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The Development of Improved Therapeutics through a Glycan- “Designer” Approach
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
There are four known types of glycosylation based on the type of carbohydrate-peptide linkage: N-linked (Asparagine), O-linked (Threonine, Serine) C-linked (via Carbon atom) and S-glycosylation (Cysteine). Glycosylation can be further diversified on the base of glycosidic linkage, glycan composition, structure, and length. The N-type glycosylation in which glycan is attached to the amine group of asparagine residue resulting in formation of amide bond, is the most prevalent naturally occurring glycosylation type. The most common glycan linkage in human body among N-glycans is N-acetylglucosamine (GlcNAcβ1-Asn) found abundant in human serum. Overall different N-glycans are prevalent structures in prokaryotic surface proteins with a unique species-specific oligosaccharide structure; for that reason the glycoproteins derived from pathogens are excellent immunogens. The synthesis of N-glycans is well described for both prokaryotes and eukaryotes. While glycosylation in prokaryotes can be easily predicted and controlled the synthesis of all eukaryotic N-glycans is more complexed.
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Published in Raj Bawa, János Szebeni, Thomas J. Webster, Gerald F. Audette, Immune Aspects of Biopharmaceuticals and Nanomedicines, 2019
Raj Bawa, János Szebeni, Thomas J. Webster, Gerald F. Audette
Glycosylation may strongly modulate immunogenicity of therapeutic protein products. Although foreign glycoforms such as mammalian xenogeneic sugars (Chung et al., 2008; Ghaderi et al., 2010), yeast mannans (Bretthauer and Castellino, 1999), or plant sugars (Gomord and Faye, 2004) may trigger vigorous innate and acquired immune responses, glycosylation of proteins with conserved mammalian sugars generally enhances product solubility and diminishes product aggregation and immunogenicity. Glycosylation indirectly alters protein immunogenicity by minimizing protein aggregation, as well as by shielding immunogenic protein epitopes from the immune system (Wei et al., 2003; Cole et al., 2004). Pegylation of therapeutic protein products has been found to diminish their immunogenicity via similar mechanisms (Inada et al., 1995; Harris et al., 2001), although immune responses to the polyethylene glycol (PEG) itself have been recognized and have caused loss of product efficacy and adverse safety consequences (Liu et al., 2011). Anti-PEG antibodies have also been found to be cross-reactive between pegylated products (Garay et al., 2012; Schellekens et al., 2013).
Contributions of Recombinant Microbes and Their Potential
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Arnold L. Demain, Akira Kimura, Atsuhiko Shinmyo
The glycosylation of a protein can be different, depending on factors such as the medium in which the cells are grown. Glycosylation influences reaction kinetics (if the protein is an enzyme), solubility, serum half-life, thermal stability, in vivo activity, immunogenicity, and receptor binding. For peptides, galactosylated enkephalins are 1,000-10,000 times more active than the peptide alone [16]. That glycosylation increases the stability of proteins was shown by cloning genes encoding bacterial nonglycosylated proteins in yeast. The yeast versions were glycosylated and were more stable [17].
Cloning, large-scale production and characterization of fusion protein (P-TUFT-ALT-2) of Brugian abundant larval transcript-2 with tuftsin in Pichia pastoris
Published in Preparative Biochemistry and Biotechnology, 2018
Rajkumar Paul, Selvarajan Karthik, Ponnusamy Vimalraj, Sankaranarayanan Meenakshisundaram, Perumal Kaliraj
P. pastoris also expressed Japanese encephalitis virus (JEV) envelope (E) protein, a recombinant protein vaccine with immunogenicity and protective efficacy in mice.[75] Asparagines at the 14th, 99th, 122nd, and 123rd position of P-TUFT-ALT-2 were predicted to be N-glycosylated. The present study also reported P-TUFT-ALT-2 as glycosylated protein in P. pastoris. The deglycosylation of 28 kDa TUFT-ALT-2 by PNGase F assay showed 3 kDa more weight due to glycosylation. The PAS staining confirmed the glycosylation. The degree, type, and position of glycosylation may further be confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Excessive glycosylation is a common problem for proteins produced in fungal hosts.[33,76] However, P-TUFT-ALT-2 was not seemed to be hyperglycosylated.
Model-guided concurrent data assimilation for calibrating cardiac ion-channel kinetics
Published in IISE Transactions on Healthcare Systems Engineering, 2023
Haedong Kim, Hui Yang, Andrew R. Ednie, Eric S. Bennett
Protein glycosylation is one of the most abundant and diverse forms of co/posttranslational modifications that impact essential protein functions, such as modulation of receptor or ion channel activities (Ednie & Bennett, 2012; Ohtsubo & Marth, 2006). A growing number of studies have shown the association between altered glycosylation and heart diseases, such as dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (Ednie et al., 2019; Ohtsubo & Marth, 2006). It is reported that up to 20% of patients with congenital disorders of glycosylation (CDG), who commonly show modest reductions in protein glycosylation, present with cardiac deficits, including idiopathic DCM (Marques-da Silva et al., 2017). However, uncovering the underlying pathological mechanisms still remains elusive. We have investigated how regulated glycosylation contributes to heart failure in the context of electrophysiology. Electrical signaling is orchestrated activities of a variety of ion channels and transporters. VGICs are heavily glycosylated, with 30% of the channel mass consisting of N-/O-linked glycans (Ednie & Bennett, 2012). Glycosylation is a multi-step process and usually ends with sialic acid added. We reported that a saturating, electrostatic effect of negatively charged sialic attached to the terminal of N-/O-glycan branches significantly altered electrical signaling in Nav (Ednie et al., 2013, 2015) as well as Kv (Ednie & Bennett, 2015). Computational modeling has been used to further investigate the functional role of reduced sialylation in Nav and Kv (Du et al., 2016, 2018).
Challenges and advancements in the pharmacokinetic enhancement of therapeutic proteins
Published in Preparative Biochemistry & Biotechnology, 2021
Farnaz Khodabakhsh, Morteza Salimian, Mohammad Hossein Hedayati, Reza Ahangari Cohan, Dariush Norouzian
Hepatic clearance could be overcome by interference in the receptor binding of proteins to hepatocytes. Genetic insertion of N- or O-linked glycosylation sites in the protein sequence could inhibit such interaction, and therefore, protect the protein from degradation.[19,20] For example, darbepoetin alfa (Aranesp®, Amgen) is a hyperglycosylated form of erythropoietin with two additional N-glycosylation sites that has a longer circulating half-life in vivo.[20] Also, a key role of sialic acid in the carbohydrate constituent of hyperglycosylated proteins has been reported for half-life extension through the kidney. The negative charge of sialic acids increases the repulsion between the hyperglycosylated protein and glomerular cell membranes, and therefore, reduces glomerular filtration in parallel.[21] Moreover, glycosylation by increasing the negative surface charge (zeta potential) of protein and shielding effect (spatial hindrance) on hydrophobic patches improves the solubility of proteins. It is also reported that glycosylation, by the same mechanism, has a preventive effect on protein aggregation and proteolytic degradation in vitro and in vivo.[22] Nonetheless, low rise in plasma half-life (2 to 4 fold), high cost of production, additional quality control tests for glycan constituent (e.g., mass spectroscopy and sialic acid content), and in some cases, a reduction in the biological activity of native protein have been reported.[1] There are also challenges about the site of glycosylation in the protein. It is reported that the insertion of glycosylation consensus sequences is not adequate for glycan addition and other parameters must be considered in designing such hyperglycosylated forms.[23] Other technologies that used natural polysaccharides like heparosan (HEPylation) and hydroxyl ethyl starch (HESylation) were proposed to enhance the pharmacokinetic parameters of therapeutic proteins such as granulocyte colony-stimulating factor (G-CSF) and human interleukin-1 receptor antagonist (IL-1Ra).[24] Although these strategies have superior features over polyethylene glycol such as biocompatibility and lower immunogenicity, however, their coupling process is often complex and needs several purification steps that are cost- and time-consuming.[25,26]