skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase
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.  more » « less
Award ID(s):
1807722 1726397
PAR ID:
10159540
Author(s) / Creator(s):
; ;
Date Published:
Journal Name:
Biomolecules
Volume:
10
Issue:
4
ISSN:
2218-273X
Page Range / eLocation ID:
624
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; (, Biomolecular NMR Assignments)
    Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction that occurs in the endoplasmic reticulum of cells during protein synthesis at the ribosome. In the central reaction, a pre-assembled high- mannose sugar is transferred from a lipid-linked donor substrate to the side-chain of an asparagine residue in an -N-X-T/S- sequence (where X is any residue except Proline). This reaction is carried by a membrane-bound multi-subunit enzyme complex, Oligosaccharyltransferase (OST). In humans, genetic defects in OST lead to a group of rare metabolic diseases collectively known as congenital disorders of glycosylation (CDG). Certain mutations are lethal for all organisms. In yeast, the OST is composed of nine non-identical protein subunits. The functional enzyme complex contains eight subunits with either Ost3 or Ost6 at any given time. Ost4, an unusually small protein, plays a very important role in the stabilization of the OST complex. It bridges the catalytic subunit Stt3 with Ost3 (or Ost6) in the Stt3-Ost4-Ost3 (or Ost6) sub-complex. Mutation of any residue from M18-I24 in the trans-membrane helix of yeast Ost4 negatively impacts N-linked glycosylation and the growth of yeast. Indeed, mutation of valine23 to an aspartate impairs OST function in vivo resulting in a lethal phenotype in yeast. To understand the structural mechanism of Ost4 in the stabilization of the enzyme complex, we have initiated a detailed investigation of Ost4 and its functionally important mutant, Ost4V23D. Here, we report the backbone 1H, 13C and 15N resonance assignments for Ost4 and Ost4V23D in DPC micelles. 
    more » « less
  2. ; ; ; (, Glycobiology)
    null (Ed.)
    Abstract Asparagine-linked glycosylation, also known as N-linked glycosylation, is an essential and highly conserved co- and post-translational protein modification in eukaryotes and some prokaryotes. In the central step of this reaction, a carbohydrate moiety is transferred from a lipid-linked donor to the side-chain of a consensus asparagine in a nascent protein as it is synthesized at the ribosome. Complete loss of oligosaccharyltransferase (OST) function is lethal in eukaryotes. This reaction is carried out by a membrane-associated multisubunit enzyme, OST, localized in the endoplasmic reticulum. The smallest subunit, Ost4, contains a single membrane-spanning helix that is critical for maintaining the stability and activity of OST. Mutation of any residue from Met18 to Ile24 of Ost4 destabilizes the enzyme complex, affecting its activity. Here, we report solution nuclear magnetic resonance structures and molecular dynamics (MD) simulations of Ost4 and Ost4V23D in micelles. Our studies revealed that while the point mutation did not impact the structure of the protein, it affected its position and solvent exposure in the membrane mimetic environment. Furthermore, our MD simulations of the membrane-bound OST complex containing either WT or V23D mutant demonstrated disruption of most hydrophobic helix–helix interactions between Ost4V23D and transmembrane TM12 and TM13 of Stt3. This disengagement of Ost4V23D from the OST complex led to solvent exposure of the D23 residue in the hydrophobic pocket created by these interactions. Our study not only solves the structures of yeast Ost4 subunit and its mutant but also provides a basis for the destabilization of the OST complex and reduced OST activity. 
    more » « less
  3. ; (, Plants)
    All living cells generate structurally complex and compositionally diverse spectra of glycans and glycoconjugates, critical for organismal evolution, development, functioning, defense, and survival. Glycosyltransferases (GTs) catalyze the glycosylation reaction between activated sugar and acceptor substrate to synthesize a wide variety of glycans. GTs are distributed among more than 130 gene families and are involved in metabolic processes, signal pathways, cell wall polysaccharide biosynthesis, cell development, and growth. Glycosylation mainly takes place in the endoplasmic reticulum (ER) and Golgi, where GTs and glycosidases involved in this process are distributed to different locations of these compartments and sequentially add or cleave various sugars to synthesize the final products of glycosylation. Therefore, delivery of these enzymes to the proper locations, the glycosylation sites, in the cell is essential and involves numerous secretory pathway components. This review presents the current state of knowledge about the mechanisms of protein trafficking between ER and Golgi. It describes what is known about the primary components of protein sorting machinery and trafficking, which are recognition sites on the proteins that are important for their interaction with the critical components of this machinery. 
    more » « less
  4. ; ; ; ; ; (, Journal of Experimental Botany)
    Abstract Complex N-glycans are asparagine (N)-linked branched sugar chains attached to secretory proteins in eukaryotes. They are produced by modification of N-linked oligosaccharide structures in the endoplasmic reticulum and Golgi apparatus. Complex N-glycans formed in the Golgi apparatus are often assigned specific roles unique to the host organism, with their roles in plants remaining largely unknown. Using inhibitor (kifunensine, KIF) hypersensitivity as read out, we identified Arabidopsis mutants that require complex N-glycan modification. Among >100 KIF-sensitive mutants, one showing abnormal secretory organelles and a salt-sensitive phenotype contained a point mutation leading to amino acid replacement (G69R) in ARFA1E, a small Arf1-GTPase family protein presumably involved in vesicular transport. In vitro assays showed that the G69R exchange interferes with protein activation. In vivo, ARFA1EG69R caused dominant-negative effects, altering the morphology of the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network (TGN). Post-Golgi transport (endocytosis/endocytic recycling) of the essential glycoprotein KORRIGAN1, one of the KIF sensitivity targets, is slowed down constitutively as well as under salt stress in the ARFA1EG69R mutant. Because regulated cycling of plasma membrane proteins is required for stress tolerance of the host plants, the ARFA1EG69R mutant established a link between KIF-targeted luminal glycoprotein functions/dynamics and cytosolic regulators of vesicle transport in endosome-/cell wall-associated tolerance mechanisms. 
    more » « less
  5. ; ; ; ; ; ; ; ; ; ; et al (, Science Advances)
    The conserved signal recognition particle (SRP) cotranslationally delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum (ER). The molecular mechanism by which eukaryotic SRP transitions from cargo recognition in the cytosol to protein translocation at the ER is not understood. Here, structural, biochemical, and single-molecule studies show that this transition requires multiple sequential conformational rearrangements in the targeting complex initiated by guanosine triphosphatase (GTPase)–driven compaction of the SRP receptor (SR). Disruption of these rearrangements, particularly in mutant SRP54 G226E linked to severe congenital neutropenia, uncouples the SRP/SR GTPase cycle from protein translocation. Structures of targeting intermediates reveal the molecular basis of early SRP-SR recognition and emphasize the role of eukaryote-specific elements in regulating targeting. Our results provide a molecular model for the structural and functional transitions of SRP throughout the targeting cycle and show that these transitions provide important points for biological regulation that can be perturbed in genetic diseases. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email