By: Richard D. Cummings
In the mid 1980s it was strongly believed that all glycosylated proteins arose within the secretory pathway, which generated both secreted and membrane-bound (plasma membrane) glycoproteins. This accepted process was taken to indicate that no glycosylated proteins occurred in the cytoplasm, nucleus, or mitochondria. This belief was shown to be dramatically incorrect, however, with the original discovery in 1984 by Gerald Hart’s laboratory that many intracellular proteins were glycosylated and N-acetylglucosamine (GlcNAc) was present in O-glycosidic β-linkage to specific Ser/Thr residues (now termed O-GlcNAc) (1,2). Soon, studies demonstrated that such O-GlcNAcylated glycoproteins with the β-linked GlcNAc are richly present in multiple proteins, including those making up the nuclear pore complexes. This latter observation explained the strong binding of the nuclear pore to the plant lectin wheat germ agglutinin (WGA), as well as the ability of WGA to inhibit import of proteins through the nuclear pore (3-5). Many years of studies have demonstrated that over 15,000 proteins occur with O-GlcNAc, and these have been identified from among 43 animal species (6). In fact, the O-GlcNAc modification is found in all multicellular organisms! (7). Amazingly, as discussed in another article in this series, it is likely that the most heavily O-GlcNAcylated protein is inside the nucleus of cells and is RNA polymerase II (Pol II) (8-10). Virtually all proteins that interact with DNA, however, including histones and transcription factors are O-GlcNAcylated glycoproteins, and this reversable modification represents a key epigenetic regulatory modification (11).
From the original discoveries of Hart’s laboratory and that of John Hanover and others, the process of O-GlcNAc addition to Ser/Thr residues was shown to be catalyzed by an O-GlcNAc-transferase (OGT) which uses the donor UDP-GlcNAc and generates UDP as a by-product, and that the modification can be removed by a specific β-hexosaminidase termed the O-GlcNAcase (OGA), which releases free GlcNAc (12-14). The OGT has a tetratricopeptide repeat (TPR) domain at its N-terminus, which regulates its interactions with substrate proteins, and the catalytic C-terminal domain uses the UDP-GlcNAc as the donor for transfer (15-17). Thus, as for protein phosphorylation, which is reversible and catalyzed by multiple types of kinases and phosphatases encoded by hundreds of genes, the O-GlcNAcylation process is catalyzed by only two major enzymes, the OGT to add it and the OGA to remove it. Interestingly, the single gene encoding the human OGT is located on the X chromosome at Xq13.1 (18). The gene encoding the OGA enzyme is at 10q24, and was first identified as the meningioma expressed antigen 5 (MGEA5) (19,20). However, there are multiple isoforms of OGT (at least three) and OGA (at least two), which may be differentially localized, e.g., mitochondria, and have different functions (7). Several heritable mutations in the OGT gene have been observed in individuals and it is associated with intellectual disabilities in male offspring (21-23).
Such activities involving O-GlcNAc addition and removal are essential to cell survival and are involved in an incredible number of biological processes, including gene expression, epigenetics, translation, cell cycle, protein degradation, and protein localization, signal transduction, and mitochondrial bioenergetics (7). Recent studies also indicate that O-GlcNAcylation is a global negative regulator of pre-mRNA splicing and positive regulator of intron retention (24). Overall, it appears that most pathways in which protein phosphorylation are important are also impacted and regulated by O-GlcNAcylation. However, while sites of Ser/Thr phosphorylation may also be reciprocally modified by O-GlcNAc, there are many sites of phosphorylation that are not O-GlcNAcylated and vice versa (14). Overall, the discovery of O-GlcNAcylation of intracellular proteins was a watershed event in the history of glycobiology, as it was the first key finding that protein glycosylation and metabolism of sugars and nucleotide sugars was intimately tied to a host of pathways regulating virtually all aspects of the life of a eukaryotic cell. Thus, glycans on proteins occur in all cellular organelles and compartments, and not only on secreted/surface glycoproteins.
Are there other types of intracellular protein glycosylation? Yes, multiple types of intracellular protein modifications are found in different organisms. While they may not be universal, they include O-GlcNAc addition to a specific glycoprotein Skp1 in Dictyostelium discoideum (25), which can be further elongated to a pentasaccharide (26), and O-Fuc on Ser/Thr residues of nuclear pore proteins, first observed in Toxoplasma gondii (27), and important in oxygen sensing in this organism (28). For T. gondii the function of SPY O-fucosyltransferase is critical for parasite proliferation in fibroblasts (29). A novel fucosyltransferase, termed FUT1, is located in the mitochondria of Leishmania protozoan parasites, and is essential to their survival (30). A similar type of pathway has also been found for the mitochondria of Trypanosoma brucei enzyme TbFUT1 (31).
Plants, as studied in Arabidopsis thaliana, have a novel O-fucosyltransferase (SPINDLY - SPY) in the nucleocytoplasmic compartments that promotes O-fucosylation of many intracellular glycoproteins. Many of the SPY substrates are nuclear proteins functioning in DNA repair, transcription, RNA splicing, and nucleocytoplasmic transport, whereas cytoplasmic SPY substrates are involved in pathways involving microtubules in cell division and protein folding (32). Many of the O-fucosylation sites are also sites for O-GlcNAcylation and SPY shares many similarities in its structure to OGT (33).
Thus, not only does intracellular protein glycosylation occur, it is a universal phenomenon and serves to regulate many biological pathways. How many more intracellular glycosylation pathways are yet to be discovered?
References
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