Physico-chemical aspects of protein glycosylation

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Physico-chemical aspects of protein glycosylation
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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.

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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
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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)
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Our model - Phytase
Phytase from Peniophora lycii 439 amino acids
Mw,pep48 kDa N-glycosylated at 10 sites When
expressed in Aspergillus oryzae, MW,gly18 kDA
(i.e. Mw66 kDa) When expressed in Saccharomyses
cerevisiae, MW,gly70 kDA (i.e. Mw110-120
kDa) When shaved by the enzyme Endo F1
(Endo-b-N-acetylglucosaminidase), MW,gly2 kDA
(i.e. Mw50 kDa)
I.e. 3 variants with 2, 25 and 60 carbohydrate
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Phytase and phytic acid
Phytase
6 Pi
inositol hexakisphosphate Main storage form for
phosphate in plants indigestible to vertebrates
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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
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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
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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) Biochemistry 42, 10295
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Vapor pressure (dew-point) osmometry
The thermodynamic activity (chemical potential)
of water in ternary (waterphytasesorbitol)
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
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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
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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?
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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.
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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 !
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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

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Hydrophilicity of glycans (II) Second virial
coefficients
SAXS measurements pH 8.0 (pI4)
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
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What phytase glycosylation doesnt 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
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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.
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Glycosylation and aggregation
Glycosylation very effectivily inhibits rapid
aggregation
Høiberg-Nielsen et al, (2006) Biochem. 45, 5057.
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Titration of critical ressidue at pH 5.1
pI3.7
Specific structurally well defined
electrostatic attractions promote aggregation
Høiberg-Nielsen et al, (2006) Biochem. 45, 5057.
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Effects of NaCl at pH 5
57?C
41?C
NaCl retards aggregation mores so for dgPhy
than for Phy Interpretation Attractive
electrostatic forces are stronger in dgPhy
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Unfolding vs. aggregation
Ag
Ag
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Aggregation pathwaysN?D?I
Aggregation of native dgPhy rate 2-4 of D?Ag
rate at Tm. This leak rate may become important
at low temperature where D0.
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Very high glycan content inhibits aggregation
further
2 Glycan
27 Glycan
60 Glycan
13 mM protein, pH 5.0, 61?C
Bagger 2007 (thesis)
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Size exclusion chromatographySmall aggregates
also for high glycan
10 min heat 50 min heat 50 min heat
Native Native Native
2 Glycan 25 Glycan 60 glycan
There is a considerable loss of monomeric enzyme
also in the glycosylated sample.
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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 doesnt help if we get many small
  (inactivated) aggregates).

• Closing remarks
• Glycosylation strongly modulates the physical of
  proteins the major mechanism is steric effects
  not favorable interactions with water.
• Steric effects include shielding of charges
  reduction of D-states flexibility (and entropy)
  entropic protein-protein repulsion.