Glycosylation refers to the post translational modification of proteins, lipids and nucleic acids in the presence of enzymes. The common post translational modifications of biological importance include N-linked glycosylation, O-linked glycosylation, phospho–serine glycosylation, as well as C-mannosylation and glypation (addition of glycophosphatidylinositol. Glycosylation reaction is mediated by glycosyltransferases that conjugate carbohydrate to proteins, lipids and nucleic acids. Glycosylation is a biological mechanism that aids the protein folding and help in protein signaling along with cell-cell interaction. Recently mass spectrometry technique is recently gained attention in quantitative estimation of glycoproteomics. In COVID-19 infection, the SARS-CoV-2 viral proteins including spike(S), envelope (C), membrane (M) and nucleocapsid (N) undergo post translational modification of glycosylation and phosphorylation that plays important role in virulence and infectivity.
Defined in the broadcast sense, glycosylation is the conjugation process of joining carbohydrate to the protein’s backbone via enzymatic reaction. Post modification after this reaction, the protein is termed as glycoprotein. In our body, the most common glycosylation reaction refers to the N-glycosylation and O-glycosylation. Among these two post modification process, the N-glycosylation is frequently occurring mechanism. This mechanism of post translation modification comes under the domain of glycobiology, which refers to the study of biosynthesis, structures and biology of saccharides (also known as sugar or carbohydrates). Glycosylation is a critical mechanism of the biosynthetic-secretory pathway that occurs in endoplasmic reticulum (ER) and Golgi apparatus . I was a known fact that nearly half of the eukaryotic proteins undergo modification, which presents the covalent conjugation of sugar moieties to particular amino acids of interest . Membrane-bound and soluble proteins along with secreted proteins, ligands, surface proteins that are transported from the Golgi to the cytoplasm also become glycosylated .
Lipids and proteoglycans are also among the susceptible targets of the glycosylation that significantly contribute in increasing the number of substrate for the post translation modification. Carbohydrate plays an important role in the regulation of multicellular organs and organism’s functions as a resultant several biological macromolecules possess covalently attached saccharides including monosaccharides and oligosaccharides which are commonly known as glycans . These glycans contribute a major role in modulating cell-to-cell interaction, development and function of cell, interaction between cell and host . Protein bound glycans are abundant in the nucleus and cytoplasm and serve as the key regulator element there .
2. N- and O-glycosylation
Several proteins undergoes post translational modification by N-glycosylation that denote with the attachment of the N-acetylglucosamine (GlcNAc) to the nitrogen atom present in the Asn side chain of the amino acid by a β-1 N linkage . These GlcNAc2 mannose (Man)3 core containing glycoconjugates shows the tendency to add/remove several monosaccharides. These conjugation reactions include galactosylation, sialylation, GlcNAclyation and fucosylation . Moreover, glycosylation occur on hydroxyl functional group of the amino acids Ser and Thr. The most common sugars showing conjugation with Ser and Thr are GlcNAc and N-acetylgalactosamine (GalNAc) . GalNAc associated glycans, often known as mucin-type O-glycans are abundantly found in extracellular spaces and secreted proteins like mucin . Mucin is characterized with high number of Pro, Ser and Thr residues that makes it susceptible to the O-linked glycosylation. The participating sugars conjugate with the protein as it moves through the cis, medial and trans Golgi apparatus. The glycopeptide O-glycans are post transnationally modified by glycosyltransferases .
C-mannosylation is different approach from glycosylation, as in this process there is reaction forms carbon-carbon bonds rather than carbon-nitrogen and carbon-oxygen bonds. C-mannosyltransferase is the key enzyme that is involved in the C-mannosylation. This enzyme conjugates the C1 of the mannose to C2 of the indole ring present in the tryptophan residue . Moreover, this enzyme recognizes the specific sequence of Trp-X-X-Trp that further ease the transfer of mannose sugar from dolichol-P-Man to the first Trp residue present in the given sequence [8, 9, 10]. In another event of the post translational modification (PTM), the phosphoglycosylation is among the PTM mechanism that is limited to the parasites including Trypanosoma and Leishmania including slime molds. The mechanism is characterized by the conjugation of glycans to the Ser and Thr residues linked by the phosphodiester bonds .
4. Glycosylation vs. glycation
Glycosylation and glycation are enzymatic and non-enzymatic reaction respectively, in which glucose and other glucose metabolites show affinity with different biological macromolecules such as proteins, lipids and nucleic acids. Increased presence of glucose both intracellular and extracellular possesses threat to the human and act as risk factor for various metabolic disorders including diabetes mellitus. Both non-enzymatic glycation and enzymatic glycosylation reaction have been shown to play important role in human health. Non-enzymatic glycation leads to the formation of advanced glycation end products (AGEs) that generates as a resultant of protein and lipid glycation with aldose sugars [12, 13]. Prior to the formation of AGEs, the early glycation reaction occurs that form Schiff bases and Amadori products. AGEs formed due to the non-enzymatic glycation generates reactive oxygen species (ROS), that further binds to the particular cell surface receptors and later forms cross link [12, 14].
5. SARS-CoV-2 and glycosylation
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is severely affecting the worldwide population. SARS-CoV-2 virulence and survival is impacted by the glycans that is covalently linked to the proteins of virus through the process of glycosylation that makes alteration in the virulence of the SARS-CoV-2 virus. It belongs to the coronavirus family which exhibit protein constituted enveloped single-stranded RNA. These viral proteins undergo post-translational modifications (PTMs) that reorganized covalent bonds and modify the polypeptides and in turn modulate the protein functions. Being viral machinery, it uses host cells system to replicate itself and make their copes, their proteins are also subject to PTMs. Glycosylation, palmitoylation of the spike and envelope proteins, phosphorylation, of the nucleocapsid protein are among the major PTMs responsible for the pathogenesis of the viral infection phase. The current knowledge of CoV proteins PTMs is limited and need to be exploring for to understand the viral pathogenesis mechanism and PTMs effect of infection phase.
The author Dr. Alok Raghav is thankful to the Department of Health Research, Ministry of Health and Family Welfare, New Delhi for providing financial support in the form of salary.
Conflict of interest
The authors declare no conflict of interest.
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Zhang X. Alterations of golgi structural proteins and glycosylation defects in cancer. Frontiers in Cell and Developmental Biology. 2021; 9:665289. DOI: 10.3389/fcell.2021.665289
Reily C, Stewart TJ, Renfrow MB, Novak J. Glycosylation in health and disease. Nature Reviews Nephrology. 2019; 15:346-366. DOI: 10.1038/s41581-019-0129-4
Smith DF, Song X, Cummings RD. Use of glycan microarrays to explore specificity of glycan-binding proteins. Methods in Enzymology. 2010; 480:417-444. DOI: 10.1016/S0076-6879(10)80033-3
Varki A, Gagneux P. Biological functions of glycans. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet]. 3rd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017. Chapter 7. DOI: 10.1101/glycobiology.3e.007
Varki A, Cummings RD, Esko JD, Stanley P, Hart GW, Aebi M, Darvill AG, Kinoshita T, Packer NH, Prestegard JH, Schnaar RL, Seeberger PH, editors. Essentials of Glycobiology [Internet]. 3rd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017
Vasudevan D, Haltiwanger RS. Novel roles for O-linked glycans in protein folding. Glycoconjugate Journal. 2014; 31:417-426
de Beer T, Vliegenthart JF, Löffler A, Hofsteenge J. The hexopyranosyl residue that is C-glycosidically linked to the side chain of tryptophan-7 in human RNase Us is alpha-mannopyranose. Biochemistry. 1995; 34(37):11785-11789. DOI: 10.1021/bi00037a016
Krieg J, Hartmann S, Vicentini A, Gläsner W, Hess D, Hofsteenge J. Recognition signal for C-mannosylation of Trp-7 in RNase 2 consists of sequence Trp-x-x-Trp. Molecular Biology of the Cell. 1998; 9(2):301-309. DOI: 10.1091/mbc.9.2.301
Doucey MA, Hess D, Cacan R, Hofsteenge J. Protein C-mannosylation is enzyme-catalysed and uses dolichyl-phosphate-mannose as a precursor. Molecular Biology of the Cell. 1998; 9(2):291-300. DOI: 10.1091/mbc.9.2.291
Hartmann S, Hofsteenge J. Properdin, the positive regulator of complement, is highly C-mannosylated. Journal of Biological Chemistry. 2000; 275(37):28569-28574. DOI: 10.1074/jbc.M001732200
Haynes PA. Phosphoglycosylation: A new structural class of glycosylation? Glycobiology. 1998; 8(1):1-5. DOI: 10.1093/glycob/8.1.1
Schmidt AM, Hori O, Brett J, Yan SD, Wautier JL, Stern D. Cellular receptors for advanced glycation end products: Implications for induction of oxidant stress and cellular dysfunction in the pathogenesis of vascular lesions. Arteriosclerosis, Thrombosis, and Vascular Biology. 1994; 14:1521-1528
Singh R, Barden A, Mori T, Beilin L. Advanced glycation end-products: A review. Diabetologia. 2001; 44:129-146
Brownlee M, Vlassara H, Cerami A. Nonenzymatic glycosylation products on collagen covalently trap low-density lipoprotein. Diabetes. 1985; 34:938-941