Physico-chemical aspects of protein glycosylation

1. Physico-chemical aspects of protein glycosylation

2. Glycosylation • Covalent attachment of oligosaccharides – glycosylation - is the most common posttranslational modification of eucaryotic protiens • Most extracellular proteins are glycosylated • N-glycosylation: Preformed (N-acetylglucosamine)2-Mannosex are attached to Asparagine at Asn-X-Ser or Asn-X-Thr in the endoplasmatic reticulum. • Glycans often modified in the Golgi apparatus. • O-Glycosylation: Attachment of oligisacchariders to some Serine and Threonine ressidues occurring in the Golgi apparatus.

3. Effects of glycosylation • Complex and highly conserved pathways – underglycosylation may be lethal. • Molecular recognition (cell-cell, trafficking, transport etc.) • Stability (anti-aggregation) • Solubility • Susceptibility to enzymatic hydrolysis. Physio-chemical mechanisms? Degree of glycosylation vs. extent of change in property Applicability as a formulation tool

4. Physico-chemical effects of glycosylation Observarions vary, but may be rationalized by distinguishing: Naturally glycosylated protein (The protein is glycosylated in vivo) Mutated glycoproteins (glycosylation sites have been introduced by protein engineering) Chemically glycosylated proteins (glycans or related compounds (e.g. PEG) has been covalently conjugated) Comparisons across these groups (e.g. regarding stability issues) may lead to doubtful conclusions Bagger 2007 (phd thesis)

5. Our model - Phytase Phytase from Peniophora lycii – 439 amino acids Mw,pep=48 kDa N-glycosylated at 10 sites When expressed in Aspergillus oryzae, MW,gly=18 kDA (i.e. Mw~66 kDa) When expressed in Saccharomyses cerevisiae, MW,gly~70 kDA (i.e. Mw~110-120 kDa) When ”shaved” by the enzyme Endo F1 (Endo-b-N-acetylglucosaminidase), MW,gly~2 kDA (i.e. Mw~50 kDa) I.e. 3 variants with ~2%, 25% and 60% carbohydrate

6. Phytase and phytic acid Phytase + 6 Pi inositol hexakisphosphate Main storage form for phosphate in plants – indigestible to vertebrates

7. Glycosylation and the peptide fold Syncrotron radiation circular dichroism Native Phytase SDS-danatured Phytase Glycosylation only marginally affect the (secondary) structure of phytase in respectively the NATIVE, HEAT-DENATURED and SDS-DENATURED states Bagger et al (2007) Biophys Chem 129, 251

8. Glycosylation and thermal (equilibrium) stability DSC of phytase variants pH5.0 Phytase is remarkably unaffected by larges differences in the glycan content. This picture has been observed for most investigated (naturally glycolylated) proteins. Mutated or chemically glycosylated proteins show wide variation. Several cases of moderate stabilization by limited glycosylation has been reported. Often problems with activity or dramatic destabilization. Bagger 2007

9. Glycans and stabilizing excipients Thermal stability: Phytase and dg-Phytase The stabilizing effect of polyols is independent on the degree of glycosylation Sorbitol Glycerol Phytase (27% glycan) Is that due to the abscence of additive-glycan interactions? Dg-Phytase Bagger et al (2003) Biochemistry42, 10295

10. Vapor pressure (dew-point) osmometry The thermodynamic activity (chemical potential) of water in ternary (water+phytase+sorbitol) systems reflects the net protein-sorbitol interaction. Ternary (sorbitol-water-dgPhy) Ternary (sorbitol-water-Phy) Binary sorbitol-water Water activity in sorbitol solutions: The protein increases the ”effective concentration” of sorbitol - hence sorbitol is preferentially excluded from the protein interface

11. Preferential interactions 1 (m3/m2) T, P, 1≈ (m3-m3*)/m2 Sorbitol Glycerol Phytase Positive contribution from glycan mantle Dg-Phytase The two polyols interacts rather strongly with the glycans But no reduced stability! Phytase Dg-Phytase

12. Interpretation of Glycan-excipient interactions Observation ”Compatible solutes” or ”organic osmolytes” appear to bind to the glycan moiety of glycoproteins. They are preferentially excluded from the peptide moiety. They do not destabilize the native protein conformation. Hypothesis Glycans are fully hydrated in the native state. Hence the glycan-osmolyte interaction does not change during denaturation and this process is unaffected. What is more hydrophilic – glycan or peptide?

13. Hydrophilicity of glycans (I): Sorption isotherms Two-channel Sorption calorimetry Out put, Channel I DH Out put, Channel II DG I II Simultaneous measurement of sorption isotherm (free energy of water binding) and sorption enthalpy. Bagger et al (2006) Eu.Biophys.J.35, 367.

14. Peptides binds water more strongly than glycans during gradual hydration Net affinity (DG) Binding energy (DH) Lyotropic changes in freeze dried protein matrix ?? At 90% RH, for example, the polypeptide binds 0.34 g H2O/g – no detectable binding of water to the glycans !

15. Hydrophilicity of glycans (II): Second virial coefficients SAXS measurements at the EMBL X33 beamline at the DORIS storage ring, DESY, Hamburg. Osmotic virial coefficient from the Zimm approximation Slopes reflect 2nd virial coefficient Guinier plots (ln I(q) vs. q2) To determine forward scattering (I(0)) for 1-15 mg protein/ml

16. Hydrophilicity of glycans (II): Second virial coefficients SAXS measurements pH 8.0 (pI~4) SLS measurements (633 nm) Peptide interacts more favorably with water than the glycans. Glycan effects are NOT due to ”stronger hydration” Høiberg-Nielsen et al (2006) Biochem. 45, 5057

17. What phytase glycosylation doesn’t do: (or does to a small extend ) • It does not change the protein fold • It does not change the enzymatic activity • It does not change the thermal stability • It does not change the stabilizing effects of (some) exipients • It does not improve hydration • It doesnt change the resistance towards SDS A remarkable non-effect of a massive covalent modification

18. Glycosylated Deglycosylated 74oC A2, 11.0  104 mL mol g-2 A2, 9.0  104 mL mol g-2 20oC A2, 10.9  104 mL mol g-2 A2, 10.9  104 mL mol g-2 Structure and interactions: SAXS • The tertiary structure of thermally denatured phytase is elongated by the glycans • The glycans are NOT particularly hydrophilic • The maximal dimensions of the native structure is hardly affected by the glycans. Høiberg-Nielsen et al, Submitted.

19. Glycosylation and aggregation Glycosylationveryeffectivilyinhibits rapid aggregation Høiberg-Nielsen et al, (2006) Biochem. 45, 5057.

20. Titration of critical ressidue at pH~ 5.1 pI~3.7 Specific – structurallywelldefined – electrostaticattractions promote aggregation Høiberg-Nielsen et al, (2006) Biochem. 45, 5057.

21. Effects of NaCl at pH 5 57C 41C NaClretardsaggregation – mores so for dgPhythan for Phy Interpretation: Attractiveelectrostatic forces arestronger in dgPhy

22. Unfolding vs. aggregation Ag Ag

23. Aggregation pathwaysNDI Aggregation of nativedgPhy – rate 2-4% of DAg rate at Tm. This ”leak rate” maybecomeimportant at lowtemperaturewhere [D]~0.

24. Very high glycan content inhibits aggregation further 2 % Glycan 27 % Glycan 60 % Glycan 13 mM protein, pH 5.0, 61C Bagger 2007 (thesis)

25. Size exclusion chromatography:Small aggregates also for high glycan Native Native Native 10 min heat 50 min heat 50 min heat 2 % Glycan 25% Glycan 60% glycan There is a considerable loss of monomeric enzyme also in the glycosylated sample.

26. Is glycosylation (and PEGylation) promising tools in protein formulation Probably so – but phytase results suggest: • It appears to work best against very fast aggregation • It appears to allow the formation of small aggregates (it doesn’t help if we get many small (inactivated) aggregates). • Closingremarks: • Glycosylationstronglymodulates the physical of proteins – the major mechanism is stericeffects; not favorable interactionswithwater. • ”Stericeffects” include: shielding of charges; reduction of D-statesflexibility (and entropy); entropic protein-protein repulsion.