Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p

Yusuf, Siti Nur Hasanah Mohd; Bailey, Ulla-Maja; Tan, Nikki Y.; Jamaluddin, M Fairuz B; Schulz, Benjamin L.

doi:10.1016/j.bbrc.2013.01.128

Cited by 17 publications

(16 citation statements)

References 37 publications

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“…distinct roles in stabilizing the complex, interacting with the Sec61 translocon or mediating glycosylation before complete folding of substrate proteins (59,60). For example, subunits of the yeast OTase complex, Ost3p/Ost6p, and the mammalian STT3B OTase complex, MagT1/TUSC3, exhibit oxidoreductase activity, which is thought to delay oxidative protein folding through mixed disulfide bond formation (61)(62)(63). Although the enzymes required to synthesize and transfer the C. jejuni heptasaccharide are encoded adjacent to each other on the chromosome, there may be other nonessential components elsewhere on the chromosome with analogous functions to the eukaryotic OTase subunits.…”

Section: Discussionmentioning

confidence: 99%

Bacterial N-Glycosylation Efficiency Is Dependent on the Structural Context of Target Sequons

Silverman

Imperiali

2016

Journal of Biological Chemistry

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Site selectivity of protein N-linked glycosylation is dependent on many factors, including accessibility of the modification site, amino acid composition of the glycosylation consensus sequence, and cellular localization of target proteins. Previous studies have shown that the bacterial oligosaccharyltransferase, PglB, of Campylobacter jejuni favors acceptor proteins with consensus sequences ((D/E)X 1 NX 2 (S/T), where X 1,2 ≠ proline) in flexible, solvent-exposed motifs; however, several native glycoproteins are known to harbor consensus sequences within structured regions of the acceptor protein, suggesting that unfolding or partial unfolding is required for efficient N-linked glycosylation in the native environment. To derive insight into these observations, we generated structural homology models of the N-linked glycoproteome of C. jejuni. This evaluation highlights the potential diversity of secondary structural conformations of previously identified N-linked glycosylation sequons. Detailed assessment of PglB activity with a structurally characterized acceptor protein, PEB3, demonstrated that this natively folded substrate protein is not efficiently glycosylated in vitro, whereas structural destabilization increases glycosylation efficiency. Furthermore, in vivo glycosylation studies in both glyco-competent Escherichia coli and the native system, C. jejuni, revealed that efficient glycosylation of glycoproteins, AcrA and PEB3, depends on translocation to the periplasmic space via the general secretory pathway. Our studies provide quantitative evidence that many acceptor proteins are likely to be N-linkedglycosylated before complete folding and suggest that PglB activity is coupled to general secretion-mediated translocation to the periplasm. This work extends our understanding of the molecular mechanisms underlying N-linked glycosylation in bacteria.

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Section: Discussionmentioning

confidence: 99%

Bacterial N-Glycosylation Efficiency Is Dependent on the Structural Context of Target Sequons

Silverman

Imperiali

2016

Journal of Biological Chemistry

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“…7B). This binding is non-covalent (18) or through a mixed disulfide (17) and depends on complementarity between the stretch of nascent polypeptide and the peptide-binding groove of Ost3p/Ost6p (Figs. 4 -6).…”

Section: Mapping Sites Of Interaction Between Substrate Andmentioning

confidence: 99%

“…7G). Incorporation of Ost3p/Ost6p into OTase would therefore enhance the glycosylation of diverse proteins, and their distinct peptide-binding specificities (17,18,25) would further increase the range of efficiently modified substrates.…”

Section: Mapping Sites Of Interaction Between Substrate Andmentioning

confidence: 99%

“…A model of Ost3p/Ost6p function in N-glycosylation site selection has been proposed (16) in which the ER-lumenal peptide-binding grooves transiently tether nascent polypeptide non-covalently or through mixed disulfides, inhibiting local protein folding and increasing the efficiency of glycosylation of nearby asparagine residues. Aspects of this model, including mixed-disulfide formation (17) and non-covalent peptide binding (18), have been tested in vitro. Although it has been established that Ost3p-OTase and Ost6p-OTase have different polypeptide substrate preferences in vivo (12)(13)(14)(15)19), the physiological relevance and details of any interactions between Ost3p/Ost6p and substrate polypeptide are unclear.…”

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confidence: 99%

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Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation

Jamaluddin

Bailey

Schulz

2014

Molecular & Cellular Proteomics

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Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptidebinding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification. Molecular & Cellular Proteomics

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“…BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0) yeast cells and the Dgas1 mutant derivative thereof (Open Biosystems) was transformed with pRS415, pRS415-GAS1 or variants using standard protocols. Cells were diluted to equal densities at OD 600 nm , serially 10-fold diluted, equal volumes spotted to minimal media agar plates and incubated at 30 o C. TOP10 E. coli cells carrying the plasmids encoding IFNb, Ost6 (pHis10-OST6L 43 ), Ost3 (pHis10-OST3L 43 ) or variants thereof were grown in LB media, and cells carrying pMAL-Gas1 (ref. 36) or pMAL-Gas2 were grown in LB-glucose media.…”

Section: Methodsmentioning

confidence: 99%

Sequence-based protein stabilization in the absence of glycosylation

et al. 2014

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Asparagine-linked N-glycosylation is a common modification of proteins that promotes productive protein folding and increases protein stability. Although N-glycosylation is important for glycoprotein folding, the precise sites of glycosylation are often not conserved between protein homologues. Here we show that, in Saccharomyces cerevisiae, proteins upregulated during sporulation under nutrient deprivation have few N-glycosylation sequons and in their place tend to contain clusters of like-charged amino-acid residues. Incorporation of such sequences complements loss of in vivo protein function in the absence of glycosylation. Targeted point mutation to create such sequence stretches at glycosylation sequons in model glycoproteins increases in vitro protein stability and activity. A dependence on glycosylation for protein stability or activity can therefore be rescued with a small number of local point mutations, providing evolutionary flexibility in the precise location of N-glycans, allowing protein expression under nutrient-limiting conditions, and improving recombinant protein production.

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Mixed disulfide formation in vitro between a glycoprotein substrate and yeast oligosaccharyltransferase subunits Ost3p and Ost6p

Cited by 17 publications

References 37 publications

Bacterial N-Glycosylation Efficiency Is Dependent on the Structural Context of Target Sequons

Bacterial N-Glycosylation Efficiency Is Dependent on the Structural Context of Target Sequons

Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation

Sequence-based protein stabilization in the absence of glycosylation

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