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Jean-Luc Pellequer
Atomic Energy and Alternative Energies Co
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In growing need of obtaining highly specific monoclonal antibodies against novel proteins, we developed new
functions implemented in the program BEPITOPE to predict continuous protein epitopes. This program
not only can compute, combine, display and print prediction profiles, but also provides a list of suggested
linear peptides to be synthesized. Novel facilities incorporated in BEPITOPE include the treatment of a
whole genome, the search for a user-defined pattern, and the combination of prediction to pattern profiles.
This latter approach is useful to remove unwanted predictions such as those including glycosylation sites.
Copyright # 2003 John Wiley & Sons, Ltd.
Keywords: antigenic site; propensity scale; epitope prediction; hybridoma; monoclonal antibodies
INTRODUCTION
PROGRAM DESCRIPTION
Standard methods
Continuous epitopes in proteins share common properties. They are exposed on the surface, often localized in
loop regions that are usually flexible and composed of
polar residues. Therefore, an ideal epitope prediction
method must reflect these properties. A protocol of the
BEPITOPE program selects putative epitopes employing
five methods described below, including one standard
and four complex ones. This consensus prediction is
made by summing the frequency of an amino acid
predicted to be located in an epitope. A user can select
either one or all of these five methods in the final
consensus prediction.
Standard methods aim at predicting a given property
encoded into a propensity scale. The computation algorithms were described elsewhere (Pellequer et al., 1991).
The BEPITOPE program adapts more than 30 propensity
scales such that the positive values in the hydrophobicity
scale correspond to hydrophilic regions, and those in a
flexibility scale represent flexible regions, etc. Consequently, putative epitopes are indicated by peaks plotted
in the prediction profile generated by the BEPITOPE
program. Each putative epitope contains a sequence of 15
residues, centered around each peak of the profile selected
above a user-defined threshold. The three complex epitope
prediction methods in BEPITOPE are based on protein
flexibility (Karplus and Schulz, 1985), protein accessibility
(Emini et al., 1985), and turns in proteins (Pellequer et al.,
1993). We modified the last method to rank predicted
epitopes according to their hydrophilicity. It should be noted
that this method is now completely automated (Plate 1). The
fourth complex epitope prediction method aims to detect
amphipathic helices by calculating the hydrophobic
moment.
Plate 1.
Plate 2.
Pattern search
Proteins in eukaryotic cells often contain post-translational
modifications. These modifications often occur in loops that
possess similar properties as epitopes. Thus, to strengthen
the efficacy of epitope prediction, BEPITOPE produces
user-defined patterns. Once the profile of a pattern is
established, it can be combined with any epitope prediction
method mentioned above to remove unwanted predictions.
For instance, a user may want to delete all predicted
glycosylation sites from an epitope prediction (Plate 2).
BEPITOPE allows one to add, subtract, multiply and divide
prediction profiles. In addition, a profile can be accentuated
by a user-defined weight.
Patterns are produced by an expression generator. The
expression generator will create the appropriate number of
profiles, depending on the number of unspecified residues.
An expression contains elements enclosed in parentheses.
An element may contain an amino acid, a group of amino
acids, a code representing a physico-chemical property or a
range of residues. For example, the expression (LIV)(X23)(LIV) generates two profiles (LIV)(XX)(LIV) and
(LIV)(XXX)(LIV). BEPITOPE generates a profile window
of size n 20, where n is the number of residues in the
pattern. Fine tuning of the profile expression can be
performed by editing weights for each residue.
21
proteins, the selected peptides must be specific. A monoclonal antibody may cross-react with proteins present in an
organism that contains the protein of interest. The antibody
may also cross-react with proteins in the host that produces
monoclonal antibodies. To alleviate this problem, BEPITOPE identifies redundancy of all predicted epitopes using
substitution matrices in an entire genome (BLOSUM62,
PAM250, or any one present in the matrix directory). As a
result, one can choose a putative epitope that has the least
chance to cross-react with unwanted proteins. By default,
the cut-off value to identify similar peptides is set to an evalue of 10 7.
Genome strategy
The purpose of predicting continuous epitopes is to
synthesize peptides that will be used to raise monoclonal
antibodies. To reduce the cross-reactivity with unrelated
Acknowledgements
We thank Olivier Pible for valuable comments during this work.
Plate 1. Graphics interface of a complex turn prediction method. Top: a user can enter a sequence accession number corresponding to the
selected database on the right-hand side. The program will download the sequence from a remote database through the Internet. Below: a
sequence name can be typed or browsed from the local disk. Information about the sequence read is shown below including the sequence
length, rst residue number and last residue number. A propensity scale can be selected using the pull-down menu. Here, the scale
4turn33 is a combination of four classical turn33 propensity scales as developed in Pellequer et al. (1993). Five graphics icon on the right
correspond to the help menu, the reset button, the paste from clipboard button, the clear button, and the set-up button, respectively.
Below in the white window is displayed the selected protein sequence. Part of the sequence can be selected using the left mouse button.
Below the sequence window, computation parameters can be set. From left to right, a user can choose to add a smoothing procedure
(Gaussian by default), the width of the Gaussian distribution, the length of the window to compute scores, the central position were the
score will be assigned, and the type of arithmetical operation to combine the four predicted curves (addition or multiplication). On the
right, four graphics icons are used to start the computation, to zoom in/out in the result window, to save the prediction prole on disk, to
print the prediction prole, respectively. On the extreme right, a check box is used to obtain secondary structure prediction through the
NPS@ remote server (Combet et al., 2000). Results are as follows: a red square corresponds to helix; a blue square to strand; an olive
square to turns or coil; and a gray square is undetermined. The red curve corresponds to a turn prediction using the multiplication
approach whereas the blue curve corresponds to the addition approach. Predicted epitopes are shown on the left window with their
associated computed values as well as on the plot as shown with red triangles.
Plate 2. Removing putative glycosylation sites from an epitope prediction plot. A standard hydrophilicity curve of the renin protein is
shown in blue and the O-glycosylation motif detection curve in red. Subtraction of these two curves produces the green curve. It shows
that two hydrophilic peaks coincide with two putative glycosylation sites. Other permitted mathematical operators are shown on the top
right. Digits are used for increasing the weight of a particular plot. Sliding bars (in green and yellow) allow a user to align prediction
proles by shifting a smaller sequence along a larger one. Label messages appear on each graphical icon pointed by the mouse.
22
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