O

Gal

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SiaT

Figure 5 Trimming and branching reactions that modify the core jV-oligosaccharides. [Adapted from Refs. (20,21).] (See color insert synthetase (DPMS) transfers four mannoses from guanidine diphosphate-Man (GDP-Man) to dolichylphospho-b-mannose (Dol-P-Man). Dolichylphospho-b-glu-cose (Dol-P-Glc) is formed by the reaction of Dol-P with UDP-Glc catalyzed by the enzyme dolicylphospho-b-glucose synthetase (DPGS). Dol-P-Glc is the donor for the final three glucoses added to the (Man)5(GlcNAc)2-PP-Dol catalyzed by the enzymes glucosyltransferase I-III (GlcT I-III). ManT VI-IX catalyze the final four mannose additions. The ManT VI-IX and GlcT I-III reactions result in the formation of (Glc)3(Man)9(GlcNAc)2-PP-Dol, the core N-oligosaccharide. OligoST transfers the (Glc)3(Man)9(GlcNAc)2 residue from (Glc)3(Man)9(GlcNAc)2-PP-Dol to the nascent protein at the exposed asparagine amino acid, resulting in the nascent glycoprotein. Macroheterogeneity results when not all of the asparagine residues have an attached core N-oligosaccharide. The complete (Glc)3(Man)9 (GlcNAc)2 residue is required for further glycosylation processes (6,11,21,24).

Trimming and Branching Reactions of N-Oligosaccharides

Once the nascent glycoprotein is formed, the trimming and branching reactions specific to the species and cell type can occur in the Golgi. Trimming and branching reactions may or may not occur at a particular glycosylation site or in a particular cell type. In general, trimming reactions are catalyzed by two types of enzymes, glu-cosidases and endomannosidases. Figure 5 shows the common trimming and branching reactions that may occur. There are three glucosidases (GI, GII, and GIII), which remove the terminal glucose residues from the nascent core glycopro-tein. Endomannosidases are able to remove one, two, or three glucose residues, plus a mannose residue, in one reaction. ER mannosidase and Golgi mannosidase I remove all four outer mannose residues. Branching reactions are very complex and may result in biantennary, triantennary, tetra-antennary, and penta-antennary oligosaccharides. Other common modifications of glycoproteins are galactosylation and sialylation. GlcNAc-TI is the key enzyme for the conversion of high-mannose type to hybrid- and complex-type N-linked oligosaccharides (Fig. 5). Other critical enzymes include GlcNAc-TII, GlcNAc-TIII, GlcNAc-TIV, GlcNAc-TV, GlcNAc-TVI, mannosidases II, IIX, and III, and a1,6-fucosyltransferase (FT). Galactosyla-tion of N-linked oligosaccharides is catalyzed by a number of species-specific and cell-specific enzymes. The most common galactosyltransferases (GalTs) are b1,4-Gal-T and b1,3-Gal-T. Another common modification of glycoproteins is sialylation. Sialylation is very important for the efficacy of many highly glycosylated proteins, such as EPO and Factor VIII. Sialyltransferases (STs) catalyze the sialylation at the terminal end of the oligosaccharide. These are many different ST enzymes. Each type of ST catalyzes the addition of a particular sialic acid with a specific terminal linkage. In humans, the most common terminal sialic acid is NeuAc. The commonly observed sialic acid linkages in mammals are a2,3-, a2,6-, and a2,8-linked sialic acids (6). These complex trimming and branching reactions, occurring and not occurring, lead to N-linked microheterogeneity (11,21,24).

The key control points for N-glycosylation of recombinant proteins are (1) ER and Golgi mannosidase I and GlcNAc transferase I, which catalyzes the conversion of high-mannose glycans to complex-type glycans; (2) GlcNAc transferases II, IV, and V, which catalyze the degree of branching; (3) GlcNAc transferase III, which catalyzes the bisection of GlcNAc attached to the core mannose; (4) a2,3- and a2,6-STs, which catalyze the addition of terminal sialic acid; and (5) CMP-NeuAc-4-hydroxylase, which catalyzes the conversion of NeuAc to NeuGc (1).

Understanding the effects of bioprocess conditions on these controlling reactions could be used to improve product quality.

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