Oligosaccharyltransferase

Introduction

Most all proteins in all living organisms are embedded with an outer carbohydrate coating. This coating of carbohydrates has the ability to perform many different roles within a cell and is necessary for homeostasis. The outer coating is often bulky and serves to protect the protein, even having the ability to distinguish what can interact with that protein. Many of the times different organisms have their own specialized carbohydrates. An example being human blood types, and how often with the blood transfusions it is necessary to ensure certain glycosylated blood types are avoided. These carbohydrate coatings can even aid in the solubility of certain proteins. The protein that performs the action of adding these carbohydrate coats to other proteins is the oligosaccharyltransferase (OST). This multimeric compound finds itself within the membrane of the endoplasmic reticulum. OST is classified as a transferase and functions to add carbohydrates to asparagines to protein chains during their synthesis. They do this through N-glycosylation, attaching the carbohydrate to the nitrogen in asparagine.^[1]

Structure and Glycosylation

Glycosylation, the addition of carbohydrates to proteins, is done in two different ways. The carbohydrate can be attached at the oxygen of a serine or threonine amino acid (O-glycosylation), or the carbohydrate can be attached to an asparagine amino acid at its amine group (N-glycosylation). The OST complex performs the N-glycosylation method. During this process a carbohydrate is first synthesized, sugar by sugar, on a lipid lipid carrier. The OST complex then transfers this carbohydrate from the lipid to the asparagine of the desired protein.^[1] This process takes place at the membrane of the endoplasmic reticulum. Following the protein is then acted upon by several different enzymes in the ER and golgi body before heading to the cell surface or leaving the cell.

The OST complex varies between prokaryotic and eukaryotic organisms. In eukaryotic cells this complex consists of several non-identical subunits. Where in single celled organisms such as bacteria, this complex exists as a single unit. In mammalian cells the OST complex either contains an STT3A (OST-A) or an STT3B (OST-B) subunit, both being paralogues of the Stt3 subunit. Recent cryo-EM of both of these structures have led to new discoveries on their differences. OST-B is bounded by an acceptor peptide and donor substrate (dolichylphosphate) compared to the OST-A complex which is only bound by dolichylphosphate. This difference suggests different affinities of both complexes in relation to the acceptor substrates.^[2] Given this there still is not enough information to fully understand the difference in the roles between the two complexes. It remains a future goal to study these non-identical subunits and their roles through N-glycosylation to further understand the mechanics of these proteins.

It is the STT3 subunit that performs the catalytic activity for N-glycosylation. The OST-A complex which contains STT3A is responsible for the majority of the glycosylation. This complex is exposed directly to the unfolded polypeptide interacting with the Sec61 translocon. The acceptor region of the polypeptide emerging from the ribosome makes contact with the STT3A which carries out glycosylation. This initial contact of the polypeptide is absent with the OST-B complex. The STT3B unit glycosylates any missed acceptor sites that weren’t initially glycosylated by STT3A. This unit acts more as a proofreading complex to ensure all regions are glycosylated. Efficiency of the STT3B unit is determined by diffusion rate of the substrate after being skipped and the folding rate of glycoproteins^[3].

Within all three domains of life, the N-glycosylation by the OST complex is very similar. In Unicellular bacteria the complex is a single unit compared to the eukaryotic multiple non-identical units. Organisms with a multitude of these subunits have the ability to glycosylate a variety of acceptor peptides. The increase in peptides glycosylated indicates that the noncatalytic subunits of these complexes are involved in some way with increasing the efficiency of the catalytic unit, STT3. However many of these units, especially those with larger domains still require further investigation to further understand their roles^[2].

Tumor Progression and Cancer Treatment

While the OST complex is necessary for the survival and function of most proteins, and deficiency in genes that encode for this complex often result in genetic disorders, this protein still has its faults. It’s been determined the subunits of OST are often cell context-dependent and are frequently altered in cancerous cells to contribute in tumor progression. Given this research is on the way to finding ways to use this to our benefit. Studies are being done to target and inhibit the OST complex to treat drug-resistant tumors.

Tumor cells have been able to alter the subunits of OST to use N-glycosylation to their advantage for immune evasion. Tumor cells have the ability to inactive T cells having a programmed death-ligand 1 (PD-L1) which binds to the programmed death (PD-1) receptor of the T Cell. This allows the tumor cell to evade the PD-L1 immune response checkpoint going unnoticed. The PD-L1 is a transmembrane protein with 4 sites available for N-glycosylation. The glycosylation of these sites prevents phosphorylation of the protein ensuring the expression of the protein on the outside of tumor cells. Tumor cells also have a process, epithelial-to-mesenchymal transition (EMT), which helps the cell acquire invasive properties by altering protein expression profiles^[4] This EMT process has the ability to upregulate the expression of PD-L1 along the catalytic subunits of OST, STT3A and STT3B. The increased regulation of PD-L1 allows for more of these proteins to undergo glycosylation further ensuring their cell surface expression. EMT also has the ability to promote glycosyltransferases in their formation of poly-N-acetyllactosamine thich proves to be essential in the binding of PD-L1 to PD-1 of T cells. This discovery suggests EMT reprograms OST to carry out this upregulation establishing PD-L1-mediated immune escape^[4].

Although the cell context-dependent abilities of the OST make the complex favorable for tumor progression, this proves to be a possible route for inhibition of tumor growth. Currently there are no known drugs which targets N-glycosylation. There are current studies that have developed N-glycosylation inhibition which has progressed what we know about the relevance of OST in cancer treatment. An inhibitor called N-glycosylation inhibitor 1 (NGI-1) has been discovered which has the ability prevent N-glycosylation stopping the function of an inactivated form of a luciferase mutant (ERLucT)^[4]. This inhibitor has the ability to inhibit the catalytic function of both catalytic subunits of OST favoring STT3B. Studying NGI-1 further will hopefully lead to the development of treatments which can prevent tumor growth where current drugs and medications fail.

Viruses

We currently are amidst a never ending war. This war exists with viruses and has been occurring since the start of all living things and is centered greatly around glycosylated proteins. Our cells are constantly evolving to develop different features to protect against viruses, while it is their goal to develop abilities to evade our defenses. One way our cells had prevented viral pathogens from infection was the use of cell surface carbohydrates to, in a sense, fly under their radar. However viruses had evolved to recognize these carbohydrates and target these cells for infection. Thankfully our immune system is equally up to speed as it has since evolved to recognize certain glycosylated lipids and proteins that the virus has acquired after hijacking a host cell. in text citation.^[1]

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