Project
Update - June 2001
Task 2: Identification of structures and activities common to plant food
allergens.
Why identify common structures and activities?
Several decision trees have been proposed to facilitate the
assessment of the allergenic risks posed by novel foods, including GMOs (Figure
1). These involve:
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Comparing the amino acid sequence of a candidate
transgene or novel food protein with known food allergens to identify
homologies.
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Determining the IgE-reactivity of the protein using sera
from individuals with a demonstrated reactivity to a homologous protein.
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Confirming a negative IgE-reactivity by skin-prick
testing in allergic humans
Whilst it was originally suggested that an IgE-reactive novel
protein should then be labelled as such, many biotechnology and food companies
have now adopted a stance which halts all further product development using the
gene. The use of allergic individuals in such risk assessment procedures is not
acceptable and hence the decision trees are in the process of being revised.
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| Figure 1: The decision tree developed for assessing the
allergenic potential of novel proteins originally proposed by Metcalfe and
co-workers (1996) |
One of the problems with protein sequence comparisons to
identify allergens is that they will only identify close homologues. One of the
tasks of Protall is engaged in is identifying structures and activities, which
are common to food allergens, using plant food allergens as an example. This
article describes our progress to date.
Plant protein allergens
There are three main types of plant protein, based on their
function within the plant. These are
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| Figure 2: Classification of seed proteins on the basis of
their solubility |
Many of the highly allergenic plant foods, such as peanuts,
are actually derived from seeds. Seed proteins have been classified on the basis
of their solubility, which does not necessarily represent molecular
relationships (see Figure 2). We have focused on two main superfamilies of plant
food allergens, which appear to sensitise individuals via the gastrointestinal
tract namely:
Prolamin superfamiliy: families of proteins belonging
to this superfamily include the storage prolamins of cereals, 2S albumins, a
-amylase/trypsin inhibitors and non-specific lipid-transfer proteins. Certain
proteins in all these families are allergens
Cupin superfamily: the families that belong to this
superfamily include the germins and the 11S and 7S storage globulins. Food
allergens are generally found in the storage globulin family, although there is
an instance a member of the germin family from bell peppers also being an
allergen.
The structural attributes common to plant food allergens will
be illustrated with examples from the prolamin and cupin superfamilies.
PROLAMIN SUPERFAMILY
The dendrogram in Figure 3 shows the relationship between the
various members of the prolamin superfamily, as defined by their amino acid
sequences.
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| Figure 3: Dendrogram showing the relationship between
albumins from a number of plant species |
Proteins known to be albumins were retrieved from the NIH
database. The translation of the nucleotide sequences and the alignment of the
protein sequences were obtained using the "Vector NTI" software
package that employs a ClustalW algorithm for sequence comparison. Those
proteins known to be plant food allergens are shown in boxes; The barley
inhibitor Hor v 1 is known to be a respiratory allergens but not as a
food allergen.
2S albumins
These proteins have a conserved cysteine skeleton, and are
generally synthesised as a single polypeptide which is cleaved to give a 3kD and
9kD subunits linked by disulphide bonds. A number of well characterised plant
food allergens belong to this family including the Brazil nut (Ber e 1)
and yellow mustard (Sin a 1) allergens. A model of the three dimensional
structure of Sin a 1 is shown in Figure 4.The proteins are rich in a
-helix and held together by a number of disulphide bonds. They can make up a
large proportion of seed protein, appear to be stable to proteolysis and can
bind lipids.
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| Figure 4: The three-dimensional homology model for the 2S albumin allergen
from yellow mustard Sin a 1 |
Non-specific lipid transfer proteins (LTPs).
The LTP family is made up of low molecular weight (7-9kDa)
monomeric proteins, which are very basic. They are able to catalyse the transfer
of lipids between vesicles and membranes in vitro and there is increasing
evidence that their role in vivo may be in cutin biosynthesis. The
proteins are made up of a bundle of four a -helices with a lipid-binding cavity
in the centre (see Figure 5). There are indications that bound lipid increases
resistance to proteolysis; these proteins also survive thermal treatments, and
can refold to their native structure on cooling.
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| Figure 5: A ribbon diagram of a model of the three-dimensional structure of
the non-specific lipid transfer protein from peach. (Pru p 3)
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The initial model was calculated using Swiss-Model (Guex and
Peitsch, 1997) and improved manually by including the disulphide bridges seen in
the homologous structures. The ribbon is coloured from blue at the N-terminus to
red at the C-terminus using ICMlite Version 2.7 (Abagyan et al., 1994).
Common properties of the 'α-class' of plant proteins
On the basis of the information on the 2S albumins and LTPs it can be seen
that they share a number of common structural features:
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Generally small with Mr of less than 30,000
daltons
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Contain a high proportion of cysteine residues and hence
are highly disulphide bonded
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Are compact and hence resistant to attack by proteolytic
enzymes
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Despite their compactness they contain a number of flexible
loops, which maybe important antibody recognition sites
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Many are able to bind lipid which can increase resistance
to proteolysis
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They are abundant in the seed. However, some allergenic
fruit LTPs are not abundant but are potent allergens perhaps because of their
exceptional stability to proteolysis.
Future discussions in the project will focus modifying this list to
accommodate information on other classes of plant allergens such as the globulin
storage proteins, which are major allergens in peanuts and soya. Identifying
common properties of allergens will pave the way for using bioinfomatics more
effectively for predicting the allergenic potential of novel proteins in the
first steps of the decision tree.
Contributors: ENC Mills1, MJC Alcocer2,
J Jenkins1, D Marion3, R I Monsalve4, PR Shewry5
1 Institute of Food Research, Norwich Laboratory, Norwich Research Park,
Colney, Norwich NR4 7UA, UK. (Partner 1)
2 School of Biological Sciences, University of Nottingham, Nottingham,
NG7 2RD, UK.
3 INRA-Laboratoire de Biochimie et Technologies des Proteines, BP
71627, Rue de la Geraudiere, Nantes 44316, CEDEX 03, France.(Partner 4)
4 Departamento de Bioquimica y Biologia Molecular, Faculdad de Quimica,
Universidad Complutense, Madrid 28040, Spain.(Partner 19)
5 IACR-Long Ashton Research Station, Department of Agricultural
Sciences, University of Bristol, Long Ashton, Bristol, BS18 9AF, UK.(Partner 3)
References
Abagyan, R.A., Totrov, M.M., and Kuznetsov, D.N. (1994). ICM - a new method
for protein modeling and design. Applications to docking and structure
prediction from the distorted native conformation. J.Comp.Chem. 15, 488-506.
Guex, N. and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer: An
environment for comparative protein modelling. Electrophoresis 18:2714-2723.
Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L.,
Fuchs R.L. (1996) Assessment of the allergenic potential of foods derived from
genetically engineered crop plants. Crit. Rev. Food Sci. Nutr. 36, S165-S186.
THE CUPINS SUPERFAMILY
What are cupins?
The cupin superfamily is comprises proteins possessing a
common b -barrel structure thought to originate in a prokaryotic ancestor. The
term ‘cupin' was derived from the Greek for ‘barrel', and relates to the
b -sheet barrel-like structure characteristic of these proteins. This motif is
found as a single domain in fungal spherulins, fern sporulins and the plant
germins and oxalate oxidases (Figure 6). There are also two domain cupins, the
globular storage proteins of plants termed legumins (11S) and euvicilins (7S).
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Figure 6: The evolution of the cupin superfamily |
Most of the food allergens belonging to the cupin superfamily that have been
characterised are primarily globulin storage proteins. This includes the
Legumins (11S): Soya glycinin, peanut arachin (Ara h)
Vicilins (7S): Soya b -conglycinin, peanut conarachin (Ara h 1),
walnut (Jug r1)
Structure of Seed Storage globulins
The 11S globulins are hexameric heteroligomeric proteins of
Mr ~ 360,000, each subunit comprising an acidic 30-40,000 dalton polypeptide
disulphide linked to a 20,000 dalton basic polypepide (Figure 2). Subunits are
generally coded by multiple genes and are synthesised as a single precursor
protein, which is postranslationally cleaved at a conserved protease site (Gly-Leu-Glu-Glu-Thr).
The 7S proteins are trimeric, of Mr ~ 180,000, made up of N-glycosylated 50,000
dalton subunits in some plant species; in others such as soya and peanut
subunits are present which have an 170 residue N-terminal insert (Figure 7).
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| Figure 7: Schematic diagram of the legumin-like 11S and
euvicilin-like 7S globulin subunits. |
Thermostability of globulins
The globulins are thermostable proteins, showing only partial
loss of secondary and tertiary structure at temperatures between 75-94ºC. Thus
differential scanning calorimetry (DSC) of the allergenic soya globulin b -conglycinin
shows the main thermal transition occurring at around 75° C. Similarly far-UV
CD analysis of the protein shows that whilst there is some loss of secondary
structure following heating a substantial amount of the b -structure remains.
These proteins also form large aggregates, many millions of Daltons in size,
following heating, which at high protein concentrations (5-10%) form heat-set
gels, an important property widely exploited by the food industry in adding soya
proteins to foods.
Proteolytic intermediates
On partial proteolysis the globulins retain much of their
quaternary structure, forming large proteolytic intermediates, which are held
together by non-covalent interactions. Thus peanut 7S globulin, Ara h 1, retains
both its trimeric structure and IgE reactivity following pepsin digestion. Soya
glycinin forms a stable intermediate of Mr ~ 280,000 on trypsinolysis known as
glycinin T and results from clipping of the acidic subunits to form a 13,000 and
a 16,000 molecular weight fragments.
Conclusions
Plant food allergens, which belong to the 11S and 7S globulin
families of the cupin superfamily share a number of common structural features
which may be important in predisposing them to becoming potent allergens. These
include
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Thermostable, able to form large aggregates and hence
able to retain multiple IgE-binding sites following cooking
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Ability to form large, stable proteolytic
intermediates, which also retain sufficient size to interact with the immune
system.
If you are interested in exploring plant protein families in
greater depth try visiting these websites:
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