Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Glycosylation is a very crucial post-translational modification observed in proteins. Approximately more than 50% of proteins are found in their glycosylated forms in eukaryotes. N– and O-glycosylation are the two major types of glycosylation^1,2. O-linked glycans (O-glycans) are covalently attached to proteins via N-acetylgalactosamine to the hydroxyl group of a serine (Ser) or threonine (Thr) amino acid residues. N-linked glycans (N-glycans) are complex oligosaccharides, which are covalently attached to asparagine (Asn) amino acid residue of the proteins through N-acetylglucosamine (GlcNAc) in a particular amino acid sequence AsN-X-Ser/Thr and a less common one, AsN-X-Cys (cysteine) (where X might be any amino acid except proline)^3,4. The basic N-glycan core consists of two HexNAc and three mannose residues. Further elongation of this common core with other monosaccharides via glycosyltransferase and glycosidase enzymes determines the type of N-glycans based on the degree of branching and the type of linkage⁵. N-glycans are generally grouped into three main classes: high mannose (HM), complex type (CT), and hybrid (HY)⁶.

N-glycans are indigestible compounds by the host organisms due to the lack of glycoside hydrolase enzymes. These compounds reach the small/large intestine in an undigested form where thousands of different bacterial species utilize them, and they can act as prebiotics by promoting specialized gut microbes, especially Bifidobacterium species⁷. Recent findings showed that N-glycans selectively stimulate the growth of certain bacterial species^8,9. N-glycans released from bovine milk glycoproteins selectively stimulated the growth of Bifidobacterium longum subspecies infantis (B. infantis), which is a crucial Bifidobacterial species in the infant's gut, but other bifidobacterial species such as Bifidobacterium animalis (B. animalis) did not utilize these compounds⁹. In addition, a recent in vivo study demonstrated that 19 unique N-glycans from milk lactoferrin and immunoglobulins selectively stimulate the growth of B. infantis⁸. Especially, B. infantis possess a genomic capability for glycan cleavage and metabolism. An Endo-β-N-acetylglucosaminidase (EndoBI-1), which belongs to glycosyl hydrolase family 18, recombinantly produced from B. infantis ATCC 15697 showed a high activity on milk glycoproteins in in vitro conditions^9,10. This novel glycoside hydrolase enzyme can cleave the N-N′-diacetylchitobiose parts found in the N-glycans^10,11. The activity of EndoBI-1 is not affected by core fucosylation and different reaction conditions such as high temperature, pH, reaction time, etc^3,11,12. This unique characteristic of Bifidobacterial glycoside hydrolases provides a promising tool for producing N-glycans from glycoprotein-rich substrates such as bovine colostrum^13,14.

Several chemically and enzymatically developed deglycosylation methods have been widely used to obtain N-glycans and O-glycans from glycoproteins^2,15. Chemical methods are widely used in glycobiology for deglycosylation of glycoproteins because of their ease of use, low cost, and high substrate specificity¹⁶. The most common chemical deglycosylation methods are β-elimination and hydrazination¹⁷. Among these methods, β-elimination is based on the principle of cleavage of glycans from glycoproteins by exposure of glycoproteins to alkaline conditions. The released glycans can be degraded during the process due to the β-elimination reactions, but this problem can be prevented using reducing agents such as sodium borohydride (NaBH₄)^18,19,20. There are different limitations in the β-elimination method. The reductive agents convert glycans to alditols, prevent them from binding a fluorophore or chromophore. Thus, challenging to monitor glycan release becomes difficult^19,20. Because of the high salt content in the cleaning step of the method, elution might result in sample losses²⁰. Another method for releasing glycan from glycoproteins is the hydrazine method based on the principle of the hydrolysis reaction following the addition of anhydrous hydrazine to the glycoprotein. Since it allows for controlling the isolation of glycans by changing reaction conditions such as temperature, the hydrazination method has been widely used in glycobiology²¹. Chemical deglycosylation can also be carried out using the anhydrous formulation of hydrogen fluoride and trifluoroacetic acid, in addition to other chemical deglycosylation methods^16,22,23. The enzymatic release of N-glycans from glycoproteins is commonly performed by peptidyl-N-glycosidases (PNGases) that generally release N-glycans, regardless of their size and charge^24,25,26,27. Similar to the chemical deglycosylation methods, the enzymatic deglycosylation process has different challenges. PNGases show activity in the presence of several detergents used, which increase the enzyme accessibility to the glycans. However, these harsh treatments might disrupt the native glycans and the remaining polypeptide structures²⁸. PNGases may not cleave the glycans when there is a fucose linked to N-acetylglucosamine²⁹. Various endoglycosidases such as F1, F2, and F3 show more activity on the native proteins than PNGases. These endoglycosidases have low activity on the multiple-antennary glycans, whereas heat-resistant novel EndoBI-1 is effective in all types of N-glycans^10,11,28. Regarding the limitations of the current methods, it is obvious that novel enzymes are still required for an effective glycan release without any restrictions. For this purpose, Bifidobacterial species, which have a large genomic island encoding various glycoside hydrolases enzymes, enable cleaving N-glycans from glycoproteins^30,31. Within the scope of this context, the overall aim of this study is to discover new glycosidases from the various Bifidobacterial species. To recombinantly produce these enzymes, different fusion tags are intended to enhance their production as well as their activity.