Comparative site-specific N-glycoproteome analysis reveals aberrant N-glycosylation and gives insights into mannose-6-phosphate pathway in cancer

Abstract

N-glycosylation is implicated in cancers and aberrant N-glycosylation is recognized as a hallmark of cancer. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 displays hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells. This comparative site-specific N-glycoproteome analysis provides a pool of potential N-glycosylation-related cancer biomarkers, but also gives insights into the M6P pathway in cancer.

Mass Spectrometry Analysis of Glycopeptides Enriched by Anion Exchange-Mediated Methods Reveals PolyLacNAc-Extended N-Glycans in Integrins and Tetraspanins in Melanoma Cells

“…Each of the approaches resulted in comparable profiles of high mannose, complex, truncated, and modified glycan types (Figure S1), excluding potential enrichment biases based on the glycosylation structure. Melanoma cells were found to predominantly express high-mannose-type N -glycans, in agreement with previous reports. , To investigate whether the higher number of glycopeptides represents a similar increase in unique glycoprotein identifications, or rather a more detailed characterization of same glycoproteins, we compared the numbers of unique glycoprotein identifications among the enrichment approaches. SOLA and RAX resulted in a higher number of unique glycoproteins, albeit the differences were less pronounced compared to unique glycopeptide identification, indicating that SOLA and RAX provide a more comprehensive coverage of peptide glycoforms (Figure B).…”

“…Melanoma cells were found to predominantly express high-mannose-type Nglycans, in agreement with previous reports. 33,34 To investigate whether the higher number of glycopeptides represents a similar increase in unique glycoprotein identifications, or rather a more detailed characterization of same glycoproteins, we compared the numbers of unique glycoprotein identifications among the enrichment approaches. SOLA and RAX resulted in a higher number of unique glycoproteins, albeit the differences were less pronounced compared to unique glycopeptide identification, indicating that SOLA and RAX provide a The percentage of polyLacNAc-containing peptides among identified glycopeptides was calculated using results obtained by Glyco-Decipher and Byonic.…”

Defining the filarial N-glycoproteome by glycosite mapping in the human parasitic nematode Brugia malayi

“…PNGase F cleavage to study the intact N-glycopeptides and correlate glycan structures with speci c glycosites 75 . As well as con rming our N-glycosite mapping and occupancy study, characterizing the N-glycans structures and their variability at each site for male worms, female worms, and micro lariae will yield a comprehensive understanding of the N-glycosylation of larial parasite proteins.…”

Defining the filarial N-glycoproteome by glycosite mapping in the human parasitic nematode Brugia malayi

“…Those N-glycoproteins identified without host orthologs are promising therapeutic or biomarker candidates. As a follow up to this work, we plan to characterize the Fbs1 enriched peptides without PNGase F cleavage to study the intact N-glycopeptides and correlate glycan structures with specific glycosites 81 . As well as confirming our N-glycosite mapping and occupancy study, characterizing the N-glycan structures and their heterogeneity at each site for male worms, female worms, and microfilariae will yield a comprehensive understanding of the N-glycosylation of filarial parasite proteins.…”