Introductory Chapter: Glimpses of Glycosylation

Molecular Biotechnology, 1994

Journal of Biotechnology, 1996

The glycosylation pathway is the most important post-translational modification of a protein and is moreover a highly specific process. The majority of proteins of pharmaceutical interest are glycoproteins. Therefore, it is necessary to identify the composition, the structure, the function and the biosynthesis of the glycoproteins. The present knowledge is described here. In addition, the performed studies about structure-function relationship of the glycoproteins have shown that the oligosaccharide part of a glycoprotein confers important and specific biological roles. Thus, the modification of the structure of the glycan chains can lead to a modification of the activity of the glycoprotein. This phenomenon is encountered at the time of the production of recombinant glycoprotein in a heterologous system. Indeed, the glycosylation profile of a protein is specific to both the host cell and the culture conditions of this cell. Thus, the advantages and the drawbacks of the different host cells used for the glycosylation engineering are presented. In this way, the identification of the different specific enzymes glycosyltransferases and glycosidases involved in the glycosylation pathway is now necessary to improve the production of recombinant glycoprotein. The structure and the characteristics of these enzymes, and more particularly the oligosaccharyltransferase and the galactosyltransferase, are also described.

Central European Journal of Biology, 2011

A host of bacteria and viruses are dependent on O-linked and N-linked glycosylation to perform vital biological functions. Pathogens often have integral proteins that participate in host-cell interactions such as receptor binding and fusion with host membrane. Fusion proteins from a broad range of disparate viruses, such as paramyxovirus, HIV, ebola, and the influenza viruses share a variety of common features that are augmented by glycosylation. Each of these viruses contain multiple glycosylation sites that must be processed and modified by the host post-translational machinery to be fusogenically active. In most viruses, glycosylation plays a role in biogenesis, stability, antigenicity and infectivity. In bacteria, glycosylation events play an important role in the formation of flagellin and pili and are vitally important to adherence, attachment, infectivity and immune evasion. With the importance of glycosylation to pathogen survival, it is clear that a better understanding of the processes is needed to understand the pathogen requirement for glycosylation and to capitalize on this requirement for the development of novel therapeutics. © Versita Sp. z o.o. D.J. Vigerust their surface in a host-specific fashion which ultimately impacts the viral glycoproteins in roles such as stability, antigenicity, and host cell invasion. O-linked glycosylation is a post-translational modification of secreted and membrane bound proteins that take place in the cis-Golgi compartment. O-linked glycans play important roles in protein localization and trafficking, protein solubility, antigenicity and cell-cell interactions. Modification of the oxygen in serine or threonine on proteins occurs with the addition of N-acetyl galactosamine (GalNAc). O-glycans can extend into long chains similar to N-glycan, but are usually less branched and can result in the formation of mucinlike molecules. O-glycans can function in lectin-ligand interactions, which may facilitate interaction with cellular proteins used as receptors of entry, and viral attachment proteins such as O-linked RSV-G protein. Clinked modifications representing the third type of glycosylation are much less common and novel. Clinked modifications result in the attachment of an α-mannopyranose to tryptophan via a carbon [4]. Until 2007, the only described glycosylation of virus proteins was of the N or O-linked varieties. Falzarano et al. demonstrated Clinked modification of Ebola soluble glycoprotein representing the first description of a C-mannosylation of a viral protein [5]. The role of this modification in eukaryotic systems has not been fully clarified; however, it was first described in human RNase-2 and interleukin-12, the modification does also exist in several innate complement components (C6, C7, C8, and C9) and in the complement regulator properdin suggesting a role in innate defense [6]. The final modification is S-glycosylation which is a new and exceedingly rare post-translational modification found in glycopeptide bacteriocins where sulfur in the amino acid cysteine is modified by N-acetylhexosmine [3]. The biological role for this modification is still under investigation but may prove to be unique to bacteriocins. Given the scope and breadth of protein modifications and the role that these modifications play in the physiology of the cell, it is not surprising that potential pathogens subvert these processes to promote their replication and survival.

NATO Science for Peace and Security Series A: Chemistry and Biology, 2008

Recently, we reported the characterization of the glycans attached at the 11 N-glycosylation sites of Hepatitis C virus E2 envelope glycoprotein by tandem mass spectrometry. Infections caused by Hepatitis C virus represent the main cause of liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma. The N-linked sugars consist primarily of high mannose glycans, with structures ranging from the minimal core structure, Man3GlcNAc2 (Man3) up to 12 hexose residues attached to the GlcNAc-ß(1-4)-GlcNAc core (depicted as Hex3Man9GlcNAc2). Furthermore, the site N41 (N423) was observed to contain complex type glycans with the structures Man3-GlcNAc and Man3-GlcNAcFuc, in addition to the high mannose population Man3 through Man6, while the site N48 (N430) was occupied exclusively with complex type glycans (Man3-Fuc, Man3-GlcNAcFuc and Man3-GlcNAc2Fuc). The present contribution summarizes our experimental observations upon the factors which may have an impact on the CID tandem mass spectra of glycopeptides.

Analytical Chemistry, 2007

The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In this study, we identify the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster on SDS-PAGE gels, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site that cannot be recovered by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by elimination of the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small FLEEL pentapeptide was used as a substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but is important for protein folding and stability.

Cells

The protein glycosylation is a post-translational modification of crucial importance for its involvement in molecular recognition, protein trafficking, regulation, and inflammation. Indeed, abnormalities in protein glycosylation are correlated with several disease states such as cancer, inflammatory diseases, and congenial disorders. The understanding of cellular mechanisms through the elucidation of glycan composition encourages researchers to find analytical solutions for their detection. Actually, the multiplicity and diversity of glycan structures bond to the proteins, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies make their detection much trickier than other kinds of biopolymers. An overview of the most prominent techniques based on mass spectrometry (MS) for protein glycosylation (glycoproteomics) studies is here presented. The tricks and pre-treatments of samples are discussed as a crucial step prodromal to the MS analy...

Current Opinion in Structural Biology, 1991

Recent studies confirm the importance of glycosylation in the physical and functional properties of proteins. Glycosylation results in the structural and functional diversification of a single protein to yield a set of glycosylation variants or glycoforms. Recent studies reviewed herein are placed in the context of a general model for rationalizing the structural effects of protein glycosylation.

Glycobiology, 2003

N-Glycosylation, the most common and most versatile protein modification reaction, occurs at the b-amide of the aspargine of the Asn-Xaa-Ser/Thr sequon. For reasons that are unclear, not all such sequons are glycosylated. To find patterns that affect glycosylation, we examined the amino acid residues from the 20th preceding the sequon to the 20th residue following it, using bioinformatics tools. A clean data set of annotated, experimentally verified, glycosylated and nonglycosylated sequons derived from 617 well-defined nonredundant N-and N-,O-glycoproteins listed in SWISS-PROT (June 2002) was used. NXS and NXT sequons were analyzed separately. Although no overt patterns were found to explain sequon occupancy or nonoccupancy, trends for over-or underrepresentation of certain amino acids at particular positions were statistically significant and different in NXS and NXT sequons. In extension of earlier reports, none of the 80 Asn-Pro-Ser/Thr found were glycosylated, and a markedly low level of glycosylation was seen in sequons with Pro at the position following the Ser/Thr. In addition, a general observation was made that the considerable number of glycosylated sequons in the C-terminal 10 residues of glycoproteins suggests that N-glycosylation in these cases may be posttranslational and not cotranslational, as widely accepted.

Biochemical Journal, 2003

The alteration of proteins by post-translational modifications, including phosphorylation, sulphation, processing by proteolysis, lipid attachment and glycosylation, gives rise to a broad range of molecules that can have an identical underlying protein core. An understanding of glycosylation of proteins is important in clarifying the nature of the numerous variants observed and in determining the biological roles of these modifications. Deglycosylation with TFMS (trifluoromethanesulphonic acid) Anal. Biochem. 118, 131-137] has been used extensively to remove carbohydrate from glycoproteins, while leaving the protein backbone intact. Glycosylated proteins from animals, plants, fungi and bacteria have been deglycosylated with TFMS, and the most extensively studied types of carbohydrate chains in mammals, the N-linked, O-linked and glycosaminoglycan chains, are all removed by this procedure. The method is based on the finding that linkages between sugars are sensitive to cleavage by TFMS, whereas the peptide bond is stable and is not broken, even with prolonged deglycosylation. The relative susceptibility of individual sugars in glycosidic linkage varies with the substituents at C-2 and the occurrence of amido and acetyl groups, but even the most stable sugars are removed under conditions that are sufficiently mild to prevent scission of peptide bonds. The post-translational modifications of proteins have been shown to be required for diverse biological functions, and selective procedures to remove these modifications play an important role in the elucidation of protein structure and function.

Academia Quantum, 2024

In their 2022 study, Kuang et al. introduced the Multivariable Polynomial Public Key (MPPK) cryptography, a quantum-safe public key cryptosystem leveraging the inversion relationship between multiplication and division. MPPK uses multiplication for key pair construction and division for decryption, generating public multivariate polynomials. Kuang and Perepechaenko expanded this into the Homomorphic Polynomial Public Key (HPPK), transforming product polynomials over large hidden rings using homomorphic encryption. Initially designed for key encapsulation mechanism (KEM), HPPK ensures security through homomorphic encryption of public polynomials over concealed rings.This paper extends HPPK KEM to a digital signature scheme. To adapt HPPK KEM for digital signatures, we introduce an extension of the Barrett reduction algorithm, which transforms modular multiplications over hidden rings into divisions in the verification equation. This extension non-linearly embeds the signature into public polynomial coefficients, employing the floor function of large integer divisions.Our proposed scheme addresses forgery attacks observed in previous MPPK/DS schemes by leveraging dual hidden rings and the Barrett reduction algorithm. This method provides non-linear encryption for the HPPK public key, preventing shortcuts other than brute-force searches. Integrating signature elements into public polynomial coefficients adds complexity to forged signature attacks, with the non-linear Barrett transformation significantly enhancing security. A toy example illustrates the functionality of the HPPK DS scheme, and security analysis indicates it achieves exponential complexity for both private key recovery and forged signature attacks. Future research will benchmark performance and compare it with NIST-standardized algorithms.