Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase

Abstract

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.

Sweet Modifications Modulate Plant Development

“…The HAPLESS (HAP) genes have been identified in Arabidopsis and represent a series of genes which are of importance for the development of pollen grains and germination as well as pollen tube elongation [ 126 ]. One of the HAP proteins, HAP6, also known as ribophorin 2, is a subunit of the OST which facilitates en bloc transfer of polypeptides from the cytosol to the ER lumen [ 184 ] ( Figure 2 ). Mutant hap6 plants were characterized by shorter pollen tubes which could not exit the style, leading to impaired pollen tube elongation.…”

Oxidoreductases in Glycoprotein Glycosylation, Folding, and ERAD

“…OST is a multi-subunit membrane–protein complex composed of one catalytic subunit (either STT3A or STT3B) and at least six of the following accessory subunits in mammalian cells: Ribophorin 1 and 2 (RPN1, RPN2), DAD1, OST48, TMEM258, OST4, KCP2, DC2/OSTC, and either TUSC3/N33 or IAP/MAGT1 [ 19 ]. Each subunit spans the membrane and possesses a significant luminal domain.…”

Platelets and Defective N-Glycosylation

“…Attachment of N-linked glycans (sugar "trees") to polypeptides takes place in the endoplasmic reticulum, where the oligosaccharyltransferase (OST) complex catalyzes the transfer of a high mannose oligosaccharide onto asparagine residues within the primary protein sequence of Asn-X-Ser or Asn-X-Thr (NXS/T), where X is any amino acid except proline [1,2]. The OST complex in humans is a protein complex composed of several subunits, including STT3 oligosaccharyltransferase complex catalytic subunit A (STT3A), subunit B (STT3B), tumor suppressor candidate 3 (TUSC3), magnesium transporter 1 (MAGT1), transmembrane protein 258 (TMEM258), keratinocyte-associated protein 2 (KCP2), ribophorins (RPN1-2), defender against cell death 1 (DAD1), oligosaccharyltransferase complex subunit 4 and 48 (OST4 and OST48), and oligosaccharyltransferase complex non-catalytic subunit (DC2) proteins [2][3][4][5] (Figure 1). So far, two OST complexes have been identified in mammalians with catalytic subunits STT3A or STT3B associated with different non-catalytic subunits [2].…”

“…The OST complex in humans is a protein complex composed of several subunits, including STT3 oligosaccharyltransferase complex catalytic subunit A (STT3A), subunit B (STT3B), tumor suppressor candidate 3 (TUSC3), magnesium transporter 1 (MAGT1), transmembrane protein 258 (TMEM258), keratinocyte-associated protein 2 (KCP2), ribophorins (RPN1-2), defender against cell death 1 (DAD1), oligosaccharyltransferase complex subunit 4 and 48 (OST4 and OST48), and oligosaccharyltransferase complex non-catalytic subunit (DC2) proteins [2][3][4][5] (Figure 1). So far, two OST complexes have been identified in mammalians with catalytic subunits STT3A or STT3B associated with different non-catalytic subunits [2]. They couple oligosaccharides to create a specific sugar tree on the protein surface [2].…”

“…So far, two OST complexes have been identified in mammalians with catalytic subunits STT3A or STT3B associated with different non-catalytic subunits [2]. They couple oligosaccharides to create a specific sugar tree on the protein surface [2]. RPNs link ribosomes to the ER surface during protein translation and present substrates to the catalytic center and additionally function as a chaperone that recognizes misfolded proteins [5,6].…”