Identification of serum glycoprotein ligands for the immunomodulatory receptor blood dendritic cell antigen 2

Blood dendritic cell antigen 2 (BDCA-2) is a C-type lectin found on the surface of plasmacytoid dendritic cells. It functions as a glycan-binding receptor that downregulates the production of type I interferons and thus plays a role in oligosaccharide-mediated immunomodulation. The carbohydrate recognition domain in BDCA-2 binds selectively to galactose-terminated bi-antennary glycans. Because the plasmacytoid dendritic cells function in a plasma environment rich in glycoproteins, experiments have been undertaken to identify endogenous ligands for blood dendritic cell antigen 2. A combination of blotting, affinity chromatography and proteomic analysis reveals that serum glycoprotein ligands for BDCA-2 include IgG, IgA and IgM. Compared to binding of IgG, which was previously described, IgA and IgM bind with higher affinity. The association constants for the different subclasses of immunoglobulins are below and roughly proportional to the serum concentrations of these glycoprotein ligands. Binding to the other main serum glycoprotein ligand, α2-macroglobulin, is independent of whether this protease inhibitor is activated. Binding to all of these glycoprotein ligands is mediated predominantly by bi-antennary glycans in which each branch bears a terminal galactose residue. The different affinities of the glycoprotein ligands reflect the different numbers of these galactose-terminated glycans and their degree of exposure on the native glycoproteins. The results suggest that normal serum levels of immunoglobulins could downmodulate interferon stimulation of further antibody production.

Serum glycoprotein biomarker validation for esophageal adenocarcinoma and application to Barrett’s surveillance

SUMMARYBACKGROUND & AIMSEsophageal adenocarcinoma (EAC) is thought to develop from asymptomatic Barrett’s esophagus (BE) with a low annual rate of conversion. Current endoscopy surveillance for BE patients is probably not cost-effective. Previously, we discovered serum glycoprotein biomarker candidates which could discriminate BE patients from EAC. Here, we aimed to validate candidate serum glycoprotein biomarkers in independent cohorts, and to develop a biomarker panel for BE surveillance.METHODSSerum glycoprotein biomarker candidates were measured in 301 serum samples collected from Australia (4 states) and USA (1 clinic) using lectin magnetic bead array (LeMBA) coupled multiple reaction monitoring mass spectrometry (MRM-MS). The area under receiver operating characteristic curve was calculated as a measure of discrimination, and multivariate recursive partitioning was used to formulate a multi-marker panel for BE surveillance.RESULTSDifferent glycoforms of complement C9 (C9), gelsolin (GSN), serum paraoxonase/arylesterase 1 (PON1) and serum paraoxonase/lactonase 3 (PON3) were validated as diagnostic glycoprotein biomarker candidates for EAC across both cohorts. A panel of 10 serum glycoproteins accurately discriminated BE patients not requiring intervention [BE+/-low grade dysplasia] from those requiring intervention [BE with high grade dysplasia (BE-HGD) or EAC]. Tissue expression of C9 was found to be induced in BE, dysplastic BE and EAC. In longitudinal samples from subjects that have progressed towards EAC, levels of serum C9 glycoforms were increased with disease progression.CONCLUSIONSFurther prospective clinical validation of the confirmed biomarker candidates in a large cohort is warranted. A first-line BE surveillance blood test may be developed based on these findings.AbbreviationsAALAleuria aurantia lectin%CV% Co-efficient of variationAUROCArea under receiver operating characteristics curveBEBarrett’s esophagusBE-HGDBarrett’s esophagus with high-grade dysplasiaBE-IDBarrett’s esophagus which is indefinite for dysplasiaBE-LGDBarrett’s esophagus with low-grade dysplasiaBMIBody mass indexC1QBComplement C1q subcomponent subunit BC2Complement C2C3Complement C3C4BComplement C4-BC4BPAC4b-binding protein alpha chainC4BPBC4b-binding protein beta chainC9Complement component C9CFBComplement factor BCFIComplement factor ICIConfidence intervalCPCeruloplasminEACEsophageal adenocarcinomaEPHAErythroagglutinin from Phaseolus vulgarisFFPEFormalin-fixed, paraffin-embeddedGERDGastroesophageal reflux diseaseGSNGelsolinJACJacalin from Artocarpus integrifoliaLeMBALectin magnetic bead arrayMRM-MSMultiple reaction monitoring-mass spectrometryNPLNarcissus pseudonarcissus lectinNSENon-specialized epitheliumOROdds ratioPGLYRP2N-acetylmuramoyl-L-alanine amidasePON1Serum paraoxonase/arylesterase 1PON3Serum paraoxonase/lactonase 3RBP4Retinol-binding protein 4SERPINA4KallistatinSISStable isotope-labeled internal standard

Complement component C9 as a new biomarker for esophageal adenocarcinoma.

19 Background: Esophageal adenocarcinoma (EAC) continues an upward trend. Mortality rate and treatment costs for EAC are very high overall and significantly increase with disease stage. Diagnosis at early stage (T1) compared to advanced stage (T3) improves 5 year survival from 20% to 80-90%. Endoscopic surveillance has been ineffective in reducing EAC. Therefore the need for better diagnostic tools for early detection.Previously we reported validation of a list of serum glycoprotein biomarker candidates for EAC in an Australian cohort (Shah et al. 2015 Mol Cell Proteomics. 14:3023-39.). Here we further evaluate the serum and tissue expression of top candidate C9 in an independent cohort from the USA. Methods: Serum samples (n=38) and FFPE tissue sections (n=34) were collected from participants with IRB approved protocol, from the Ochsner Clinic undergoing endoscopic surveillance for Barrett’s and esophageal cancer. Serum glycoprotein candidates were measured using lectin magnetic bead array-coupled multiple reaction monitoring assay (Shah et al. 2015 Mol Cell Proteomics. 14:3023-39.). Immunohistochemistry was optimised for complement component C9 antibody. Initial evaluation of 2 different antibodies gave similar results, thereafter, a polyclonal antibody from Sigma Aldrich was used. Staining was assessed by a gastrointestinal pathologist. Results: Specific glycosylated forms of 3 candidate serum biomarkers were confirmed to be significantly different between EAC and benign groups (p<0.05, Kruskal-Wallis). The diagnostic value for the 3 individual markers, complement component C9, gelsolin and alpha-1-antichymotrypsin (Serpin A3) was 0.71 to 0.82 area under the receiver operating curve (AUROC). A multivariate panel consisting of 8 glycoprotein biomarkers achieved AUROC of 0.96, indicative of high diagnostic value. Immunohistochemistry of tissues detected high levels of C9 in BE and EAC compared to normal squamous epithelium. Infiltrated lymphocytes showed variable staining. Conclusions: We propose a novel candidate biomarker complement component C9 which could add to current endoscopic surveillance strategy for early detection of adenocarcinoma.