Assessment of Hepatocellular Carcinoma Metastasis Glycobiomarkers Using Advanced Quantitative N-glycoproteome Analysis

Abstract

Hepatocelluar carcinoma (HCC) is one of the most common malignant tumors with high incidence of metastasis. Glycosylation is involved in fundamental molecular and cell biology process occurring in cancer including metastasis formation. In this study, lectin microarray, lectin blotting, lectin affinity chromatography and tandem 18O stable isotope labeling coupled with liquid chromatography-mass spectrometer (LC-MS) analysis were applied to quantify the changes in N-glycosite occupancy for HCC metastasis serum. Firstly, lectin microarray was used to screen glycoforms and Phaseolus vulgaris Leucoagglutinin (PHA-L) reactive structure (β1,6-GlcNAc branched N-glycan) was found to be increased significantly in HCC patients with metastasis compared with those with non-metastasis. Then, PHA-L affinity glycoproteins were enriched followed by N-glycosite occupancy measurement with strategy of tandem 18O stable isotope labeling. 11 glycoproteins with significantly changed N-glycosite occupancy were identified, they were associated with cell migration, invasion and adhesion through p38 mitogen-activated protein kinase signaling pathway and nuclear factor kappa B signaling pathway. Quantification of N-glycosite occupancy for PHA-L reactive glycoproteins could help to discover important glycoproteins of potential clinically significance in terms of HCC etiology. Also, understanding of N-glycosite occupancy alterations will aid the characterization of molecular mechanism of HCC metastasis as well as establishment of novel glycobiomarkers.

The Continuum of Aging and Age-Related Diseases: Common Mechanisms but Different Rates

“…The relative quantification of the N-glycan species that constitute the sugar shell of circulating proteins is a wealthy source of reliable biomarkers. The characterization of circulating N-glycans from sera or plasma, hereafter referred as glycomics, has provided markers in several clinical fields such as hepatology ( 247 – 249 ), type 2 diabetes ( 250 – 254 ), RA ( 255 – 258 ), and cancer ( 259 – 262 ).…”

1H-NMR Based Serum Metabolomics Highlights Different Specific Biomarkers between Early and Advanced Hepatocellular Carcinoma Stages

“…Protein glycosylation, an enzymatic process by which saccharide groups are added to the maturing proteins, is involved in fundamental molecular and cell biology processes occurring in cancer, including metastasis formation. It has also been established that especially the N-glycosite occupancy of a protein is associated with the enzymatic activity and the physical stability of glycoproteins [47], which might contribute to the metastasis of HCC.…”

Application of Lectin Microarrays for Biomarker Discovery

“…As PHA−E levels showed a significant decrease in HCC patients with early HE, they concluded that 26 PHA−E‐associated glycoproteins might be involved in the occurrence of early HE . In another study by Liu et al., 11 PHA−L reactive glycoproteins with a significantly altered N ‐glycosite occupancy (β1,6‐GlcNAc branched N ‐glycan) were identified in HCC patients with metastasis, using a lectin microarray, lectin affinity chromatography, and stable isotope labeling coupled with LC‐MS, which suggested their involvement in HCC metastasis (Table ) …”