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Oligosaccharyltransferase subunits bind polypeptide substrate to locally enhance N-glycosylation.

ABSTRACT: 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 peptide-binding 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.

SUBMITTER: Jamaluddin MF

PROVIDER: S-EPMC4256483 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Oligosaccharyltransferase subunits bind polypeptide substrate to locally enhance N-glycosylation.

Jamaluddin M Fairuz B MF Bailey Ulla-Maja UM Schulz Benjamin L BL

Molecular & cellular proteomics : MCP 20140812 12

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 peptide-binding g ...[more]

PMID: 25118247

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Project description:BackgroundAsparagine (N)-linked glycosylation is one of the most crucial post-translational modifications, which is catalyzed in the lumen of the endoplasmic reticulum (ER) by the oligosaccharyltransferase (OST) in eukaryotic cells. Biochemical and genetic assay leads to the identification of the nine subunits (Ost 1-6, Stt3, Swp1 and Wbp1) of the yeast OST and in which Stt3p is proposed playing a central and conserved role in N-glycosylation. Two STT3 isoform genes, STT3A and STT3B, exist in the plant and mammal genomes. OST with different catalytic STT3 isoforms has different enzymatic properties in mammals. The mutation of STT3A in Arabidopsis thaliana causes a salt hypersensitive phenotype the inhibited root growth and swollen root tips suggesting protein N-glycosylation is indispensable for plant growth and development. Spartina alterniflora is widely used for shoreline protection and tidal marsh restoration due to the strong salt tolerance although the exact molecular mechanism is little known. To explore the possible biological roles of N-glycosylation in plant adaptive resistance to salinity stress, we cloned the STT3 genes from S. alterniflora and heterogenously expressed them in Arabidopsis mutant to observe the functional conservation.ResultsSaSTT3A and SaSTT3B genes were cloned from Spartina alterniflora. SaSTT3A genomic sequences spanned over 23 exons and 22 introns, while SaSTT3B had 6 exons and 5 introns. The gene structures of both genes were conserved among the analyzed plant species. Subcellular localization and transmembrane structure prediction revealed that these two genes had 13 and 11 transmembrane helices respectively. The functional complementation in which the cDNA of SaSTT3A and SaSTT3B driven by CaMV 35S promoter completely or partially rescued Arabidopsis stt3a-2 mutant salt-sensitive phenotype, indicating STT3A functions conservatively between glycophyte and halophyte and N-glycosylation might be involved in plant resistance to salinity.ConclusionsTwo STT3 isoform genes, SaSTT3A and SaSTT3B, were cloned from S. alterniflora and they were evolutionally conserved at gene structure and coding sequences compared with their counterparts. Moreover, SaSTT3 genes could successfully rescue Arabidopsis stt3a-2 salt-sensitive phenotype, suggesting there exists a similar N-glycosylation process in S. alterniflora. Here we provided a first piece of evidence that the N-glycosylation might be involved in salt tolerance of halophyte.

Project description:Asparagine-linked glycosylation, also known as N-linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all three domains of life. This modification is essential for specific molecular recognition, protein folding, sorting in the endoplasmic reticulum, cell-cell communication, and stability. Defects in N-linked glycosylation results in a class of inherited diseases known as congenital disorders of glycosylation (CDG). N-linked glycosylation occurs in the endoplasmic reticulum (ER) lumen by a membrane associated enzyme complex called the oligosaccharyltransferase (OST). In the central step of this reaction, an oligosaccharide group is transferred from a lipid-linked dolichol pyrophosphate donor to the acceptor substrate, the side chain of a specific asparagine residue of a newly synthesized protein. The prokaryotic OST enzyme consists of a single polypeptide chain, also known as single subunit OST or ssOST. In contrast, the eukaryotic OST is a complex of multiple non-identical subunits. In this review, we will discuss the biochemical and structural characterization of the prokaryotic, yeast, and mammalian OST enzymes. This review explains the most recent high-resolution structures of OST determined thus far and the mechanistic implication of N-linked glycosylation throughout all domains of life. It has been shown that the ssOST enzyme, AglB protein of the archaeon Archaeoglobus fulgidus, and the PglB protein of the bacterium Campylobactor lari are structurally and functionally similar to the catalytic Stt3 subunit of the eukaryotic OST enzyme complex. Yeast OST enzyme complex contains a single Stt3 subunit, whereas the human OST complex is formed with either STT3A or STT3B, two paralogues of Stt3. Both human OST complexes, OST-A (with STT3A) and OST-B (containing STT3B), are involved in the N-linked glycosylation of proteins in the ER. The cryo-EM structures of both human OST-A and OST-B complexes were reported recently. An acceptor peptide and a donor substrate (dolichylphosphate) were observed to be bound to the OST-B complex whereas only dolichylphosphate was bound to the OST-A complex suggesting disparate affinities of two OST complexes for the acceptor substrates. However, we still lack an understanding of the independent role of each eukaryotic OST subunit in N-linked glycosylation or in the stabilization of the enzyme complex. Discerning the role of each subunit through structure and function studies will potentially reveal the mechanistic details of N-linked glycosylation in higher organisms. Thus, getting an insight into the requirement of multiple non-identical subunits in the N-linked glycosylation process in eukaryotes poses an important future goal.

Dataset Information

Oligosaccharyltransferase subunits bind polypeptide substrate to locally enhance N-glycosylation.

Publications

Oligosaccharyltransferase subunits bind polypeptide substrate to locally enhance N-glycosylation.

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