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Introduction
N-linked glycosylation is an essential process in protein modification. This form of glycosylation is important in the folding and sorting of proteins in the endoplasmic reticulum (ER) and the interaction between proteins and cells. [1] In humans, N-linked glycosylation is catalyzed co-translationally by an enzyme complex called oligosaccharyltransferase complex A (OST-A) in the rough ER. This means that the peptide chain is glycosylated by this complex as it is synthesized by the ribosome and enters the ER lumen through translocon protein Sec61.[2] As suggested by the name, this enzyme complex transfers the high mannose fourteen-sugar chain from a lipid-linked oligosaccharide donor containing dolichol pyrophosphate to the peptide chain containing the Asn-X-Thr (N-X-T) sequence, where X is any amino acid but not Proline.[1] In addition, this enzyme complex is also part of the glycosyltransferase-C (GT-C) fold, which is a protein that has a transmembrane helical domain and a mix of α/β soluble domains.[3] On this page, the structure of the OST-A and its components; its mechanism, and diseases associated with this complex are discussed.
Structure
The human OST-A complex is a transmembrane protein that has 27 transmembrane helices integrated into the endoplasmic reticulum (ER) outer membrane with soluble domains on both the cytosolic side and the luminal side of the membrane.[4] However, most of the functional sites of the complex are found on the luminal side. The OST-A complex consists of three sub-complexes with a total of nine subunits. All subunits have a transmembrane domain and soluble domains. The subcomplex I consists of two subunits: transmembrane protein 258 (TMEM258) and robophorin-1 (RPN-1). The subcomplex II consists of four subunits: , OST 4 kDa subunit (OST4), keratinocyte-associated protein 2 (KCP2), and DC2. Lastly, the subcomplex III consists of three subunits: defender against cell death 1 (DAD1), OST 48 kDa subunit (OST48), and ribophorin-2 (RPN-2).[5] The transmembrane domains of TMEM258 and RPN-1 are also in close proximity to a protein called malectin, which is believed to be involved in quality control in protein synthesis.[4] In addition, the OST-A complex is associated with a translocon protein in the ER membrane called Sec61. The C-terminal of the RPN-1 subunit also forms a 4-helix bundle that specifically binds to the ribosome in the cytosol.[4]
The subunit consists of thirteen transmembrane helices and a mixture of alpha-helices and beta-sheet on their ER-luminal side. The N-terminal of this subunit is on the cytosolic side while the C-terminal is on the luminal side.[4] The catalytic of the OST-A complex is in this subunit, making it the most conserved and central subunit of the whole complex. It is homologous to the oligosaccharyltransferase in other species, such as PglB in some bacteria and AglB in archaea.[6] The TMEM258 has two transmembrane helices with both N- and C-terminals on the luminal side. The RPN-1 and OST48 have a similar structure with one C-terminal transmembrane helix and N-terminal anti-parallel beta-sheet on the luminal side. The difference between the two subunits is that RPN-1 has a C-terminal helix bundle on the cytosolic side while OST48 has two helices on their luminal side. The OST4 subunit only consists of one transmembrane helix with N-terminal on the luminal side and the C-terminal on the cytosolic side. DC2 and DAD1 both have three transmembrane helices with the N-terminal from the cytosolic side and the C-terminal from the luminal side. However, DAD1 has a C-terminal helix on the cytosolic side. The RPN-2 subunit has three transmembrane helices and the anti-parallel beta-sheet at the N-terminal on the luminal side.[4] Lastly, the KCP2 subunit has four transmembrane helices with both C- and N-terminals on the cytosolic side.[5]
Active Site
The of this complex is in the soluble domain on the luminal side of the subunit. The active pocket consists of the (residues 543-546) from the external loop 5 (EL5) between TM9 and TM10 of the STT3A packing against the ER-luminal domain of this subunit. This forms a binding groove for lipid-linked oligosaccharide (LLO) donor substrate in the form of dolichol pyrophosphate (DolPP) and the divalent magnesium ion.[4] The magnesium ion will form hydrogen bonds with the oxygen from each phosphate group of DolPP. The active site also has a , consisting of three residues Trp525-Trp526-Asp527, for the recognition of acceptor peptide Asn-X-Thr (N-X-T), where X is any amino acid except for Proline. The residues are also part of the active site and are involved in the catalytic reaction of the OST-A complex.[5]
Function
The OST-A complex performs N-linked glycosylation or Asparagine-linked glycosylation co-translationally. This means that the glycosylation is done on the newly synthesized polypeptide chain. The complex must bind with the translocon protein Sec61 and the ribosome physically through the subunit DC2 and KCP2.[3] Without the association with the translocon, the OST-A complex is inactive because it can only glycosylate the newly synthesized unfolded polypeptide chain.
There are two substrates of the OST-A complex: the newly synthesized (nascent) polypeptide acceptor and the lipid-linked oligosaccharide (LLO) donor.[4] The complex will transfer the oligosaccharide molecule from the lipid-linked donor to the nascent peptide acceptor. The LLO consists of dolichol pyrophosphate and a fourteen-sugar chain. The fourteen-sugar chain consists of three glucose (Glc) molecules, nine mannose (Man) molecules, and two N-acetylglucosamine (GlcNAc) molecules. The sugar chain is assembled onto the DolPP on the cytosolic side with the two N-acetylglucosamine molecules first binding to the oxygen on a phosphate group of the DolPP. Then five mannose molecules are added to the chain. At this point, the LLO is inverted to the luminal side. From there, four more mannose molecules are added to the existing chain followed by the addition of three glucose molecules, making the chain (Glc3Man9GlcNAc2-DolPP).[5] The glycosylate sequence of the acceptor peptide substrate has an Asn-X-Thr (N-X-T) sequence, where X is any amino acid except Proline. The oligosaccharide molecule will be transferred to the Asparagine (Asn) residue of this sequence.[5] The exclusion of Proline in the +1 position of this sequence is due to its structure. The five-membered ring of Proline restricted the phi dihedral angle of the peptide chain, which prevents the oligosaccharide to bind to the Asparagine residue since it is sterically hindered. In addition, the Proline residue is lack of hydrogen bond on its sidechain to work as a hydrogen bond donor when the peptide chain is bound to the active site.[7]
The nascent polypeptide is synthesized by the ribosome on the rough endoplasmic reticulum (ER) and enters the ER lumen through the translocon protein Sec61. The OST-A complex is bound to this translocon protein and scans the peptide chain for the N-X-T sequence. The WWD motif (residue 525-527) at the active site of the STT3A subunit forms hydrogen bonds with the +2 Threonine residue of the glycosylate sequence. The sidechain hydroxyl group of Threonine will form hydrogen bonds with the amide group on the two Tryptophan residues while the backbone amide group of the sequence forms a hydrogen bond with the sidechain hydroxyl group of the Aspartate residue.[5] This interaction between the Threonine residue and the WWD motif holds the acceptor sequence in place and ready for glycosylation. The LLO donor, Glc3Man9GlcNAc2-DolPP, is held in the LLO binding groove with its phosphate groups forming ionic interaction with the magnesium ion. The magnesium ions also interact with the carboxyl group of the Glu351 and Asp49 residues in the active site of the STT3A subunit. The amine sidechain of the Asparagine residue of the accepter sequence forms hydrogen bonds with both Glu351 and Asp49 residues.[5] This causes the rotation of the C-N bond of the Asparagine sidechain, exposing the lone-pair electrons of the nitrogen atom. This makes this nitrogen atom become more reactive in the nucleophilic attack to the C1 carbon of the N-acetylglucosamine on the LLO donor, cleaving the dolichol pyrophosphate. In this reaction, the dolichol pyrophosphate is acting as a leaving group in the nucleophilic reaction.[7] At the end of the reaction, the oligosaccharide molecule (Glc3Man9GlcNAc2) is transferred from the LLO donor to the Asparagine residue of the nascent peptide chain. This process can happen at multiple places on the newly synthesized peptide chain if the complex finds the N-X-T sequence. In addition, the OST-A complex required the acceptor peptide chain to be linear due to the specific structure of its active site.[5] Therefore, it can only glycosylate unfolded protein, and the formation of the disulfide bridge of folded protein will inhibit the protein entry to the complex.
Disease
Defect in the OST-A complex causes the inherited congenital disorder of glycosylation (CDG). This is a multi-organ disorder since mistakes in N-linked glycosylation can affect many cellular processes such as folding of the protein and cell recognition and communication. The phenotype of this disorder includes microcephaly, dysmorphic facies, congenital heart defect, infantile spasm, and skeletal dysplasia.[8] Other phenotypes of this disorder are mental retardation, development delay, liver dysfunction, dysmorphic feature, anorexia, and gastrointestinal disorders.[5]
Recently, the RPN-1, RPN-2, and STT3A subunits of the OST-A complex are found to associate with the development of breast cancer. Defective OST-A complex produces misfolded proteins leading to ER stress by the accumulation of these proteins. ER stress is associated with the development of cancer cells. The research found that knock-out RPN-1 cells have a poorer proliferation rate and a lower rate of migration and invasion of cancer cells. It also found that the defective genes of RPN-1, RPN-2, and STT3A subunits are significantly up-regulated.[9] This allows the cells to produce misfolded proteins and persist ER stress.
N-linked glycosylation is also relevant in inhibiting the infection of SARS-CoV-2 and its variants. The virus has four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). Out of these four proteins, the S, E, and M proteins require N-linked glycosylation in the host cell. The tested molecule NGI-1 targets the STT3A subunit and inhibits the glycosylation of the spike protein.[10] This can prevent the spread of the assembly of the virus in the host cell and it is effective for all variants of this virus.
