Role Of Glycosylation In Function Research Articles

Lectins as versatile tools to explore cellular glycosylation.

Lectins are naturally occurring carbohydrate-binding proteins that are ubiquitous in nature and highly selective for their, often incompletely characterised, binding partners. From their discovery in the late 1880s to the present day, they have provided a broad palette of versatile tools for exploring the glycosylation of cells and tissues and for uncovering the myriad functions of glycosylation in biological systems. The technique of lectin histochemistry, used to map the glycosylation of tissues, has been instrumental in revealing the changing profile of cellular glycosylation in development, health and disease. It has been especially enlightening in revealing fundamental alterations in cellular glycosylation that accompany cancer development and metastasis, and has facilitated the identification of glycosylated biomarkers that can predict prognosis and may have utility in development of early detection and screening, Moreover, it has led to insights into the functional role of glycosylation in healthy tissues and in the processes underlying disease. Recent advances in biotechnology mean that our understanding of the precise binding partners of lectins is improving and an ever-wider range of lectins are available, including recombinant human lectins and lectins with enhanced, engineered properties. Moreover, use of traditional histochemistry to support a broad range of cutting-edge technologies and the development of high throughout microarray platforms opens the way for ever more sophisticated mapping - and understanding - of the glycome.

Activation and activity of glycosylated KLKs 3, 4 and 11.

Human kallikrein-related peptidases 3, 4, 11, and KLK2, the activator of KLK3/PSA, belong to the prostatic group of the KLKs, whose major physiological function is semen liquefaction during the fertilization process. Notably, these KLKs are upregulated in prostate cancer and are used as clinical biomarkers or have been proposed as therapeutic targets. However, this potential awaits a detailed characterization of these proteases. In order to study glycosylated prostatic KLKs resembling the natural proteases, we used Leishmania (LEXSY) and HEK293 cells for secretory expression. Both systems allowed the subsequent purification of soluble pro-KLK zymogens with correct propeptides and of the mature forms. Periodic acid-Schiff reaction, enzymatic deglycosylation assays, and mass spectrometry confirmed the glycosylation of these KLKs. Activation of glycosylated pro-KLKs 4 and 11 turned out to be most efficient by glycosylated KLK2 and KLK4, respectively. By comparing the glycosylated prostatic KLKs with their non-glycosylated counterparts from Escherichia coli, it was observed that the N-glycans stabilize the KLK proteases and change their activation profiles and their enzymatic activity to some extent. The functional role of glycosylation in prostate-specific KLKs could pave the way to a deeper understanding of their biology and to medical applications.

Effect of O-Linked Glycosylation on the Equilibrium Structural Ensemble of Intrinsically Disordered Polypeptides.

Glycosylation is one of the most common post-translational modifications (PTMs), which provides a large proteome diversity. Previous work on glycosylation of globular proteins has revealed remarkable effects of glycosylation on protein function, altering the folding stability and structure and/or altering the protein surface which affects their binding characteristics. Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) of large proteins are also frequently glycosylated, yet how glycosylation affects their function remains to be elucidated. An important open question is, does glycosylation affect IDP structure or binding characteristics or both? In this work, we particularly address the structural effects of O-linked glycosylation by investigating glycosylated and unglycosylated forms of two different IDPs, tau174-183 and human islet amyloid polypeptide (hIAPP), by all-atom explicit solvent simulations. We simulate these IDPs in aqueous solution for O-linked glycosylated and unglycosylated forms by employing two modern all-atom force fields for which glycan parameters are also available. We find that O-linked glycosylation only has a modest effect on equilibrium structural ensembles of IDPs, for the cases studied here, which suggests that the functional role of glycosylation may be primarily exerted by modulation of the protein binding characteristics rather than structure.

The role of protein glycosylation in laccases from Lentinus sp.

Laccases are blue multicopper oxidases that can oxidize various organic and inorganic compounds by reducing O2 to water. Despite the decades’ research, the roles of protein glycosylation in laccases are still unclear. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated lcc4) from a white‐rot fungus Lentinus sp. in which two N‐glycosylated sites at Asn75 and Asn458 were identified. We observed the activity of native laccase was decreased significantly after α‐mannosidase digestion, suggesting the importance of glycosylation in laccase activity. To address the structural and functional role of glycosylation, we cloned laccase genes and created a number of mutant enzymes with the Asn→Asp mutation on predicted glycosylation residues, Asn75, Asn236, Asn399, and Asn458. The recombinant mutant enzymes expressed in Pichia pastoris all showed much lower activities compared to wild‐type counterpart. On the other hand, hyperglycosylation observed in the yeast expressed recombinant laccases may interfere the protein folding giving a decrease in the enzyme catalytic activity. This study provides some possible structural and functional roles of glycosylation in laccase enzymes. NSC‐97–3114‐P‐001–001, Taiwan

Functional Role of Glycosylation in a Human IgG4 Antibody Assessed by Surface Plasmon Resonance Technology

Fc glycosylation of immunoglobulins is necessary for antibody effector functions. These glycans of immunoglobulins are often referred as glycoforms and they can be heterogeneous due to the variations in glycosylation machinery present in the endoplastic reticulum (ER) and in the Golgi. In the absence of a generic culturing protocol that can render a consistent glycosylation pattern, monitoring glycoforms of monoclonal antibodies from cultured cells is becoming essential. Accordingly, quantification of glycosylated and deglycosylated heavy chains of an IgG4 monoclonal antibody was accomplished using an Agilent Bioanalyzer Lab-On-the-Chip electrophoresis system. In addition to the native antibody, completely deglycosylated antibody prepared by treating with PNGase F and a F(ab’)2 fraction were evaluated for their antigen binding kinetics using Biacore surface Plasmon resonance (SPR). The equilibrium binding constants KD are found to be comparable at 1.81E-09M, 1.96E-09M, for the native and deglycosylated antibody, respectively, and 5.79E-10M for the F(ab’)2. An in vitro biological activity employing a competition binding assay was also developed to demonstrate the role of the Fc glycan. The results confirm that for a neutralizing antibody therapeutic the biological activity of the native MAb-1 and deglycosylated antibody are comparable, thus indicating that the Fc glycan does not contribute to the antigen binding or the biological function. The kinetics and competitive assays performed on an SPR instrument are quick and reliable. Combined with the on-chip electrophoresis method they can be used as monitoring methods for process development and quality control.

A computational investigation on the role of glycosylation in the binding of alpha1 nicotinic acetylcholine receptor with two alpha‐neurotoxins

Based on the crystal structure of the extracellular domain (ECD) of the mouse nicotinic acetylcholine receptor (nAChR) alpha1 subunit bound to α-bungarotoxin (α-Btx) we have generated in silico models of the human nAChR α1 bound to α-Btx and α-cobratoxin (α-Cbtx), both in the presence and in the absence of the N-linked carbohydrate chain. To gain further insight into the structural role of glycosylation molecular dynamics (MD) simulations were carried out in explicit solvent so as to compare the conformational dynamics of the binding interface between nAChR α1 and the two toxins. An interesting observation during the course of the MD simulations is the strengthening of the receptor-toxin interaction in the presence of the carbohydrate chain, mediated through a shift in the position of the sugars towards the bound toxin. Critical protein-sugar interactions implicate residues Ser187 and Trp184 of nAChR and Thr6, Ser9, and Thr15 of α-Btx, as well as Thr6 and Pro7 of α-Cbtx. Analysis of the predicted residue-specific intermolecular interactions is intended to inspire biophysical studies on the functional role of glycosylation in the gating mechanism.

Role of Glycosylation in Function of Follicle-Stimulating Hormone

The oligosaccharide structures of heterodimeric glycoprotein hormones, such as follicle-stimulating hormone (FSH), have been shown to play an important role in the biosynthesis, secretion, metabolic fate, and regulation of potency of the hormone. The oligosaccharide structures attached to each subunit of the protein seem to exhibit distinct roles in some of these functions. Glycans attached to the alpha-subunit are critical for dimer assembly, integrity, and secretion, as well as for signal transduction; although beta-subunit glycans are also important for dimer assembly and secretion, they play a crucial role in clearance of the dimer from the circulation. Alternative glycosylation on FSH and other glycoprotein hormones not only may affect the metabolic clearance and net in vivo biopotency of the hormone, but also offers the interesting possibility that some glycosylation variants of the hormone may provoke differential or even unique effects at the target cell level. Glycosylation of FSH is regulated by hypothalamic and/or end products from the glands under the control of this hormone. In particular, estrogens regulate terminal sialylation and thus some functional properties of the gonadotropin influenced by sialic acid. Through these extrapituitary inputs, the gonadotroph may regulate not only the amount but also the intensity of the gonadotropin signal to be secreted by the pituitary in a given physiological condition.

Age-related variations in glycosylation of hydroxylysine in human and rat skin collagens

The extent of glycosylation of hydroxylysine in human skin collagen rapidly decreased during maturation and then gradually increased in proportion to the age. This decrease of glycosylation observed during maturation was also confirmed in whole, soluble and insoluble collagens from rat skin. These findings may contribute to the investigations on the functional role of glycosylation and also on the mechanism of maturational as well as senile processes.