Diversity and functions of protein glycosylation in insects

Walski, Tomasz; Schutter, Kristof De; Damme, Els J.M. Van; Smagghe, Guy

doi:10.1016/j.ibmb.2017.02.005

Cited by 91 publications

(71 citation statements)

References 146 publications

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“…Few methods currently exist to produce recombinant proteins incorporating the Man 5 and Man 9 glycans that are present in the HIV gp120 envelope protein. Expression of gp120 in yeast results in the incorporation of long chain high-mannose glycans (68), and insect expression systems produce a preponderance of paucimannose forms (Man 3–4 ) (69). Glycosidase inhibitors (e.g.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

Byrne

O’Rourke

Alexánder

et al. 2018

Preprint

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Over the last decade multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein, gp120, have been described. Surprisingly many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of HIV envelope (Env) proteins compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan dependent epitopes has been challenging. Here we report the development of a stable suspension adapted CHO cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the CRISPR/Cas9 gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase (MGAT1). Monomeric gp120s produced in the MGAT1- CHO cell line exhibit improved binding to prototypic glycan dependent bN-mAbs directed to the V1/V2 domain (e.g. PG9) and the V3 stem (e.g. PGT128 and 10–1074) while preserving the structure of the important glycan independent epitopes (e.g. VRC01). The ability of the MGAT1-CHO cell line to limit glycosylation to early intermediates in the N-linked glycosylation pathway, without impairing the doubling time or ability to grow at high cell densities, suggest that it will be a useful substrate for the biopharmaceutical production of HIV-1 vaccine immunogens.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

Byrne

O’Rourke

Alexánder

et al. 2018

Preprint

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“…These modifications enhance the molecular and functional diversity of the proteome to large degree, providing biological access to vast information space at minimum genetic cost . Protein glycosylation is involved in various cellular or molecular mechanisms, including protein folding and stability, molecular functional switching, signaling cascades, enzymatic activity, cancer metastasis, cell adhesion, and cell‐cell interactions . A recent review highlights the multifunctional role of glycosylation in organismal diversity, involving structures, modulation, extrinsic recognition, intrinsic recognition, and molecular mimicry .…”

Section: Introductionmentioning

confidence: 99%

Mass spectrometry for protein sialoglycosylation

Zhang

Wang

et al. 2017

Mass Spectrometry Reviews

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Sialic acids are a family of structurally unique and negatively charged nine-carbon sugars, normally found at the terminal positions of glycan chains on glycoproteins and glycolipids. The glycosylation of proteins is a universal post-translational modification in eukaryotic species and regulates essential biological functions, in which the most common sialic acid is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid) (Neu5NAc). Because of the properties of sialic acids under general mass spectrometry (MS) conditions, such as instability, ionization discrimination, and mixed adducts, the use of MS in the analysis of protein sialoglycosylation is still challenging. The present review is focused on the application of MS related methodologies to the study of both N- and O-linked sialoglycans. We reviewed MS-based strategies for characterizing sialylation by analyzing intact glycoproteins, proteolytic digested glycopeptides, and released glycans. The review concludes with future perspectives in the field.

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“…Our results are in agreement with the insect glycans analyses. In insects and insect cells, glycoproteins were mainly found to carry paucimannosidic glycans (Walski, De Schutter, Van Damme, & Smagghe, 2017). However, it has been demonstrated that the most common substitution of the terminal mannose residues is a terminal GlcNAc on the 1,3-linked mannosyl residues added by the GlcNActransferase I (Katoh & Tiemeyer, 2013;Léonard et al, 2006;Walski et al, 2017).…”

Section: Resultsmentioning

confidence: 99%

In vitro evidence for the participation of Drosophila melanogaster sperm β‐N‐acetylglucosaminidases in the interactions with glycans carrying terminal N‐acetylglucosamine residues on the egg's envelopes

Intra

Veltri

et al. 2017

Arch Insect Biochem Physiol

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Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm-associated β-N-acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid-phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well-defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β-N-acetylglucosamine, but not core β-N-acetylglucosamine residues. The capacity of sperm β-N-acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β-N-acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β-N-acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm-egg interactions in Drosophila.

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Diversity and functions of protein glycosylation in insects

Cited by 91 publications

References 146 publications

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

Mass spectrometry for protein sialoglycosylation

In vitro evidence for the participation of Drosophila melanogaster sperm β‐N‐acetylglucosaminidases in the interactions with glycans carrying terminal N‐acetylglucosamine residues on the egg's envelopes

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