Site-specific N-glycosylation of HeLa cell glycoproteins

Abstract

We have characterized site-specific N-glycosylation of the HeLa cell line glycoproteins, using a complex workflow based on high and low energy tandem mass spectrometry of glycopeptides. The objective was to obtain highly reliable data on common glycoforms, so rigorous data evaluation was performed. The analysis revealed the presence of a high amount of bovine serum contaminants originating from the cell culture media – nearly 50% of all glycans were of bovine origin. Unaccounted, the presence of bovine serum components causes major bias in the human cellular glycosylation pattern; as is shown when literature results using released glycan analysis are compared. We have reliably identified 43 (human) glycoproteins, 69 N-glycosylation sites, and 178 glycoforms. HeLa glycoproteins were found to be highly (68.7%) fucosylated. A medium degree of sialylation was observed, on average 46.8% of possible sialylation sites were occupied. High-mannose sugars were expressed in large amounts, as expected in the case of a cancer cell line. Glycosylation in HeLa cells is highly variable. It is markedly different not only on various proteins but also at the different glycosylation sites of the same protein. Our method enabled the detailed characterization of site-specific N-glycosylation of several glycoproteins expressed in HeLa cell line.

Structure and anticancer activity of a new lectin from the cultivated red alga, Kappaphycus striatus

“…Sugahara et al [ 17 ] verified that the cell viability induced by ESA lectins was decreased after the treatment of Colo201 cells with α-mannosidase, β-mannosidase and endoglycosidase H to cleave high mannose type sugar chains on cell surface. These data combine with the recent reports on the presence of the high-mannose structures on the surface of Hela and MCF-7 cells [ 19 – 21 ] corroborate that the lectins interacted with cancer cells through their surface mannose branches. Therefore, the anticancer activity of KSL is able to specifically bind between lectin and high-mannose oligosaccharides present on the surface of the cancer cells.…”

Mucinomics as the Next Frontier of Mass Spectrometry

“…Byonic has been used widely to characterize N-glycosylation, as this is the most commonly studied type of glycopeptide. To be sure, Byonic has been used in the analysis of human milk glycoproteins, virus-receptor interactions of the SARS-CoV-2 Spike protein and human ACE2 receptor, , the proteome profiling of blood–brain barrier perturbation by group B streptococcus (GBS), and the quantification of the exercise-regulated plasma peptidome, among other projects. , Additionally, Byonic contains a very useful “Viewer” that can annotate peptide spectral matches. That said, Byonic struggles with the assignment of O-glycosylation, often relying on HCD spectra for site-localization .…”

Glycosylation in Cervical Cancer: New Insights and Clinical Implications

“…A high degree of fucosylation, a median level of sialylation, and possible site-specific N-glycosylation were well characterized in HeLa cervical cancer cell line, using acetone precipitation and subsequent high and low energy tandem mass spectrometry measurements (14). Although high mannose or complex glycans were principal in most samples, the site-specific pattern and importance of these glycans in patients with cervical cancer remains to be described (4,14).…”

“…A high degree of fucosylation, a median level of sialylation, and possible site-specific N-glycosylation were well characterized in HeLa cervical cancer cell line, using acetone precipitation and subsequent high and low energy tandem mass spectrometry measurements (14). Although high mannose or complex glycans were principal in most samples, the site-specific pattern and importance of these glycans in patients with cervical cancer remains to be described (4,14). The importance of glycosylation in cervical cancer has been highlighted by the fact that glycosylation changes could regulate the cancer development and progression, acting as important cervical cancer biomarkers and contributing to the deciphering of fundamental processes underlying the clinical behaviors of cancer (10,11,15).…”