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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. [3] 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.[4] 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.[3] 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.[5] 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.[6] 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: STT3A, 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).[7] 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.[6] 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.[6]
The STT3A 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.[6] 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.[8] 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.[6] Lastly, the KCP2 subunit has four transmembrane helices with both C- and N-terminals on the cytosolic side.[7]
Active Site
The active site of this complex is in the soluble domain on the luminal side of the STT3A subunit. The active pocket consists of the DNNT loop (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.[6] The magnesium ion will form hydrogen bonds with the oxygen from each phosphate group of DolPP. The active site also has a WWD motif, 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 Glu351 and Asp49 are also part of the active site and are involved in the catalytic reaction of the OST-A complex.[7]
Function
Disease
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