SnapShot: N-Glycosylation Processing Pathways across Kingdoms

Chung, C.‐S.; Majewska, Natalia I.; Wang, Qiong; Paul, Jackson T.; Betenbaugh, Michael J.

doi:10.1016/j.cell.2017.09.014

Cited by 67 publications

(63 citation statements)

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“…A). Excellent reviews have summarised the structure, localisation, biosynthesis and functions of the canonical types of N ‐glycoproteins, their biological involvement in human health and disease, and their phylogenetic differences across species (Kreisman & Cobb, ; Breitling & Aebi, ; Chung et al ., ; Varki, ).…”

Section: Introductionmentioning

confidence: 98%

“…Asparagine ( N )‐linked glycosylation, the focus of this review, encompasses a broad class of glycoproteins that exists across most multi‐ and unicellular eukaryotic species and in some prokaryotes (Chung et al ., ; Corfield, ; Eichler & Koomey, ; Nothaft & Szymanski, ; Weerapana & Imperiali, ). Often limited sets of monosaccharide building blocks are utilised, usually in preferred anomeric and linkage configurations, during the biosynthesis of N ‐glycoproteins across species including α‐L‐fucose (Fuc), N ‐acetyl‐α‐D‐galactosamine (GalNAc), N ‐acetyl‐β‐D‐glucosamine (GlcNAc), α‐D‐glucose (Glc), α/β‐D‐galactose (Gal), α/β‐D‐mannose (Man) and sialic acid e.g.…”

Section: Introductionmentioning

confidence: 99%

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Human protein paucimannosylation: cues from the eukaryotic kingdoms

et al. 2019

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Paucimannosidic proteins (PMPs) are bioactive glycoproteins carrying truncated α-or β-mannosyl-terminating asparagine (N )-linked glycans widely reported across the eukaryotic domain. Our understanding of human PMPs remains limited, despite findings documenting their existence and association with human disease glycobiology. This review comprehensively surveys the structures, biosynthetic routes and functions of PMPs across the eukaryotic kingdoms with the aim of synthesising an improved understanding on the role of protein paucimannosylation in human health and diseases. Convincing biochemical, glycoanalytical and biological data detail a vast structural heterogeneity and fascinating tissue-and subcellular-specific expression of PMPs within invertebrates and plants, often comprising multi-α1,3/6-fucosylation and β1,2-xylosylation amongst other glycan modifications and non-glycan substitutions e.g. O-methylation. Vertebrates and protists express less-heterogeneous PMPs typically only comprising variable core fucosylation of bi-and trimannosylchitobiose core glycans. In particular, the Manα1,6Manβ1,4GlcNAc(α1,6Fuc)β1,4GlcNAcβAsn glycan (M2F) decorates various human neutrophil proteins reportedly displaying bioactivity and structural integrity demonstrating that they are not degradation products. Less-truncated paucimannosidic glycans (e.g. M3F) are characteristic glycosylation features of proteins expressed by human cancer and stem cells. Concertedly, these observations suggest the involvement of human PMPs in processes related to innate immunity, tumorigenesis and cellular differentiation. The absence of human PMPs in diverse bodily fluids studied under many (patho)physiological conditions suggests extravascular residence and points to localised functions of PMPs in peripheral tissues. Absence of PMPs in Fungi indicates that paucimannosylation is common, but not universally conserved, in eukaryotes. Relative to human PMPs, the expression of PMPs in plants, invertebrates and protists is more tissue-wide and constitutive yet, similar to their human counterparts, PMP expression remains regulated by the physiology of the producing organism and PMPs evidently serve essential functions in development, cell-cell communication and host-pathogen/symbiont interactions. In most PMP-producing organisms, including humans, the N -acetyl-β-hexosaminidase isoenzymes and linkage-specific α-mannosidases are glycoside hydrolases critical for generating PMPs via N -acetylglucosaminyltransferase I (GnT-I)-dependent and GnT-I-independent truncation pathways. However, the identity and structure of many species-specific PMPs in eukaryotes, their biosynthetic routes, strong tissue-and development-specific expression, and diverse functions are still elusive. Deep exploration of these PMP features involving, for example, the characterisation of endogenous PMP-recognising lectins across a variety of healthy and N -acetyl-β-hexosaminidase-deficient human tissue types and identification of microbial adhesins reactive to human PMPs, are amongs...

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Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Human protein paucimannosylation: cues from the eukaryotic kingdoms

et al. 2019

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“…In spite of such structural constraints, N ‐glycoproteins often display considerable heterogeneity due to structural variations in the glycan antenna region . Three prominent classes, the oligomannosidic, hybrid, and complex type N ‐glycoproteins, dominate the human glycobiological literature and are often studied in cancer research …”

Section: Introductionmentioning

confidence: 99%

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

et al. 2019

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While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics‐centric study investigates a possible link between protein paucimannosylation, an under‐studied class of human N‐glycosylation [Man1‐3GlcNAc2Fuc0‐1], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non‐cancerous specimens are profiled from 467 published and unpublished PGC‐LC‐MS/MS N‐glycome datasets collected over a decade. PMGs, particularly Man2‐3GlcNAc2Fuc1, are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0–50.2%). Analyses of paired (tumor/non‐tumor) and stage‐stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N‐acetyl‐β‐hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.

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“…The glycosylation process initiates in the lumen of the endoplasmic reticulum followed by elongation and completion in the Golgi apparatus (Spiro, ; Varki & Schauer, ). As an enzyme‐directed, site‐specific process, the activity and availability of the relevant glycosyltransferases, and the metabolite fluxes through nucleotide sugar synthesis and transportation pathways are critical to the final glycan profile of recombinant proteins (Chung, Majewska, Wang, Paul, & Betenbaugh, ). Numerous process effects, such as the composition and nutrients in the culture media, harvest time, culture temperature, pH, CO 2 , and dissolved oxygen level can also affect the final glycoforms of therapeutic proteins (Hossler et al, ).…”

Section: Introductionmentioning

confidence: 99%

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

Wang

Yang

Wang

et al. 2019

Biotech & Bioengineering

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With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N-and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N-and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc Nglycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi-and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1-4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N-and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest. K E Y W O R D S CHO-GS cells, culture media, EPO-Fc protein, intact glycopeptide analysis, site-specific glycosylation SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section.

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SnapShot: N-Glycosylation Processing Pathways across Kingdoms

Abstract: Post-translational modification of proteins with carbohydrates shapes their localization and function. This SnapShot presents the core pathways from different organisms that install these complex and highly variable structures.

Cited by 67 publications

References 0 publications

Human protein paucimannosylation: cues from the eukaryotic kingdoms

Human protein paucimannosylation: cues from the eukaryotic kingdoms

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

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

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