Brief discussion on Protein Glycosylation

1. Brief discussion on Protein Glycosylation Glycosylation is the attachment of the sugar moieties to proteins. It is the post-translational modification that offers greater proteomic diversity than any other PTMS. This process is highly crucial for the wide range of biological processes like cell attachment to the extracellular matrix and protein-ligand interactions present in the cell. It is also characterized by various glycosidic linkages like C-, O- and N- linked glycosylation, and phospho glycosylation. This can be identified, purified and analyzed in different stages like visualization and staining, glycan crosslinking to agarose or magnetic resin for purification or labelling. Types of glycosylation The Glycopeptide bonds can be categories into several specific groups depending on the nature of the sugar peptide bond and the oligosaccharide attached like N-, O- and C-linked glycosylation, phosphoglycerate and glypiation. This is because N-and O-glycosylation and glypiation are the

2. most commonly detected types of glycosylation. So, the different types of glycosylation include, N-linked: glycan binds to the amino group of asparagine in the ER O-linked: These are the monosaccharides bound to the hydroxyl group of the serine or threonine in the ER, cytosol, Golgi and nucleus. C-linked: It is the mannose blinds to the indole ring of tryptophan Phospho Glycosylation: Glycan binds to serine through the phosphodiester bond Glypiation: Glycan core links a phospholipid and the protein Generally, the proteins are restricted to any particular type of glycosylation biologically. Indeed, proteins are often glycosylated at multiple sites with various types of the glycosidic linkage which will be based on multiple factors as mentioned here.

3. 1. Availability of the enzyme Glycosylation is controlled by moving to various areas with different enzymes concentration. The cell sequesters enzymes in certain compartments to regulate various activities. For instance, after the protein is N-glycosylated in the ER, the glycan processing will occur in several step-wise functions. This happens by traffic for the proteins to distinct Golgi cisternae that will contain a high amount of high concentration of some specific Gtfs and Glycosidases. 2. Amino acid sequence Apart from the requirement of the right amino acids for various purposes, several enzymes have consensus sequences or motifs that will enable the formation of the glycosidic bond. 3. Protein conformation As protein is synthesized, they begin to fold their nascent secondary structure that can assist in preparing some specific amino acids inaccessible for the glycosidic binding. Thus, the target amino acids will be accessible, and this increases the chance of glycosylation.

4. Common methods to detect and analyze glycoproteins The influence that glycoproteins will have on the biological process and the disease states will continue to expand. It is because the glycoproteins are a combination of the protein and oligosaccharides they are more complex to analyse the common non-glycosylated proteins. Besides, the vast diversity in glycan structure and composition add a level of complexity to the glycoprotein analysis. 1. Glycan staining or labelling Due to the structure, the glycan sugar moieties are not reactive to the labelling and staining molecules. Aiming to overcome this problem, the sugar groups can be chemically restructured with this type of periodic acid that oxidizes the vicinal hydroxyls on the sugar. It helps in aldehydes or ketones that are reactive to various dyes. The periodic acid-Schiff stain uses the reaction to detect and quantify the glycoprotein in several biological samples. 2. Glycoprotein purification or enrichment

5. Lectins can be used to determine and analyze the functioning of the glycoprotein. These glycan-binding proteins will highly specificity for many distinct sugar moieties. Like antibodies, the lectins can be conjugate to certain problems like fluorophores, horseradish peroxidase, and biotin. This will immobilize solid support like Thermo Scientific NeutrAvidin Protein and streptavidin. So, the most common uses include  Glycoprotein identification  Glycoprotein purification/enrichment  Glycosylation mutant generation  Gtf and glycosidase activity analysis Many lectins are commercially available for various purposes. One of the most famous ones is concanavalin A from the Jackbean, but other lectins like Jacaalin, lentil lectin are also widely popular. 3. Glycoprotein and glycome analyses by the mass spectrometry Glycoproteins are unique from another post-translationally modified protein as they are in a combination of the glycan and protein. Both are composed of a significant proportion of the molecular weight of a particular molecule. So glycoprotein can be analyzed as the whole individual components. There are also several subsets of proteomics that will differ from glycomics. With other proteomic analysis glycoprotein identification and quantization is performed with the mass spectrometer. The basic pipeline for the analysis will include  Data analysis through the bioinformatics  Tandem mass spectrometry  Glycoprotein or glycopeptides enrichment  Multidimensional separation by liquid enrichment This approach can be commonly performed before and after the enzymatic coverage of the glycans through the endoglucanase. So, these are the vital aspects with the array glycan. Endnote

6. By now, you might have got some idea of the protein glycosylation process. Use them effective to enjoy the benefits. For more info, visit: www.arrayglycan.com