Comparative N-Glycan Profiling of Colorectal Cancer Cell Lines Reveals Unique Bisecting GlcNAc and α-2,3-Linked Sialic Acid Determinants Are Associated with Membrane Proteins of the More Metastatic/Aggressive Cell Lines

Abstract

Advances in colorectal cancer (CRC) diagnosis will be enhanced by development of more sensitive and reliable methods for early detection of the disease when treatment is more effective. Because many known disease biomarkers are membrane-bound glycoproteins with important biological functions, we chose to compare N-glycan profiles of membrane proteins from three phenotypically different CRC cell lines, LIM1215, LIM1899, and LIM2405, representing moderately differentiated metastatic, moderately differentiated primary, and poorly differentiated (aggressive) primary CRC cell lines, respectively. The N-glycan structures and their relative abundances were determined as their underivatized reduced forms, using porous graphitized carbon LC-ESI-MS/MS. A key observation was the similar N-glycan landscape in these cells with the dominance of high mannose type glycan structures (70-90%) in all three cell lines, suggesting an incomplete glycan processing. Importantly, unique glycan determinants such as bisecting N-acetylglucosamine were observed at a high level in the metastatic LIM1215 cells, with some expressed in the moderately differentiated LIM1899, while none were detected in the poorly differentiated LIM2405 cells. Conversely, α-2,3-sialylation was completely absent in LIM1215 and LIM1899 and present only in LIM2405. RNA-Seq and lectin immunofluorescence data correlated well with these data, showing the highest upregulation of Mgat3 and binding with PHA-E in LIM1215. Downregulation of Man1α1 and Mgat1 in LIM1215 also coincided with the higher degree of incomplete N-glycan processing and accumulation of high mannose type structures as well as bisecting N-glycans when compared to the other two cell lines. This study provides a comprehensive analysis of the membrane N-glycome in three CRC cell lines and identifies N-glycosylation differences that correlate with the histological and pathological features of the cell lines. The unique glycosylation phenotypes may therefore serve as a molecular feature to differentiate CRC disease stages.

Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation, and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels

“…Further, our mass spectrometry analysis showed that the sialic acids on bisected N-glycans were almost in an ␣2,6-linkage. Consistently, a recent study on the N-glycan profiling of a GnT-III highly expressed colorectal cancer cell line using the method of porous graphitized carbon LC-ESI-MS/MS indicated that the bisecting GlcNAc structures accounted for 38% of total relative abundance of complex and hybrid glycans, but no ␣2,3-linked sialic acids were detected on the bisected N-glycans (17). All these results suggest that the biantennary or hybrid bisecting structures may not favor the subsequent transfer of ␣2,3-linked sialic acids as compared with the nonbisecting chains, but it does not mean that ␣2,3-sialyltransferases could not use the bisecting structures as substrates at all because traces of the bisecting N-glycans that carry ␣2,3-linked sialic acids have been detected recently in ovarian cancer cell lines, although they are much less than ␣2,6-sialylated bisecting N-glycans (16).…”

“…Recently, the expression of both GnT-III and ST6GAL1 was found up-regulated in the human ovarian cancer and knockdown of ST6GAL1 significantly inhibited cell migration (16,40). One group last year showed a highly metastatic colorectal cancer cell line, which contained an abundance of bisecting GlcNAc glycans (17). This made them somewhat surprised because GnT-III is usually considered to be a tumor metastasis suppressor.…”

N-glycan MALDI Imaging Mass Spectrometry on Formalin-Fixed Paraffin-Embedded Tissue Enables the Delineation of Ovarian Cancer Tissues

“…The increase in high mannose structures have been previously reported in breast cancer progression in mouse models, colorectal cancer cell lines and hepatocellular carcinoma, among others (42)(43)(44). The increased expression of highmannose glycans could suggest a premature termination of the glycosylation pathway during glycan synthesis.…”

Human Neutrophils Secrete Bioactive Paucimannosidic Proteins from Azurophilic Granules into Pathogen-Infected Sputum

“…Intrigued by the temporal and compartment-specific biosynthetic route for paucimannosylation in neutrophils proposed here, we are investigating whether paucimannosylation is unique to neutrophils or common across cell types. We have recently reported that cultured human colon and breast cancer epithelial cells produce paucimannosidic epitopes (6,60), albeit in lower quantities (typically less than 15-20% of the total N-glycome), supporting a possible oncofetal antigen potential of paucimannosylation (12) and the possible molecular and functional similarity of neutrophils and epithelial cells (58). However, the lack of azurophilic granules in epithelial cells implies that production, storage, and secretion of paucimannosidic proteins may be facilitated by other mechanisms in these systems.…”