&lt;i&gt;N&lt;/i&gt;-Glycan structures of glycoproteins in suspension-cultured &lt;i&gt;Arabidopsis thaliana&lt;/i&gt; T87 cells

Kajiura, Hiroyuki; Tanaka, R.; Kato, Ko; Hamaguchi, Yuichi; Seki, Tatsuji; Fujiyama, Kazuhito

doi:10.5511/plantbiotechnology.11.0709a

Cited by 2 publications

(4 citation statements)

References 28 publications

(29 reference statements)

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“…N -acetylglucosamine is commonly present in N -glycans of plant cell walls [ 11 , 20 ] and is important for N- glycan formation, since it is the first monosaccharide attached to glycoproteins [ 25 ]. Therefore metabolic click-mediated labeling of Arabidopsis cell walls was investigated with a N -acetyl-glucosamine analog containing a clickable azide (Ac 4 GlcNAz; Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We investigated five glycans: N -acetyl- d -glucosamine, l -fucose and l- arabinose - which are all known to be present in the glycocalyx of Arabidopsis [ 11 , 20 ] - and N -acetyl- d -galactosamine (GalNAc) and N -acetyl- d -mannosamine. While the latter two glycans are not known to be present in plant glycans, it was recently discovered that UDP-GalNAc is present and transported in the ER of Arabidopsis [ 21 ], indicating that GalNAc is metabolized by plants.…”

Section: Introductionmentioning

confidence: 99%

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Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides

et al. 2016

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BackgroundCarbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques.ResultsArabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, l-fucose, and l-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions.ConclusionsOur results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands the molecular toolbox for direct glycan imaging in plants, from three to eight glycan analogs, which enables more extensive future studies of spatiotemporal glycan dynamics in a wide variety of plant tissues and species. We also show, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods that are bio-orthogonal and lead to more uniform labeling. These improved labeling methods can be generalized and extended to already existing and future click chemistry-enabled monosaccharide analogs in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0907-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides

et al. 2016

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“…The related M3X (Glycan #8) was also a significant, albeit less abundant (2–15%), PMG expressed by A. thaliana . It remains unknown why M3FX (Glycan #24) as opposed to the more truncated mono‐ and bimannosylated PMGs (Glycans #21 and #22a/b) dominates the N ‐glycome of A. thaliana and more widely in Plantae (Henquet et al ., ; Liebminger et al ., ; Kajiura et al ., ).…”

Section: Surveying Pmps Across the Eukaryotic Kingdoms And Phylamentioning

confidence: 97%

“…S1B). Surprisingly, M3FX‐peptides (Glycan #24) were not reported despite this PMG being a prominent A. thaliana N ‐glycome signature (Kajiura et al ., ) (see Table S1). Ma et al .…”

Section: Surveying Pmps Across the Eukaryotic Kingdoms And Phylamentioning

confidence: 99%

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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<i>N</i>-Glycan structures of glycoproteins in suspension-cultured <i>Arabidopsis thaliana</i> T87 cells

Cited by 2 publications

References 28 publications

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides

Human protein paucimannosylation: cues from the eukaryotic kingdoms

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