb18 antibody suppressed cell motility by rearrangement of actin cytoskeleton, which was probably induced by decreasing the phosphorylation of focal adhesion kinase, phosphatidylinositide-3 kinase (PI3K), Akt, and Girdin in the integrin signaling pathway. In an orthotopic model of hepatocellular carcinoma in BALB/c nude mice, cHAb18 treatment effectively reduced the tumor metastasis in liver and prolonged the survival. These findings reveal new therapeutic potential for cHAb18 antibody targeting CD147 on tumor therapy.

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.