In-depthN-glycome profiling of paired colorectal cancer and non-tumorigenic tissues reveals cancer-, stage- and EGFR-specific protein N-glycosylation

Abstract

Glycomics may assist in uncovering the structure-function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-liquid chromatography-tandem mass spectrometry-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N-Glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.6 ± 13.1 y.o.) suffering from colorectal adenocarcinoma. The CRC tissues were typed according to their epidermal growth factor receptor (EGFR) status by western blotting and immunohistochemistry. Detailed N-glycan characterization and relative quantitation identified an extensive structural heterogeneity with a total of 91 N-glycans. CRC-specific N-glycosylation phenotypes were observed including an overrepresentation of high mannose, hybrid and paucimannosidic type N-glycans and an under-representation of complex N-glycans (P < 0.05). Sialylation, in particular α2,6-sialylation, was significantly higher in CRC tumors relative to non-tumorigenic tissues, whereas α2,3-sialylation was down-regulated (P < 0.05). CRC stage-specific N-glycosylation was detected by high α2,3-sialylation and low bisecting β1,4-GlcNAcylation and Lewis-type fucosylation in mid-late relative to early stage CRC. Interestingly, a novel link between the EGFR status and the N-glycosylation was identified using hierarchical clustering of the N-glycome profiles. EGFR-specific N-glycan signatures included high bisecting β1,4-GlcNAcylation and low α2,3-sialylation (both P < 0.05) relative to EGFR-negative CRC tissues. This is the first study to correlate CRC stage and EGFR status with specific N-glycan features, thus advancing our understanding of the mechanisms causing the biomolecular deregulation associated with CRC.

Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function

“…Differences in primary structure do not account for the observed differences in N-glycan processing: the CD16a polypeptide sequence and presence of the coexpressed Fc e receptor g-chain were the same in both NK cells and that used in HEK293F cells to express frCD16a. Cell-type specific differences in the glycosylation of a specific protein are known (40)(41)(42)(43)(44) and are justifiable given the complexity of gene expression and activity for glycan-processing enzymes in different tissues (45)(46)(47)(48)(49)(50).…”

Identification of IgM as a contaminant in lectin-FLISA assays for HCC detection

“…Glycosylation changes have been observed in many cancers, including but not limited to liver[13–16], pancreas[17–20], lung [21, 22], breast [23], ovarian[24], colon[25] and prostate cancer[26, 27]. In many, proteins that contain these glycan changes have been identified.…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…1B) (20). Recently, protein paucimannosylation was detected in other cell and tissue types including human fetal lung fibroblasts (103), saliva (176), epithelial colorectal (44,177,178) and breast (179) cancer cells, and in the mouse brain (19) also using glycoproteomics and other -omics technologies. Glycoproteomics was furthermore used to identify core and noncore fucosylated full and truncated chitobiose core type N-glycans from mouse synaptosome (7) and liver (13).…”

“…These include site-specific analysis of N-glycoproteins isolated to relative purity rather than in complex mixtures (11, 12, 20, 34 -42), N-glycomics analyses of glycans released from glycoproteins (18,38,(43)(44)(45)(46)(47), and identification and quantification of previously glycosylated sites on de-N-glycosylated proteins ("deglycoproteomics") after removal of the entire glycan or with remnant N-glycan core remaining (48 -57). Although these studies per se do not qualify under our definition of glycoproteomics, (site-specific analysis of the glycoproteome at the intact glycopeptide level), they still provide useful information in conjunction with glycoproteomics for the glycobiologist, provided correct experimental design is applied (58).…”