Glycosylation In Plant Made Recombinant Protein Biology Essay

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Glycosylation is a process in which glycosyl donor, which is a carbohydrate, attaches to a glycosyl acceptor which can be any other functional group. It can be also defined as attachment of glycans to proteins. Glycosylation helps in many post-translational & co-translational modifications and also important in membrane proteins and secreted proteins [2]. There are various types of glycosylations. N-type glycosylation is very common type of glycosylation, occurs in lumen of endoplasmic reticulum of eukaryotes, also present in archae but rarely in bacteria. N-linked glycans attaches to the argenine side chain [3]. O-linked glycosyltion occurs in Golgi apparatus of eukaryotes, also in archae and bacteria. O-linked glycans attach to serine, threonine, tyrosine, hydroxylysine or hydroxylproline side chains [4].

Post translational modifications (PTMs) of the recombinant proteins are important in therapeutics. Targeting glycosylation pathway is one way of production of recombinant proteins. At the same time mechanism of glycosylation of recombinant proteins should be similar to that present naturally in human system to minimise immunological effect and to increase half-life [5]. Most of the times downstream processing, i.e. isolation and purification of proteins determines the cost of production [6]. Plants are cost effective for both production and purification of glycosylated recombinant proteins [7]. Plants are advantageous because they show high level of accumulation of proteins, storage of proteins in different plant organs and compartmentalization inside the cell [6]. Plants are also biologically safe, gives fast production, have protein folding assembly [7] and they are free from human pathogens [9], this makes plants as an attractive bioreactor for recombinant protein production. But it is important to "humanize" plant glycosylation process in order to avoid immune reactivity against them [7].

N-glycoengineering is a vast and well understood branch of glycosylation [8].N-glycosylation is known to be important for stability, reactivity and biological activity of therapeutic proteins [10]. It prevents proteolytic degradation and thermal stability of glycoproteins [11]. N-glycosylation starts in Endoplasmic reticulum, where oligosaccharide is co-transferred to aspargine residue of polypeptide chain. Oligosaccharide processing occurs which moves towards the maturation of these glycoproteins. Matured glycoproteins shows absence of sialic acid and presence of β(1,2)-xylose & α(1,3)-fucose, which is not in case of human system. Glycosyltransferases and glycosidases are the enzymes involved in maturation of glycoproteins in plants [12]. Hence inhibition of endogenous Golgi glycosyltransferases & addition of human specific glycosyltransferase are important factors involved in humanization of plant glycosylation system [10]. Another strategy to overcome limitations in plant glycosylation system is to retain recombinant protein in ER [10], to prevent glycan modification occurring in Golgi compartment, which is specific to plants [13]. This retention of glycoproteins in ER can be carried out by KDEL-mediated retention. KDEL is an amino acid sequence (K-lysine, D-aspartate, E-glutamate, L-leucine), which is responsible for retrieval of ER proteins from Golgi apparatus [13].

To minimize difference between plat and mammalian oligosaccharides produced in glycosylation, various steps can be taken, like transfer of oligosaccharide precursors used in natural glycosylation process to N-glycosylation sites. Processing of oligosaccharide side chains can also serve this purpose. One more advantage of N-glycosylation is that, it is not organ specific; hence the recombinant protein purification can be carried out from whole plant [11]. The branched glycoproteins are important in cancer metastasis and regulation of T cell activation. Branched glycoproteins are not produced in plant system, but by targeting Galactose-1-phosphate uridyl transferase (GalT), which converts galactose to glucose, a branched recombinant glycoproteins can be produced [9]. N-glycosylation gives structural stability to Fc arm and it is also linked with the Fab fragment of immunoglobulin. It is found that GUY's 13 monoclonal antibody which acts against dental caries, has heavy chain which is attached to N-linked glycans. But in plants sialic acid is not introduced on glycoproteins as humans. Modification of N-glycosylation to introduce sialic acid gives GUY's 13 antibody which can prevent dental caries caused due to Streptococcus mutans [14].

N-glycoengineering is already established branch of production of recombinant proteins. Hence O-glycosylation is a new and upcoming branch [8]. O-glycosylation is very complex and less understood PTM of proteins [5].Modification of serine and threonine occurs in O-glycosylation. O-glycans are found in higher amounts on tumours, so it is suggested that O-glycans can be used as anti-cancer vaccines, to induce immunity against tumour. Plant does not have machinery for production of mammalian O-glycans. To initiate mammalian type O- glycosylation in plants, GalNAc transferase enzyme, which transfers GalNAc to serine/threonine residue, has to be expressed by plants [8]. This requires introduction of 2 components of O- glycosylation mechanism, they are e4-epimerase and GalNAc-T. some scientists reported success in production of stable O- glycosylation system in plants. Tobacco BY2 suspension having secretion ability & soil grown Arabidopsis are examples of these success. Also modification by genetic engineering in O- glycosylation related gene can be carried out. Co-expression of the genes for UDP-GalNAc, N-acetyl-α-galactosaminyltransferase, UDP-GlcNAc 4-epimerase & UDP-GalNAc transporter made O-glycosylation possible in plants [5]. Mucin type glycosylation takes place in Golgi apparatus. Conversion of UDP-GlcNAc to UDP-GalNAc in cytosol is important for transport of UDPGalNAc into Golgi lumen, here with the help of GalNAc-T it attaches to O- glycosylation site. This is achieved in N. benthamiana and production of Erythropoietin-Fc was reported.

Although a little success has been obtained in O-glycosylation, there are many hurdles which have to be crossed into the future. In plants, O- glycosylation results in conversion of prolein to hydroxyprolein and addition of arabinose in maturation step. This arabinose linked to hydroxyprolein can serve as an epitope for IgE binding and hence can induce allergic reactions. Production of many recombinant proteins requires both O & N- glycosylation, lack of complete understanding of O- glycosylation comes between the successful productions of these types of proteins. Hence the steps towards simultaneous modification of both the pathways should be taken. Detailed understanding of positioning of glycosylation enzymes is very important so that they can be positioned correctly for complete processing of proteins [8].

Recent studies are moving towards the use of plant cell culture for glycosylation linked recombinant protein production [16]. Also use of seed is being thought for production of recombinant proteins as they provide good and long term storage system [17]. Carrot cells which produce β-glucocerebrosidase are already being used as enzyme replacement therapy for Gaucher's disease [15]. In all we observed that N & O glycosylation in plants is efficient and cost effective way of producing recombinant proteins. More research should be carried out for O- glycosylation pathway for decreasing differences between plant and mammalian glycosylation mechanisms.

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