Influence of media composition on recombinant monoclonal IgA1 glycosylation analysed by lectin-based protein microarray and MALDI-MS

Pažitná, Lucia; Nemčovič, Marek; Pakanová, Zuzana; Baráth, Péter; Алиев, Т. К.; Долгих, Д. А.; Argentova, V. V.; Katrlı́k, Jaroslav

doi:10.1016/j.jbiotec.2020.03.009

Cited by 17 publications

(14 citation statements)

References 41 publications

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“…To release N‐glycans, samples were incubated with 1 unit of the peptide‐N‐glycosidase F (Roche, Basel, Switzerland) at 37°C for 16 h. N‐glycans were isolated by non‐porous graphitised carbon solid‐phase extraction (SPE; Supelclean ENVI‐Carb SPE), and desalted by reverse‐phase SPE (LiChroprep RP18, 25−40 µm; Hykollari et al ., 2017). To increase signal intensities, free N‐glycans were permethylated (Palmigiano et al ., 2018) and analysed by the UltrafleXtreme MALDI ToF/ToF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) in the reflectron positive ion mode using as a matrix solution 2% (w/v) 2,3‐dihydroxybenzoic acid in 30% (v/v) acetonitrile with the addition of 10 m m NaOH to unify the adduct formation (Pažitná et al ., 2020). Raw data were processed by FlexAnalysis (Bruker Daltonics) and GlycoWork Bench software (Ceroni et al ., 2008).…”

Section: Methodsmentioning

confidence: 99%

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin

et al. 2020

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We report on the homo-and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [a(1-4)GalAp] 5 acceptorthe homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [a(1-4)GalAp] 5 acceptor substrates at the À4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[a(1-4)GalAp] 5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[a(1-4)GalAp] 5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and remodelling , and affect the structural features of walls.

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Section: Methodsmentioning

confidence: 99%

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin

et al. 2020

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“…Lectins such as Con A are routinely applied to isolate glycoproteins by affinity chromatography [123]. Lectin-based microarrays help assess the structural alterations of glycans and enable screening and assessment of the glycosylation profiles of therapeutic proteins [124].…”

Section: Discussionmentioning

confidence: 99%

Plant‐Derived Lectins as Potential Cancer Therapeutics and Diagnostic Tools

Mazalovska

Kouokam

2020

BioMed Research International

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Cancer remains a global health challenge, with high morbidity and mortality, despite the recent advances in diagnosis and treatment. Multiple compounds assessed as novel potential anticancer drugs derive from natural sources, including microorganisms, plants, and animals. Lectins, a group of highly diverse proteins of nonimmune origin with carbohydrate-binding abilities, have been detected in virtually all kingdoms of life. These proteins can interact with free and/or cell surface oligosaccharides and might differentially bind cancer cells, since malignant transformation is tightly associated with altered cell surface glycans. Therefore, lectins could represent a valuable tool for cancer diagnosis and be developed as anticancer therapeutics. Indeed, several plant lectins exert cytotoxic effects mainly by inducing apoptotic and autophagic pathways in malignant cells. This review summarizes the current knowledge regarding the basis for the use of lectins in cancer diagnosis and therapy, providing a few examples of plant-derived carbohydrate-binding proteins with demonstrated antitumor effects.

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“…Analysis of N-glycans was performed as described before [ 39 ]. Briefly, after the release of N-glycans with PNGase F (Roche), the sample was subjected to PGC chromatography.…”

Section: Methodsmentioning

confidence: 99%

Production of Recombinant Human Ceruloplasmin: Improvements and Perspectives

Patti

Cutone

Nemčovič

et al. 2021

IJMS

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The ferroxidase ceruloplasmin (CP) plays a crucial role in iron homeostasis in vertebrates together with the iron exporter ferroportin. Mutations in the CP gene give rise to aceruloplasminemia, a rare neurodegenerative disease for which no cure is available. Many aspects of the (patho)physiology of CP are still unclear and would benefit from the availability of recombinant protein for structural and functional studies. Furthermore, recombinant CP could be evaluated for enzyme replacement therapy for the treatment of aceruloplasminemia. We report the production and preliminary characterization of high-quality recombinant human CP in glycoengineered Pichia pastoris SuperMan5. A modified yeast strain lacking the endogenous ferroxidase has been generated and employed as host for heterologous expression of the secreted isoform of human CP. Highly pure biologically active protein has been obtained by an improved two-step purification procedure. Glycan analysis indicates that predominant glycoforms HexNAc2Hex8 and HexNAc2Hex11 are found at Asn119, Asn378, and Asn743, three of the canonical four N-glycosylation sites of human CP. The availability of high-quality recombinant human CP represents a significant advancement in the field of CP biology. However, productivity needs to be increased and further careful glycoengineering of the SM5 strain is mandatory in order to evaluate the possible therapeutic use of the recombinant protein for enzyme replacement therapy of aceruloplasminemia patients.

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Influence of media composition on recombinant monoclonal IgA1 glycosylation analysed by lectin-based protein microarray and MALDI-MS

Cited by 17 publications

References 41 publications

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin

Plant‐Derived Lectins as Potential Cancer Therapeutics and Diagnostic Tools

Production of Recombinant Human Ceruloplasmin: Improvements and Perspectives

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