# Phytoglycoproteins and Human Health: Current Knowledge and Future Applications
## 2 Structural Features and Occurrence of Phytoglycoprotein
The second residue after Asn could be serine (Ser) or threonine (Thr), whereas the mid residue (represented as \"X\" in the sequon) can be any amino acid except proline [19]. The asparagine sequence must be on the surface of the three-dimensional folded nascent polypeptide rather than embedded in the structure. Finally, the asparagine should be in the lumen of the ER [18]. After the attachment of the matured glycan to the asparagine of the polypeptide, the final glycoprotein product is further processed, first in the ER and then in the Golgiapparatus (Figure 1) by the removal of one or two glucose moieties based on the folding pattern of the protein [19]. Like N-glycosylation that attach glycan molecules to Asn, O-glycosylation attaches the glycan moieties to mainly hydroxylated form of proline (hydroxylproline; HyP) and Ser residues. O-glycoprotein have two major subclasses named arabingalactan-proteins (AGPs) and extensins. Extensions have regular structures involving glycosylation of a pentapeptide residue, Ser-HyP-HyP-HyP-HyP. Hydroxyproline (HyP) is produced as a post-translation modification in the lumen of the ER by hydroxylation of Cg of proline by propyl hydroxylase [20]. Peptide sequences with 3 or 5 HyP are common sites for O-glycosylation [21]. In the Ser residue, monogalactoosylation takes place whereas in the HyP residue oligoarabinosylation occurs to produce structurally relevant extensins. In contrast, AGPs are the most bulky and complex structured glycoproteins and have a disproportionate arrangement of their oligopeptides attached to several uninformed cross-linkages of polysaccharides. AGPs have major structural relevance in cells [21]. Glycoproteins have been studied extensively in the past using several methodologies, which mainly involve hydrolysis of the glycoprotein into its oligoglycan and polypeptide, and maybe further into its monosaccharides [22]. In most studies, crude isolation of the glycoproteins is done using affinity chromatography [7; 23; 24] or ammonium sulphate precipitation [25; 26; 27] and dialysis. The existence of proteins and carbohydrates in the extract is essentially confirmed by checking absorbances at 280 and 490 nm for protein-rich and carbohydrate-rich fractions, respectively. Thereafter, the existence of a band with [PERSON]'s reagent on sodium dodecyl sulfate-polyacrylamideamide gel electrophoresis (SDS-PAGE) confirms the existence of glycoproteins. More recent approaches have applied advanced chromatographic techniques to analyze intact glycoproteins without hydrolysis. The techniques separate glycoprotein mixtures on the basis of their molecular weights, which are further used for identification and classification [28]. This approach presents ambiguity particularly when identifying glycoproteins with identical molecular weight and different atomic arrangements and stereo-conformations. Since several biologically active compounds are stereo-specific, varying results of the biological activities may be achieved if the stereo-specificity of isolated phytoglycoproteins is taken into consideration. A recent review by [PERSON] et al. [29] described the stereospecificity in both simple and complex carbohydrates and their diverse roles. Despite the paucity of studies on the relevance of stereospecificity on bulky macromolecules, the chirality of monosaccharides that make up the matured glycans may influence the properties of the nascent glycoprotein, and hence its biological activities. Therefore, further work is needed in developing comprehensive workflow targeted towards eliminating ambiguity in identifying plant-specific glycop  
Figure 1: The process of N-glycosylation of proteins in plant endoplasmic reticulum. teins. Advanced techniques such as NMR, mass spectroscopy and X-ray crystallography could be adopted for accurate structural elucidation of specific glycoprotein isomers.