Asparagine linked glycosylation of proteins is an essential protein modification reaction in most eukaryotic organisms. Metazoan organisms express two oligosaccharyltransferase complexes that are ...composed of a catalytic subunit (STT3A or STT3B) assembled with a shared set of accessory subunits and one to two complex specific subunits. siRNA mediated knockdowns of STT3A and STT3B in HeLa cells have shown that the two OST complexes have partially non-overlapping roles in N-linked glycosylation. However, incomplete siRNA mediated depletion of STT3A or STT3B reduces the impact of OST complex loss, thereby complicating the interpretation of experimental results. Here, we have used the CRISPR/Cas9 gene editing technology to create viable HEK293 derived cells lines that are deficient for a single catalytic subunit (STT3A or STT3B) or two STT3B-specific accessory subunits (MagT1 and TUSC3). Analysis of protein glycosylation in the STT3A, STT3B and MagT1/TUSC3 null cell lines revealed that these cell lines are superior tools for investigating the in vivo role and substrate preferences of the STT3A and STT3B complexes.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Asparagine linked glycosylation of proteins is an essential protein modification reaction in most eukaryotic organisms. N-linked oligosaccharides are important for protein folding and stability, ...biosynthetic quality control, intracellular traffic and the physiological function of many N-glycosylated proteins. In metazoan organisms, the oligosaccharyltransferase is composed of a catalytic subunit (STT3A or STT3B) and a set of accessory subunits. Duplication of the catalytic subunit gene allowed cells to evolve OST complexes that act sequentially to maximize the glycosylation efficiency of the large number of proteins that are glycosylated in metazoan organisms. We will summarize recent progress in understanding the mechanism of (a) cotranslational glycosylation by the translocation channel associated STT3A complex, (b) the role of the STT3B complex in mediating cotranslational or posttranslocational glycosylation of acceptor sites that have been skipped by the STT3A complex, and (c) the role of the oxidoreductase MagT1 in STT3B-dependent glycosylation of cysteine-proximal acceptor sites.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Asparagine-linked glycosylation of proteins by the oligosaccharyltransferase (OST) occurs when acceptor sites or sequons (N-x≠P-T/S) on nascent polypeptides enter the lumen of the rough endoplasmic ...reticulum. Metazoan organisms assemble two isoforms of the OST that have different catalytic subunits (STT3A or STT3B) and partially non-overlapping cellular roles. Potential glycosylation sites move past the STT3A complex, which is associated with the translocation channel, at the protein synthesis elongation rate. Here, we investigated whether close spacing between acceptor sites in a nascent protein promotes site skipping by the STT3A complex. Biosynthetic analysis of four human glycoproteins revealed that closely spaced sites are efficiently glycosylated by an STT3B-independent process unless the sequons contain non-optimal sequence features, including extreme close spacing between sequons (e.g. NxTNxT) or the presence of paired NxS sequons (e.g. NxSANxS). Many, but not all, glycosylation sites that are skipped by the STT3A complex can be glycosylated by the STT3B complex. Analysis of a murine glycoprotein database revealed that closely spaced sequons are surprisingly common, and are enriched for paired NxT sites when the gap between sequons is less than three residues.
Protein synthesis, transport, and N-glycosylation are coupled at the mammalian endoplasmic reticulum by complex formation of a ribosome, the Sec61 protein-conducting channel, and ...oligosaccharyltransferase (OST). Here we used different cryo-electron microscopy approaches to determine structures of native and solubilized ribosome-Sec61-OST complexes. A molecular model for the catalytic OST subunit STT3A (staurosporine and temperature sensitive 3A) revealed how it is integrated into the OST and how STT3-paralog specificity for translocon-associated OST is achieved. The OST subunit DC2 was placed at the interface between Sec61 and STT3A, where it acts as a versatile module for recruitment of STT3A-containing OST to the ribosome-Sec61 complex. This detailed structural view on the molecular architecture of the cotranslational machinery for N-glycosylation provides the basis for a mechanistic understanding of glycoprotein biogenesis at the endoplasmic reticulum.
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BFBNIB, NMLJ, NUK, ODKLJ, PNG, SAZU, UL, UM, UPUK
Asparagine-linked glycosylation of polypeptides in the lumen of the endoplasmic reticulum is catalyzed by the hetero-oligomeric oligosaccharyltransferase (OST). OST isoforms with different catalytic ...subunits (STT3A versus STT3B) and distinct enzymatic properties are coexpressed in mammalian cells. Using siRNA to achieve isoform-specific knockdowns, we show that the OST isoforms cooperate and act sequentially to mediate protein N-glycosylation. The STT3A OST isoform is primarily responsible for cotranslational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The STT3B isoform is required for efficient cotranslational glycosylation of an acceptor site adjacent to the N-terminal signal sequence of a secreted protein. Unlike STT3A, STT3B efficiently mediates posttranslational glycosylation of a carboxyl-terminal glycosylation site in an unfolded protein. These distinct and complementary roles for the OST isoforms allow sequential scanning of polypeptides for acceptor sites to insure the maximal efficiency of N-glycosylation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Stabilization of protein tertiary structure by disulfides can interfere with glycosylation of acceptor sites (NXT/S) in nascent polypeptides. Here, we show that MagT1, an ER-localized thioredoxin ...homologue, is a subunit of the STT3B isoform of the oligosaccharyltransferase (OST). The lumenally oriented active site CVVC motif in MagT1 is required for glycosylation of STT3B-dependent acceptor sites including those that are closely bracketed by disulfides or contain cysteine as the internal residue (NCT/S). The MagT1- and STT3B-dependent glycosylation of cysteine-proximal acceptor sites can be reduced by eliminating cysteine residues. The predominant form of MagT1 in vivo is oxidized, which is consistent with transient formation of mixed disulfides between MagT1 and a glycoprotein substrate to facilitate access of STT3B to unmodified acceptor sites. Cotranslational N-glycosylation by the STT3A isoform of the OST, which lacks MagT1, allows efficient modification of acceptor sites in cysteine-rich protein domains before disulfide bond formation. Thus, mammalian cells use two mechanisms to achieve N-glycosylation of cysteine proximal acceptor sites.
In metazoan organisms, the STT3A isoform of the oligosaccharyltransferase is localized adjacent to the protein translocation channel to catalyze co-translational N-linked glycosylation of proteins in ...the endoplasmic reticulum. The mechanism responsible for the interaction between the STT3A complex and the translocation channel has not been addressed. Using genetically modified human cells that are deficient in DC2 or KCP2 proteins, we show that loss of DC2 causes a defect in co-translational N-glycosylation of proteins that mimics an
phenotype. Biochemical analysis showed that DC2 and KCP2 are responsible for mediating the interaction between the protein translocation channel and the STT3A complex. Importantly, DC2- and KCP2-deficient STT3A complexes are stable and enzymatically active. Deletion mutagenesis revealed that a conserved motif in the C-terminal tail of DC2 is critical for assembly into the STT3A complex, whereas the lumenal loop and the N-terminal cytoplasmic segment are necessary for the functional interaction between the STT3A and Sec61 complexes.
Metazoan organisms assemble two isoforms of the oligosaccharyltransferase (OST) that have different catalytic subunits (STT3A or STT3B) and partially nonoverlapping roles in asparagine-linked ...glycosylation. The STT3A isoform of the OST is primarily responsible for co-translational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The C-terminal 65-75 residues of a glycoprotein will not contact the translocation channel-associated STT3A isoform of the OST complex before chain termination. Biosynthetic pulse labeling of five human glycoproteins showed that extreme C-terminal glycosylation sites were modified by an STT3B-dependent posttranslocational mechanism. The boundary for STT3B-dependent glycosylation of C-terminal sites was determined to fall between 50 and 55 residues from the C terminus of a protein. C-terminal NXT sites were glycosylated more rapidly and efficiently than C-terminal NXS sites. Bioinformatics analysis of glycopeptide databases from metazoan organisms revealed a lower density of C-terminal acceptor sites in glycoproteins because of reduced positive selection of NXT sites and negative selection of NXS sites.
Oligosaccharyltransferases (OSTs) N-glycosylate proteins by transferring oligosaccharides from lipid-linked oligosaccharides (LLOs) to asparaginyl residues of Asn-Xaa-Ser/Thr acceptor sequons. ...Mammals have OST isoforms with STT3A or STT3B catalytic subunits for cotranslational or posttranslational N-glycosylation, respectively. OSTs also hydrolyze LLOs, forming free oligosaccharides (fOSs). It has been unclear whether hydrolysis is due to one or both OSTs, segregated from N-glycosylation, and/or regulated. Transfer and hydrolysis were assayed in permeabilized HEK293 kidney and Huh7.5.1 liver cells lacking STT3A or STT3B. Transfer by both STT3A-OST and STT3B-OST with synthetic acceptors was robust. LLO hydrolysis by STT3B-OST was readily detected and surprisingly modulated: Without acceptors, STT3B-OST hydrolyzed Glc₃Man₉Glc-NAc₂-LLO but not Man₉GlcNAc₂-LLO, yet it hydrolyzed both LLOs with acceptors present. In contrast, LLO hydrolysis by STT3A-OST was negligible. STT3A-OST however may be regulatory, because it suppressed STT3B-OST–dependent fOSs. TREX1, a negative innate immunity factor that diminishes immunogenic fOSs derived from LLOs, acted through STT3B-OST as well. In summary, only STT3B-OST hydrolyzes LLOs, depending upon LLO quality and acceptor site occupancy. TREX1 and STT3A suppress STT3B-OST–dependent fOSs. Without strict kinetic limitations during posttranslational N-glycosylation, STT3B-OST can thus moonlight for LLO hydrolysis. In contrast, the STT3A-OST/translocon complex preserves LLOs for temporally fastidious cotranslational N-glycosylation.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
A critical event in protein translocation across the endoplasmic reticulum is the structural transition between the closed and open conformations of Sec61, the eukaryotic translocation channel. ...Channel opening allows signal sequence insertion into a gap between the N- and C-terminal halves of Sec61. We have identified a gating motif that regulates the transition between the closed and open channel conformations. Polar amino acid substitutions in the gating motif cause a gain-of-function phenotype that permits translocation of precursors with marginally hydrophobic signal sequences. In contrast, hydrophobic substitutions at certain residues in the gating motif cause a protein translocation defect. We conclude that the gating motif establishes the hydrophobicity threshold for functional insertion of a signal sequence into the Sec61 complex, thereby allowing the wild-type translocation channel to discriminate between authentic signal sequences and the less hydrophobic amino acid segments in cytosolic proteins. Bioinformatic analysis indicates that the gating motif is conserved between eubacterial and archaebacterial SecY and eukaryotic Sec61.
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