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The hydrolysis pattern of procasomorphin by gut proteases from plant parasite
Heliothis Zea determined by sequence analyses performed on the unfractionated
digestion mixtures
Pasquale Petrilli 1, Carlo Caporale 2 *and Carla Caruso 2
1 Istituto di Industrie Agrarie, Universita' di Napoli, Parco Gussone 80005 Portici, Italy.
2 Dipartimento di Agrobiologia e Agrochimica, Universita' della Tuscia, Via S. Camillo
de Lellis 01100 Viterbo, Italy.
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Abstract
The digestion pattern of procasomorphin, a putative precursor of ß-casomorphins (ßcasein-derived opioid peptides) by gut proteases from plant parasite insect Heliothis
Zea, has been determined to evaluate the possibility of introducing in plants the gene
encoding for this peptide in order to confer resistance to the insect damage. The method
we used is based on the possibility of deducing the sequence of the fragments
produced by proteases at different times of digestion by means of automatic Edman
degradation performed on the fragment mixtures without any purification step. This
approach can be considered of general utility in studying the digestion of peptides by a
mixture of proteases.
KEY WORDS: casomorphin, opioid peptides, plant parasites, sequence.
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Introduction
Protection of plants is a very important task and some approaches have been
proposed to defend plant against insect damage such as the introduction in the plant of
(a) genes encoding for proteinase inhibitors in order to affect the insect digestion
process (1-3) or (b) genes encoding for neuropeptides able to interfere with insect
metabolism (4). These approaches require the knowledge of the peptide digestion
process of the insect. In particular, in the second case, it is important to know to what
extent the neuropeptide is digested by gut proteases and the pathway of the
digestion. This may help in designing homologous peptides more resistant to the action
of proteases. Procasomorphin (5) is a putative precursor of casein-derived opioid
peptides (casomorphins) (6), which seem to have effects on the nervous system of
insects (7, Pennacchia et al., unpublished results). It consists of 10 amino acid residues,
while casomorphin-9 is a peptide of 9 amino acid residues, lacking the N-terminal valine
in comparison with procasomorphin. We determined the digestion pathway of
procasomorphin by gut proteases from the plant parasite insect Heliothis Zea, in order
to verify the possibility of using this peptide against pest attack.
The study of peptide digestion is usually achieved by the analysis of purified
fragments (5, 8) or by Fast Atom Bombardment Mass Spectrometry analysis
performed on the unfractionated fragment mixtures (9, 10). In the present case, we
used an alternative simple approach to study the digestion of procasomorphin. The
method does not require any purification step and is based upon the possibility of
deducing the sequence of the fragments produced at different times of digestion b y
means of automatic Edman degradation performed on the unfractionated fragment
mixtures. In fact, with the advent of the modern pulsed-liquid phase sequencers
equipped on-line with phenylthiohydantoine amino acid derivatives analysers, it is very
simple to identify several amino acid residues at each step of degradation. Due to the
optimized chemistry of the Edman reaction occurring on modern sequencers, the
sequence analysis of peptide mixtures can be currently used as a general strategy to
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resolve very quickly specific problems. For example, since 1968 Gray theorized that
this approach can be applied to the determination of the amino acid sequence of
proteins by using an appropriate algorithm working on the sequence data obtained from
peptide mixtures generated by different methods of protein hydrolysis [11]. In a more
general view, if the sequence of a protein (or peptide) is known, the automatic
sequence analysis of its fragment mixtures generated by one or more hydrolysis
methods can be used to complement or replace Fast Atom Bombardment Mass
spectrometry data in assessing protein features such as disulfide bridge localization,
post-translational modifications and sequence of homologous proteins. The problem
consists, from time to time, of designing the right strategy utilizing an algorithm suited for
interpreting data. In this paper we report an example of such strategy applied to the
determination of the hydrolysis pattern of a peptide by a mixture of proteases.
Materials and Methods
Extraction of proteases from Heliothis Zea gut
Guts from five insects Heliothis Zea were suspended in 2 ml of 0.5% ammonium
bicarbonate, pH 8.0, and gently stirred for 30 min. The supernatant obtained after
centrifugation at 10,000 rpm for 15 min at 4 °C was utilized to digest procasomorphin.
The supernatant was tested for tryptic activity to verify the presence of general
proteolytic enzymes. It contained about 33 µg/ml of trypsin-like activity measured
according to (12).
Digestion of procasomorphin
Procasomorphin (0.05 mg), prepared as described in (5), was incubated at 37 ° C
with an amount of protease mixture corresponding to 0.3 µg of trypsin-like activity in 4
ml of 0.5% ammonium bicarbonate, pH 8.0. Aliquots of the incubation mixture,
corresponding to 500 pmol of procasomorphin, were withdrawn at different times,
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acidified and freeze-dried. The freeze-dried samples were then dissolved in water (0.2
ml) and lyophilised twice.
Sequence analyses
Sequence analyses were performed by using a pulsed liquid-phase sequencer
(Applied Biosystems model 477A) equipped on-line with a phenylthiohydantoine
amino acid derivatives analyser (Applied Biosystems model 120A). Samples were
dissolved in aqueous 0.1% trifluoroacetic acid (20-30 µl) and loaded onto a trifluoroacetic
acid-treated glass-fiber filter, coated with polybrene and washed according to
manufacturer instructions. The sequencing reagents were from Applied Biosystems.
Results and Discussion
Procasomorphin was incubated with proteases extracted from gut of plant parasite
insect Heliothis Zea and aliquots of the incubation mixture withdrawn at different times
were submitted to automatic sequence analysis. In Table 1 are reported pmoles data of
all phenylthiohydantoine amino acid derivatives determined at each of ten steps of the
Edman degradation performed on the mixtures after 1h (Table 1a), 2h (Table 1b) and
3h (Table 1c) digestion. Pmoles data of the residues identified at each step are
boldfaced. In Fig. 1 are shown the sequence of procasomorphin as well as the amino
acids identified by the sequence analyses on the basis of the data reported in Table 1.
After 1 h incubation (Fig. 1a), the identification of the N-terminal valine and of two
additional N-terminal residues (Lys and Tyr) at the first step of degradation indicates the
hydrolysis of the peptide. The lysine residue can be explained by the action of a
carboxypeptidase,
while the tyrosine
residue
reveals
the presence
of
an
aminopeptidase removing the valine residue; in fact the sequence 2-9 of
procasomorphin, corresponding to the sequence 1-8 of casomorphin-9, was
completely identified as N-terminal sequence (steps 1-8). The simultaneous presence
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of the sequence 1-9 of procasomorphin (steps 1-9) indicates that the removal of the Nterminal valine was not complete. In particular, pmoles data in Table 1a show that the
fragment 2-9 was present in minor amount than the peptide 1-9, while the removal of
the C-terminal lysine was almost complete. This result suggests that the action of the
carboxypeptidase was more effective than the action of the aminopeptidase.
After 2 h digestion (Fig. 1b) the N-terminal sequence from Phe4 to Pro9 was inferred
(steps 1-6) in addition to the sequence 2-9. This result could indicate: i) the removal of
the tripeptide Val-Tyr-Pro from the intact peptide by an endopeptidase; ii) the
sequential removal of Val, Tyr, Pro residues by the aminopeptidase previously
observed; iii) the action of a dipeptidase removing the dipeptide Tyr-Pro from the
peptide 2-9. However, neither the sequence Val-Tyr-Pro (steps 1-3) nor the
appearance of proline at the first step of Edman degradation were observed, thus
indicating the action of a dipeptidase. Pmoles data in Table 1b indicate the complete
removal of the N-terminal valine by the aminopeptidase and show that the cleavage of
the dipeptide Tyr-Pro from the peptide 2-9 by the dipeptidase occurred on 50% of
molecules about, as the sequence from Phe4 to Pro9 was detectable in equal amount
at the steps 1-6 and 3-8.
The activity of the dipeptidase was confirmed by the results obtained after 3 h
digestion. In fact, the partial removal of the dipeptides Phe-Pro, Gly-Pro and Ile-Pro
from the peptide 4-9 was observed (Fig. 1c, steps 1-2) as well as the presence of the
peptide 6-9 (Fig. 1c, steps 1-4). Pmoles data in Table 1c indicate the almost complete
cleavage of the dipeptide Tyr-Pro from the peptide 2-9. The remaining peptide 4-9
was further partially digested originating both the dipeptide Phe-Pro and the peptide 69. Moreover, an aliquot of the peptide 6-9 was further digested originating the
dipeptides Gly-Pro and Ile-Pro. Finally, it should be noted that, due to the absence of
proline at the first step
of degradation after 3 h digestion (Fig. 1c), the
carboxypeptidase previously observed is not able to remove the C-terminal proline
from the produced fragments. Furthermore, the action of the dipeptidase is the most
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effective once the N-terminal valine has been removed by the action of the
aminopeptidase.
It can be pointed out that the dipeptidase which seems to be the major responsible
for procasomorphin degradation is a dipeptidyl aminopeptidase IV, since it cleaves XPRO dipeptides (8, 10). This enzyme is widely distributed in mammalian tissues and is
able to degrade ß-casomorphins (10, 13-16). Due to its presence in gut of Heliothis
Zea, it appears unlikely that casomorphin can reach concentrations of physiological
significance in the insect. In any case, procasomorphin could be more resistant in vivo
since its digestion requires the preliminary action of an aminopeptidase. This step might
slow down the subsequent degradation of casomorphin (peptide 2-9) by dipeptidyl
peptidase IV. As a fact, the complete removal of the N-terminal valine from
procasomorphin was detectable after 2h of digestion (Table 1b and Fig. 1b). We are
analyzing the possibility of introducing in plants the gene encoding for this peptide in
order to confer resistance to insect damage.
The approach presented here enabled us to identify the fragments produced from
procasomorphin hydrolysis without performing tedious and time-consuming purification
steps. This feature is commonly accepted as a prerogative of Fast Atom Bombardment
Mass spectrometry and let this technique have a great success in the last 10 years in
resolving various kind of problems regarding proteins and peptides [10, 17-22]. On the
contrary, even if some examples of sequencing more than one peptide simultaneously
have been reported in literature, the use of this approach as a systematic method was
proposed only in theory [11]. In this paper we show that automatic sequence analysis
of peptide mixtures can be a worthy alternative to mass spectrometry presenting some
advantages. In fact, it is difficult to detect by mass spectrometry small peptides such as
the dipeptides produced after 3 h incubation and identified by the Edman degradation
(Fig. 1c, steps 1-2); furthermore, the quantity of the produced fragments can b e
determined by pmoles data of the sequence analyses. The completeness of sequence
data allows to construct, from time to time, appropriate algorithms for optimizing the
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performance of a modern automatic sequencer, allowing this instrument to replace
successfully a mass spectrometer.
In conclusion, automatic sequence analysis of peptide mixtures revealed very useful
in studying the digestion pattern of peptides and may represent an example of
application of a more general idea.
Acknowledgments
This work was supported by a grant of "Progetto Finalizzato: Resistenze genetiche
delle piante agrarie agli stress abiotici e biotici" from Ministero Agricoltura e Foreste.
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* Corresponding author: Carlo Caporale, Dipartimento di Agrobiologia e Agrochimica,
Universita' della Tuscia, Via S. Camillo de Lellis 01100 Viterbo, Italy. FAX N. 0761357242