Protein Structure and Function Lecture 7 Notes: Glycosylation and Therapeutic Antibodies Part 3 Investigating Glycan Structure - Deliberate control of the glycan profile of a glycoprotein would be useful to study structure- function relationships to produce a defined product which optimises therapeutic efficacy. There are three main approaches: manipulation of the biosynthetic pathway genetically, glycan remodelling chemoenzymatic action and glycoengineering of Fc N- glycans for drug conjugation. of O H 1 H-C-OH 6 CH2OH HO- C-H 3 5 OH H-C H H = H- OH 4 OH H HO 35 H H- 5 OH OH 6 CH2OH D-Glucose (open-chain form) CH2OH OH I HA KOH H OH HO H OH a-D-Glucopyranose CH2OH H Q OH OH H HO H OH B-D-Glucopyranose - When a sugar is the cyclic form (as they were linked) there is a new stereocentre at C1 where there are two possible forms (a or B). The designation depends on the orientation at C1, relative to that at the highest stereocentre. Operationally, in D sugars OH down means a, up means B. - Glycans can be detected on electrophoresis gels or in tissues with periodate-Schiff reagent (used as a stain on protein gel electrophoresis). Hydrolysis with strong acids will liberate monosaccharides and amino acids (i.e. information is lost). The enzyme PNGaseF will liberate intact N-glycans as free oligosaccharides. Polysaccharide HO2SHN HIO4 RCH(OH) O2SHN O NH2 + 2R-CHO C =NH HO2SHN Aldehyde SO3H RCH(OH) O2SHN Schiff's Reagent Quinoid chromophore (red ~ purplish blue) - Endo-H cleaves between chitobiose cores of high mannose and hybrid (but not complex) glycans. O-glycosidase, also known as endo-a-N-acetylgalactosaminidase catalyses the removal of core 1 and core 3 o-linked disaccharides from glycoproteins. - PNGaseF removes intact N-linked glycans from the proteins as an unstable 1-amino oligosaccharide Peptide CH,OH O H HO NH CHE CH CH,OH Lo HON T R - O' NH / PNGase F NH 0 C=0 1 H?C =0 O G=O NH2 + Peptide 1 NH CHE Peptide Core GlcNAc Asparagine Residue HO - O=O CH C=0 I Peptide Aspartic Acid Residue
- Alkaline elimination will liberate intact O-glycans as free (reactive) oligosaccharides (no good with N-glycans). However, subsequent analysis of the peptide site is difficult, and the liberated sugar can degrade in alkali. - Glycans can be analysed via released glycan analysis (with enzymes or alkali), glycopeptide analysis (peptide mapping) and intact glycoprotein analysis (measure MS of intact proteins). These finish with the application of spectrophotometry. This method is used to retain the PTM site info. However, the products are complex so PTM structural information may be buried in other peptide information. - Released glycan analysis involves releasing glycans (i.e. with an enzyme or alkali treatment), separating them from the residual protein (isolation), and analysing them via methylation analysis for linkage determination. This is for investigating glycans in detail as they are more clearly revealed, however, the amino acid site information is lost. - Linkage analysis via methylation involves free OHs in the glycan being converted to stable methyl ethers. The