Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

Hall, M. Kristen; Weidner, Douglas A.; Zhu, Yong; Dayal, Sahil; Whitman, Austin A.; Schwalbe, Ruth A.

doi:10.3390/ijms17060925

Cited by 14 publications

(21 citation statements)

References 33 publications

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“…Accordingly, the organization of the secretory pathway in eukaryotic cells evolved for this purpose; limited exposure time of glycoprotein substrates to processing enzymes because of defined localization of enzymes in Golgi stacks, limiting enzyme, or substrate concentrations are properties that are the prerequisite for primary sequence-directed glycan maturation. The structural organization of the secretory pathway in eukaryotic cells therefore follows the functionality of site-specific glycan processing, generating novel properties mediated by site-specific structures (18,(51)(52)(53).…”

Section: Discussionmentioning

confidence: 99%

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/ protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity. FASEB J. 31, 4623-4635 (2017). www.fasebj.org KEY WORDS: glycoengineering • glycobiology • glycoprotein maturation • Golgi processing • enzymatic flux N-glycosylation is a major posttranslational modification of proteins that modulates folding, maturation, trafficking, and secretion, as well as specific protein functions (1-3). Contrary to many other posttranslational modifications, N-glycans are composed of many monosaccharide units, creating a large diversity of glycan structures (4, 5). In eukaryotic cells, the N-glycosylation occurs in the endoplasmic reticulum (ER) after a conserved pathway. The oligosaccharide GlcNAc 2 Man 9 Glc 3 is assembled on the lipid dolicholpyrophosphate at the membrane of the ER and then is covalently linked to the side-chain amide of the asparagine residue in the sequon N-X-S/T by oligosaccharyltransferase (OST) (1, 6, 7). Multiple sites on one glycoprotein can be modified by the OST complex and with the increasing complexity of multicellular organism, an increasing number of N-glycosylation sites in the glycoproteome are observed. It is estimated that up to 6000 different sites are modified by OST in mammalian cells (8). In the second phase of the N-glycosylation pathway, the protein-linked glycan is trimmed by glucosidases and mannosidases (1, 9) and is subsequently modified by different glycosyltransferases spatially distributed along the secretory pathway. N-glycan processing enzymes have defined substrate specificities and locations in the secretory pathway (9, 10), and their expression level can be regulated upon internal and external signals.Glycan maturation in the secretory pathway is not a template-driven process. The...

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Section: Discussionmentioning

confidence: 99%

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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“…In contrast, there was a reduction in the GNL bound to the plasma membrane of the HuNB cell line compared to the HuNB(-MGAT2) cell line. The bar graph showed that binding of L-PHA and E-PHA were at least five-fold higher for the parental cell line than the glycosylation mutant, while the binding of GNL was reduced by half ( Figure 1C).The difference in GNL binding between HuNB cells and HuNB(-MGAT2) cells was like that observed in the parental and glycosylation mutant lacking MGAT2 in rat NB [13,25] and CHO [24] cell lines. Lectin blotting verified the flow cytometry observations, such that band intensities detected by L-PHA and E-PHA were much higher in the HuNB cell line than the HuNB(-MGAT2) cell line, while they were of lighter intensity in the HuNB cell line using GNL (Figure 2A).…”

Section: Lectin Binding Assays Of Cell Linesmentioning

confidence: 72%

“…In short, CRISPR-Cas9 technology was used to silence MGAT2 in the HuNB cell line to produce HuNB(-MGAT2), as we previously described for creating the rat NB_1(-MGAT2) cell line [25]. Of note, the sgRNA oligonucleotides (5 -CACCGTTCCGCATCTACAAACGGA-3 and 5 -AAACTCCGTTTGTAGATGCGGAAC-3 ) were identical to those used to silence MGAT2 in Chinese hamster [24] and rat [25] cells. Sequencing of the genomic DNA fragment verified that the gene was silenced from nine separate cell clones.…”

Section: Cell Linesmentioning

confidence: 99%

Knockdown of N-Acetylglucosaminyltransferase-II Reduces Matrix Metalloproteinase 2 Activity and Suppresses Tumorigenicity in Neuroblastoma Cell Line

Hall

Whitman

Weidner

et al. 2020

Biology

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Neuroblastoma (NB) development and progression are accompanied by changes in N-glycans attached to proteins. Here, we investigated the role of N-acetylglucosaminyltransferase-II (GnTII, MGAT2) protein substrates in neuroblastoma (NB) cells. MGAT2 was silenced in human BE(2)-C NB (HuNB) cells to generate a novel cell line, HuNB(-MGAT2), lacking complex type N-glycans, as in rat B35 NB cells. Changes in N-glycan types were confirmed by lectin binding assays in both cell lines, and the rescued cell line, HuNB(-/+MGAT2). Western blotting of cells heterologously expressing a voltage-gated K+ channel (Kv3.1b) showed that some hybrid N-glycans of Kv3.1b could be processed to complex type in HuNB(-/+MGAT2) cells. In comparing HuNB and HuNB(-MGAT2) cells, decreased complex N-glycans reduced anchorage-independent cell growth, cell proliferation, and cell invasiveness, while they enhanced cell-cell interactions. Cell proliferation, invasiveness and adhesion of the HuNB(-/+MGAT2) cells were more like the HuNB than HuNB(-MGAT2). Western blotting revealed lower protein levels of MMP-2, EGFR and Gab2 in glycosylation mutant cells relative to parental cells. Gelatin zymography demonstrated that decreased MMP-2 protein activity was related to lowered MMP-2 protein levels. Thus, our results support that decreased complex type N-glycans suppress cell proliferation and cell invasiveness in both NB cell lines via remodeling ECM.

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“…The formation of multivalent interactions between glycoproteins/glycolipids and clustered surface glycans is favored in lipid rafts [36]. This contributes to special plasma membrane architecture and peculiar cellular properties, typical of each ABO phenotype [127].…”

Section: Glycan Reactionsmentioning

confidence: 99%

Blood Type Diets (BTD) and Aging: An Overview

Menapace¹

2019

J Aging Sci

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It has recently been proposed that glycans, being the third alphabet of life, interact intricately with endogenous biomolecules to modulate tolerance, immune and inflammatory responses. Specifically, food glycans could impact health and be a source of inflammation and age-related diseases. These special carbohydrates are present as glycoconjugates (glycoproteins or glycolipids) in and on the surface of all the cells (glycocalyx) of all organisms or are found in free form in biological fluids. Recent advances in glycobiology and glycochemistry have shown how glycans bind with naturally present human proteins (lectins), through protein-carbohydrate interactions (or PCI), but also how oligosaccharides can interact with other glycans, present throughout the human body (through carbohydrate-carbohydrate interactions, or CCI). Oligosaccharides present in food sources, which go beyond the definition of normal fibers, once ingested are then either absorbed in the bloodstream, where they are recognized by the immune system, or interact with the surface of GI epithelial cells, thus generating appropriate biochemical cascades that induce a tolerance or immune/inflammatory response. Because the ABO epitopes have been encountered on all human cells, not just erythrocytes and based on the different biotypology (A, AB, B, and O) impose morphic changes in the distribution of the glycans on the glycocalyx (lipid rafts and clustered saccharide patches), their CCI with food and microbe glycans will be different, thus, eliciting contrasting responses. This can explain the epidemiological data for blood type diets (BTD). Through continuous consumption of the wrong types of glycans, processes of chronic inflammation could be initiated and progress to accelerated aging. Four basic modes of action have been identified showing how glycans can trigger inflamm-aging. Since glycobiology is a young science, further studies with newer technologies are warranted for advancement in this field.

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Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

Cited by 14 publications

References 33 publications

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Knockdown of N-Acetylglucosaminyltransferase-II Reduces Matrix Metalloproteinase 2 Activity and Suppresses Tumorigenicity in Neuroblastoma Cell Line

Blood Type Diets (BTD) and Aging: An Overview

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