Identification of Fucosylated SERPINA1 as a Novel Plasma Marker for Pancreatic Cancer Using Lectin Affinity Capture Coupled with iTRAQ-Based Quantitative Glycoproteomics

Pancreatic cancer (PC) is an aggressive cancer with a high mortality rate, necessitating the development of effective diagnostic, prognostic and predictive biomarkers for disease management. Aberrantly fucosylated proteins in PC are considered a valuable resource of clinically useful biomarkers. The main objective of the present study was to identify novel plasma glycobiomarkers of PC using the iTRAQ quantitative proteomics approach coupled with Aleuria aurantia lectin (AAL)-based glycopeptide enrichment and isotope-coded glycosylation site-specific tagging, with a view to analyzing the glycoproteome profiles of plasma samples from patients with non-metastatic and metastatic PC and gallstones (GS). As a result, 22 glycopeptides with significantly elevated levels in plasma samples of PC were identified. Fucosylated SERPINA1 (fuco-SERPINA1) was selected for further validation in 121 plasma samples (50 GS and 71 PC) using an AAL-based reverse lectin ELISA technique developed in-house. Our analyses revealed significantly higher plasma levels of fuco-SERPINA1 in PC than GS subjects (310.7 ng/mL v.s. 153.6 ng/mL, p = 0.0114). Elevated fuco-SERPINA1 levels were associated with higher TNM stage (p = 0.024) and poorer prognosis for overall survival (log-rank test, p = 0.0083). The increased plasma fuco-SERPINA1 levels support the utility of this protein as a novel prognosticator for PC.

Glycan array analysis of Pholiota squarrosa lectin and other fucose-oriented lectins

The α(1,6)fucose residue attached to the N-glycoprotein core is suspected to play an essential role in the progression of several types of cancer. Lectins remain the first choice for probing glycan modifications, although they may lack specificity. Thus, efforts have been made to identify new lectins with a narrower core fucose (CF) detection profile. Here, we present a comparison of the classical Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA) and Aspergillus oryzae lectin (AOL) with the newer Pholiota squarrosa lectin (PhoSL), which has been described as being specific for core fucosylated N-glycans. To this end, we studied the binding profiles of the four lectins using mammalian glycan arrays from the Consortium of Functional Glycomics. To validate their glycan specificity, we probed AOL, LCA and PhoSL in western-blot assays using protein extracts from eight common colorectal cancer (CRC) lines and colorectal biopsies from a small cohort of patients with CRC. The results showed that (i) LCA and PhoSL were the most specific lectins for detecting the presence of CF in a concentration-dependent manner; (ii) PhoSL exhibited the highest N-glycan sequence restriction, with preferential binding to core fucosylated paucimannosidic-type N-glycans, (iii) the recognition ability of PhoSL was highly influenced by the presence of terminal N-acetyl-lactosamine; (iv) LCA bound to paucimannosidic, bi-antennary and tri-antennary core fucosylated N-glycans and (v) AOL and AAL exhibited broader specificity towards fucosylation. Together, our results support the choice of LCA as the most appropriate lectin for CF detection, as validated in protein extracts from CRC cell lines and tissue specimens from patients with CRC.

Assessment of Altered Fucosylation of Serum α-1-Acid Glycoprotein in Hepatocellular Carcinoma Patients by Gold-Nanoparticle Aggregation Immunoassay

Gold nanoparticles have attracted considerable attention due to their unique properties and potential applications as optical probes. When proteins that are adsorbed on gold nanoparticles subsequently get cross-linked by any interaction specific to that protein, the size of the aggregates increases and this enhancement of size have been used for sensitive, convenient and powerful tool to monitor the presence of the specific cross-linkers. Dynamic light scattering (DLS) is a technique that is routinely used for detecting aggregation in macromolecular solutions. In this work, we first applied DLS to identify specific glycoprotein–lectin interactions exclusively present in the serum of hepatocellular carcinoma patients compared to healthy controls that showed the altered fucosylation of a serum protein, Serum α-1-Acid glycoprotein. Further, based on the DLS data a simple, rapid, serological assay was developed based on antibody coated gold nanoparticle and fucose binding lectin (Aleuria aurantia lectin) as linker to asses the level of fucosylation of α-1-acid glycoprotein. As a consequence of the triggered aggregation of the GNP probes in presence of lectin, plasmon band was shifted from red to blue, which colorimetrically reported the enhanced fucosylation of α-1-acid glycoprotein and formed the basis of a rapid visual assay for hepatocellular carcinoma.

Serum leucine-rich alpha-2-glycoprotein-1 with fucosylated triantennaryN-glycan as a novel tumor marker for colorectal cancer.

595 Background: CEA and CA19-9 are measured widely in daily practice for colorectal cancer to monitor the condition of treatment, though its specificity and sensitivity is not satisfied enough. While, the innovation of mass spectrometry in this decade is dramatically. The revolutionized approach for the screening of glycomarkers provides a chance to discover more desirable tumor markers. We focused on glycan alterations of serum glycoproteins and tried to find new markers which are more specific and sensitive than the current markers. Methods: Sera of 80 CRC patients and 50 healthy volunteers were analyzed in this study. Glycopeptides were prepared from serum proteins by trypsin digestion, enriched by filtration and aleuria aurantia lectin(AAL), and then applied to liquid chromatography time-of flight mass spectrometry* (*LC-TOF-MS). Data was analyzed with our newly developed software, which calculates all of peak intensities and positions (m/z and elution time), aligns all peaks for all samples, and finally screens markers from a million of candidates. Results: Leucine-rich alpha-2-glycoprotein-1 with fucosylated triantennary N-glycan (LRG-FTG) was discovered as a new CRC marker from over 100,000 candidate glycopeptide peaks. The level of LRG-FTG in CRC patients (1.25 ± 0.973 U/ml) was much higher than that of healthy parsons (0.496 ± 0.433 U/ml, p < 10-10). Its sensitivity and specificity exceeded those of CA19-9. CEA and LRG-FTG levels were moderately correlated (r = 0.61), however their relation was independent and complementary. Combination marker of LRG-FTG and CEA showed 84% of sensitivity, 90% of specificity and 0.91 of AUC (ROC analysis), and responded well to the efficacy of chemotherapy. Conclusions: Serum LRG-FTG was significantly elevated in CRC patients compared to healthy volunteers. The combination with CEA is expected to be an alternative diagnostic marker which distinguishes CRC from healthy persons and a sensitive monitoring marker for evaluation of efficacy during chemotherapy. Further validation study is warranted to evaluate serum LRG-FTG combination test compared with conventional tumor markers.