Site-SpecificN-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

Abstract

Abstract. Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/ MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

“…This tendency is not consistent with the findings by Palmisano and co-workers: on RP, higher number of sialic acid increases retention on RP, but decreases retention on ZIC-HILIC [139]. In the analysis of N-glycosylation of recombinant pentameric and hexameric human IgM, limited number of sialylated glycopeptides were identified after enrichment on ZIC-HILIC [140]. Another review pointed out the disadvantage of HILIC separation is in low resolution of highly sialylated glycans compared to RP or WAX separations [141].…”

Ninjurin1 Assembles Into a Homomeric Protein Complex Maintained byN-linked Glycosylation

“…Numerous studies have demonstrated the regulatory role of N ‐glycosylation in protein stability and trafficking [Haga et al, ; Imjeti et al, ; Moharir et al, ]. Recently, the role of N ‐glycosylation was extended to protein assembly [Kim et al, ; Wu et al, ], and Moh et al [] reported that site‐specific N ‐glycosylation determines the pentameric or hexameric assembly of recombinant human IgM. Our results revealed that both of endogenous and exogenous Ninj1 are N ‐glycosylated.…”

Characterization of intact glycopeptides reveals the impact of culture media on site‐specific glycosylation of EPO‐Fc fusion protein generated by CHO‐GS cells

“…Intact glycopeptide profiling characterizing the site‐specific glycans has emerged as an alternative and complementary glycan analysis tool in recent years. Its application includes biomarker discovery, disease diagnosis and biopharmaceutical product quality control (Bora de Oliveira, Spencer, Barton, & Agarwal, ; Kolarich, Jensen, Altmann, & Packer, ; Moh, Lin, Thaysen‐Andersen, & Packer, ; Narimatsu et al, ). Here we adopted this glycoproteomic method for evaluating the effects of different media compositions on the glycan profile of a recombinant protein, EPO‐Fc, secreted from CHO cells.…”