Design of in vitro protein digestibility d h i l h i k assays ... - Europabio

Design of in vitro protein digestibility
assays and d their h i relevance l to the h risk ik
assessment
Clare Mills
Institute of Inflammation and Repair
The University of Manchester

Declaration of Interests
Research funding
• UK Biological and Biotechnological Sciences Research
Council
• UK Technology T h l Strategy St t BBoard dP Project j t( (collaborative ll b ti with ith
Waters Corporation, Romer Labs, The Laboratory of the
Government Chemist)
• UK Food Standards Agency (contractor to Food Allergy
Branch; member of the Advisory Committee on Novel
Foods and Processes [ACNFP]*) [ACNFP] )
• European Union –CHANCE
• Novartis, , DBV Technology gy
• European Food Safety Authority

Why is digestibility important for
food allergy? gy

Lipid
droplets
Events in the gut lumen affecting protein
release and breakdown
Lipid‐adsorbed
allergen
Impact on release
ffrom intact i t t
cells
Proteolysed
protein
aggregates
Resistant
allergen
Protein
stabilised
emulsions
How does the form of a
protein determine its
release from food and
stability to digestion?

Uptake and intracellular processing of allergens…….
HHow do d proteins i cross the h mucus
layer?......
……and how does this affect their uptake p by y the
epithelium and immune cells.............
……. making g an antigen g into an allergen? g
Maciercenka, et al 2011 Soft Matter 7 (18) 8077‐8084

This is important for
•Elicitation of allergic g reactions in sensitised
individuals
•Severity Severity of reaction and conditions like exercise‐ exercise
induced anaphylaxis
•Affecting Affecting the balance between tolerance and
sensitisation to dietary protein

Digestion also plays an important role in
antigen presentation
The endosome – a “stomach stomach inside a cell” cell is involved in
•Generating peptides taken up by a diverse range of
cells
•pH is ~2
•Proteases include aspartate and cysteine proteases
•R •Redox d enzymes such h as γ‐interferon i t f llysosomal l
thioreductase (GILT)
Hastings and Cresswell (2011) ANTIOXIDANTS & REDOX SIGNALING 15: 657‐668

Digestibility and allergenicity risk assessment
Digestibility g ystudies provide p useful data regarding g gthe
properties and characteristics of the novel protein affecting
•Gut lluminal i lprocessing i and d uptake k
•Intracellular processing and antigen presentation
These may influence properties such as tolerance induction
or sensitisation in a host.
They may also provide data on the stability and molecular
mobility bili of f polypeptide l id chains hi
MMutschlechner hl h et al l J Allergy All Cli Clin IImmunoll2010 2010;125:711‐8
125 711 8

Gastroduodenal digestion and the gut as a
bi biological l i lprocessing i plant l
Biomechanics and motility y determine
luminal flow and mixing behaviour
+
Variation in activity, content and
secretion of digestive enzymes
Determine the rate of delivery of
absorbable species to the gut wall
All of this is under tight biological control, including
gut‐brain signalling
9

Dynamic y a cGast Gastric c Model ode ( (DGM): G ): Full u ssimulation uato oof gast gastric c
forces and motility
Main Body:
Gentle 3 contraction
wave
per p min cycle y
In‐homogenously mixed
environment
AAntrum: t
High shear well mixed
environment
Shear at 10‐100 sec 1 ‐1
Phase II contraction waves
Inventors: Martin Wickham, Richard Faulks
Licensed to PBL

Dynamic Duodenal Model: combining
segmented and peristaltic flow.
Bostjan Hari, Serafim Bakalis, Peter Fryer,
UUniversity i i of f Birmingham
Bi i h

β‐Lactoglobulin β Lactoglobulin ‐ normally resists pepsinolysis
in solution but is partially digested as an
emulsion
β-Casein: Emulsification alters the digestion kinetics
of giving g g rise to Mr 6,000 , resistant peptides p p
Maceirczenka et al Soft Matter 2009;5(3):538‐550
; ( )
T=60min

Physical models of digestion are
Mimics Mimics of flow and mixing behaviour in the GI
tract
Addition Addition of digestive enzymes and pH adjustment
more like a “real” gut
Designed es g ed to dgest digest real ea foods oods aand dmeal‐sized ea s ed
portions
Easier to sample p than human volunteers
X Not necessarily validated against the human
situation
XNot adapted to analysis of small amounts of
material or purified p protein p

Modelling Digestion –Biochemical Biochemical Models
pH 2.5
I=0.15
Sh Shake, k 37ºC
Shake Shake, 37ºC
Sample over
time
pH 7.5
I=0.15
pH 6.5
I=0.15
Gastric mix:
Stop gastric
Duodenal mix:
Pepsin
digestion by Trypsin,
Phosphatidyl addition of chymotrypsin
choline h li vesicles il NOH NaOH Li Lipase/colipase / li
Amylase
Bile salts
Sample over
time
pH 6.5
I=0.15
Stop duodenal
digestion by
addition ofSBTI or
PMSF

Unfolding of proteins at gastric pH: Bet v 1
homologues unfold at low pH
AApi ig 1 partially illunfolds f ld at pH2.5 H2 5
BUT
Mal d 1 is completely unfolded
Sancho, et al 2011 Mol Nutr Food Res. 55(11):1690‐9.

Peach LTP is highly resistant to pepsinolysis
IIt has h many candidate did pepsin i cleavage l sites i but b iis
completely resistant to digestion in its native
folded form
Wijesinha‐Bettoni j et al 2010 Biochemistry. y 49(10):2130‐9. ( )

Duodenal digestion ‐ only 2 out of 14 tryptic and
chymotryptic are hydrolysed!
Chymotrypsin Trypsin yp
Three digestion products can be
Unreduced
identified – residues 1‐79 1 79, 1‐39 1 39 and
40‐79 which are only observed in
unreduced protein

Local side chain dynamics may explain preferential
cleavage of certain sites
Residues in cleavage sites are more mobile
This may allow more effective location of side
chains into protease specificity pockets

Biochemical models of digestion
Can Can be scaled down to analyse small amounts of
single proteins
Some Some collaborative trials published showing inter‐ inter
laboratory validation
Homogeneously o oge eous y mixed ed so sa sampling p g is s eas easier e
Can mimic the GI tract‐ a model system with
assumptions p and limitations [enzyme:substrate
[ y
ratios, pH titration, homogeneous mixing]
XNot well suited to analysis y of foods [sampling, [ p g,
soluble versus insoluble phases]

How to measure digestiblity?

SDS‐PAGE and IgE binding capacity:
Bet v 1 homologues susceptible to gastric
di digestion ti
MMass spectrometry t t mapping i of f digestion di ti products d t –
A way of profiling peptides
Cleavage patterns are similar for both proteins but Api g
1 is digested more slowly than Mal d 1
IE IgE epitopes it are ddestroyed t dbbut tdi digestion ti products d t can
still activate T‐cells
Sancho, et al 2011 Mol Nutr Food Res. 55(11):1690‐9.

Gastric Gast c digestion dgesto of o pea peanut ut Araa h 1 does not ot
alter its allergenic activity
PC
0 1 2 4 8 16 30 60 90 120 min
Ara h 1
Eiwegger, Rigby et al Clin Experimental Allergy 2006
releasee
is ta m in
% H
80
60
40
20
0
Ara h 1
Ara h1 Phase 1
Phase 1+2 Digestion Fluid P1
Digestion Fluid P1+2 Gastro‐duodenal
Gastric Gast c digestion dgesto
MMedium di
control
0001 0.001 001 0.01 01 0.1 1
Concentration (µg/ml)
di digestion i

Tests for resistance to gastroduodenal g digestion g
Validation is needed regarding
•Levels L l of f enzymes and d bi biosurfactants f [h [these
change with age, food composition]
•Standardisation S d di i of f mixing ii conditions di i
•Interlaboratory comparisons
•Agreed A d“ “outcome” ” measures (SDS (SDS‐PAGE, PAGE
mass spectrometry to monitor digestion of
polypeptides, l tid bi bioactivity ti it measurements t lik like
IgE binding, T‐cell reactivity)
NB –variation in outcomes of interlaboratory trials maybe
determined more by measurement and sampling than the
protocol per se!

Pepsin resistance test

1996: Astwood et al identify a correlation between
resistance to pepsin digestion and allergenicity [Nat
Biotechnol. 1996 Oct;14(10):1269‐73.]. This
•Posed the hypothesis ‘that food allergens must exhibit
sufficient gastric stability to reach the intestinal mucosa
where absorption and sensitization (development of
atopy) can occur.’
BUT
•The test is non physiological
•the pH is lower than found in vivo and changes
pepsin specificity,
•Pepsin is present in a gross excess which affects
kinetics of digestion

Is the pepsin resistance test more a biochemical
surrogate of “stability” than simulated gastric digestion?
•Pepsin, like other endoproteases (trypsin, chymotrypsin)
cleaves mobile, surface accessible sites on a substrate
•Resistance to pepsinolysis is a function of
•Resistance Resistance to low pH unfolding
•Polypeptide mobility at the cleavage site
•Resistance to pepsin maybe a surrogate measure of
endosomal processing involved in antigen presentation
•It may hhave validity ld as a correlative l test defined df dusing
panels of “allergens” and “non‐allergens” included in the
iintegrative i risk ikassessment approach
h

In vitro protein digestibility assays and their
relevance to the risk assessment
•Simulated Gastroduodenal Digestion provides
information relevant to understanding the context of
how a protein is presented to the immune system in a
physiologically relevant context
•The Pepsin Resistance Test is a distinctly different
biochemical test which provides complimentary
information on the biochemical stability of a protein
which may be predictive of allergenic potential
(of course if we had a good animal model we would
not have to rely on these tests so much!)

Manchester Team: Phil Johnson, Claire Eyers, Adnan Custovic,
Angela l Simpson, Mark k Travis, John h McLaughlan hl
IFR Team: Neil Rigby, Guisy Mandalari, Andrew Watson, Alan
MMackie ki
Past Team Members: Javier Moreno, Ana Sancho, Yuri
Al Alexeev, Emi E iVVassilopolou, il l JJohn h Jenkins J ki
Collaborators: Peter Shewry, Lorna Smith, Jean‐Michel Wal,
KKarine i ZZsolt ltSSzefalusi, f l i Nik Nikos PPapadopolous, d l BBarbara b Ballmer‐ Bll
Weber, Charlotte Madsen
Plus l numerous other h UK and d European collaborators! ll b !
Protall