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Studies on proteins in post-ecdysial nymphal cuticle of locust,
Locusta migratoria
, and cockroach,
Blaberus craniifer
Svend Olav Andersen
*August Krogh Institute, University of Copenhagen, 13 Universitetsparken, DK-2100 Copenhagen O, Denmark
Received 22 November 1999; received in revised form 28 January 2000; accepted 3 February 2000
Abstract
Proteins were extracted from the cuticle of mid-instar nymphs of locusts,Locusta migratoria, and cockroaches,Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers–Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein fromBombyx moripupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.2000 Elsevier Science Ltd. All rights reserved.
Keywords:Amino acid sequence; Cuticular proteins; Endocuticle; Mass spectrometry
1. Introduction
Insects produce a new cuticle every time they moult, and part of the new cuticle is deposited during a rela-tively brief period before ecdysis. Immediately after ecdysis this extracellular layer is expanded and some of its regions are sclerotised; the other regions remaining soft and pliable. Deposition of cuticular material often continues for an extended period after ecdysis, resulting in a layer of post-ecdysial cuticle which is thicker than the pre-ecdysial layer. The differences between post- and pre-ecdysial layers tend to be minor in soft, pliable cut-icles, as both layers stain similarly with histochemical stains, are soft and hydrated and have a similar ultra-structural architecture, characterised by helicoidally organised chitin filaments (Neville, 1975).
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E-mail address:[email protected] (S.O. Andersen).
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The differences between the two layers are more pro-nounced in stiff and solid types of cuticle, i.e. the scler-otised regions. Here the post-ecdysial layer stains differ-ently from the overlaying pre-ecdysial layer, and the chitin filaments in the two layers are often organised in different ways: the filaments in the pre-ecdysial layer are organised in a helicoidal pattern, the filaments in the post-ecdysial layer can occur either in a preferred unidi-rectional orientation or in alternating helicoidal and unidirectional orientations, where the unidirectional lay-ers all run in the same direction or have different direc-tions (Neville, 1975).
The pre- and post-ecdysial layers of soft, pliable cut-icles tend to have similar protein compositions, but a marked change in composition occurs during the late period of post-ecdysial protein deposition in a number of soft-bodied, last instar larvae, such asDrosophila mel-anogaster (Chihara et al., 1982), Manduca sexta
(Wolfgang and Riddiford, 1986) and Lucilia cuprina
drastic as the changes occurring in solid cuticle in con-nection with ecdysis.
Some types of solid cuticle show distinct differences in protein composition between the pre-ecdysial and the post-ecdysial layer. Roberts and Willis (1980) report an abrupt change in electrophoretic pattern of extractable proteins in both larvae, pupae and adults of the beetle,
Tenebrio molitor, in connection with ecdysis, and Lemo-ine et al. (1990) demonstrate by means of monoclonal antibodies that a change in deposition of cuticular pro-teins occurs in connection with adult ecdysis in Teneb-rio. Also, proteins specific for either the outer or the inner lamellar zone of the thin pupal cuticle of D. mel-anogaster were reported (Wolfgang et al., 1986), and pronounced changes occur at ecdysis in the electrophor-etic patterns of cuticular proteins from nymphs and adults of the locusts, Locusta migratoria and Schisto-cerca gregaria (Andersen and Højrup, 1987; Andersen, 1988), and of the cockroach, Blaberus craniifer(Jensen et al., 1997).
Amino acid sequences have been published for a num-ber of proteins derived from both pre- and post-ecdysial solid cuticles from several insect species (Andersen et al., 1995a; Bærnholdt and Andersen, 1998; Jensen et al., 1997; Kollberg et al., 1995; Lampe and Willis, 1994; Mathelin et al. 1995, 1998), but only in a few cases is it possible to compare pre- and post-ecdysial proteins from the same cuticular region and developmental stage of a given species. Five sequences have been reported for post-ecdysial proteins from pupal cuticle of T. moli-tor (Bærnholdt and Andersen, 1998; Mathelin et al., 1998), and nine sequences have been determined for pre-ecdysial pupal cuticle proteins from the same species (Andersen et al. 1995b, 1997; Haebel et al., 1995; Ron-dot et al., 1996). Two post-ecdysial proteins have been sequenced from cuticle of adult L.migratoria(Talbo et al., 1991; Jespersen et al., 1994) to be compared to 20 sequences of pre-ecdysial cuticular proteins from pharate adult locusts (Andersen et al., 1995a; Jensen et al., 1998; S.O. Anderson, unpublished data). Eight sequences have been reported for post-ecdysial proteins from the cuticle of the adult desert locust, Schistocerca gregaria
(Jespersen et al., 1994; Andersen, 1998); they are very similar to the corresponding proteins from adult L.
migratoria, but so far no pre-ecdysial proteins have been sequenced from S. gregaria.
Two post-ecdysial cuticular proteins have been sequenced from nymphs of L. migratoria (Nøhr et al., 1992) and three from nymphs of B.craniifer(Jensen et al., 1997), but none from pre-ecdysial cuticular proteins from such nymphs. Two-dimensional electrophoretic separation of the extractable proteins indicates that the pre-ecdysial cuticular proteins in locust nymphs are identical to the pre-ecdysial proteins in adults (Nøhr and Andersen, 1993); the patterns of pre-ecdysial cuticular proteins in nymphs and adults of Blaberus are similar,
but not completely identical (Jensen et al., 1997). Only a small part of the proteins in the extracts of the nymphal cuticles have been sequenced, and it is uncertain how representative they are for the post-ecdysial nymphal cuticles. To obtain a larger number of sequences for comparisons between different types of cuticle, five post-ecdysial proteins from nymphs ofB.craniiferand seven proteins from nymphs of L. migratoria have been pur-ified and sequenced.
2. Materials and methods
2.1. Purification of cuticular proteins
Nymphal cuticles were obtained from the laboratory stock cultures of L. migratoria and B. craniifer. Fifth instar nymphs were collected from both species 4–5 days after ecdysis and stored at218°C until a sufficient
num-ber of animals had been collected, about 100 locust nymph or ten cockroach nymphs.
Locust nymphal cuticle was prepared according to Nøhr et al. (1992) with slight modifications. After hom-ogenisation and washing in 1% K-tetraborate, the pro-teins were extracted from the cuticle by 6 M urea in 0.1% trifluoroacetic acid (TFA) and the extract was frac-tionated according to size by gel-permeation on a col-umn (2.6 cm×90 cm) of Sephacryl S200HR (Pharmacia-Biotech, Uppsala, Sweden). The fractions containing the low-molecular weight proteins were subjected to reverse phase high performance chromatography (RP-HPLC), and three proteins were obtained at sufficient purity for sequence studies. The fractions from the gel-permeation column containing the medium sized proteins were further purified on a cation exchange column (SP-Sepharose, 1.6 cm×15 cm, Pharmacia-Biotech, Uppsala, Sweden), pH 2, and were eluted by a linear gradient in 0–0.5 M ammonium chloride in 4 M urea in 0.1% TFA. Some of the major peaks were selected for further frac-tionation by RP-HPLC, and four proteins were purified for sequencing.
The abdominal cuticle from cockroach nymph was obtained by carefully peeling the cuticle from the still frozen abdominae; it was manually cleaned and washed briefly, first in 1% K-tetraborate and then in distilled water. The proteins were extracted from the cuticle by 6 M urea in 0.1% TFA, and the extract fractionated by cation exchange on SP-Sepharose as described pre-viously (Jensen et al., 1997). Proteins were purified for sequence studies by RP-HPLC of aliquots from selec-ted fractions.
2.2. Purification by RP-HPLC
Elution was performed with 0.1% TFA in ultrahigh-quality (UHQ) water (A-buffer) in combination with 90% acetonitrile in 0.08% TFA (B-buffer). To separate cuticular proteins the column was equilibrated with 20% B, during the first 2 min after application of the sample the concentration of B was increased from 20% to 30%, it was increased from 30% to 50% during the next 40 min and then kept constant at 50% for 3 min before being readjusted to 20%.
To separate peptides derived by proteolytic digestion of the proteins the same eluants were used with the elu-tion scheme: 0–10% B for 2 min, 10–50% for 40 min, constant at 50% for 3 min, and thereafter returned to 0% for reequilibration.
2.3. Enzymatic digestion
To obtain peptides of a reasonable size for sequence studies the proteins were digested with one or more of the following proteolytic enzymes: trypsin and endoprot-einase Glu-C from Promega (Madison, WI), chymotryp-sin and endoproteinase Asp-N from Boehringer (Mannheim, Germany). The digestions were performed as described previously (Andersen, 1998).
2.4. Mass spectrometry
Matrix-assisted laser ionisation mass spectrometry (MALDI-MS) was performed at the Institute of Molecu-lar Biology, Odense University, as described previously (Jensen et al., 1998).
Plasma desorption mass spectrometry (PDMS) was performed on a BioIon 20 time-of-flight mass spec-trometer (BioIon, Uppsala, Sweden). Peptides or small proteins were dissolved in 0.1% TFA to a concentration of approximately 50 pmol/µl, and 5µl applied to a nitro-cellulose covered target (Roepstorff, 1993). Spectra were acquired for 0.5–1×106 fission events at 15 kV
acceler-ation voltage and calibrated based on the H+ and Na+ ions.
2.5. Sequence determination
The intact proteins and selected peptides were sequenced by Edman degradation with an Applied Bios-ystems 476A Protein Sequencer. Degradation, conver-sion and identification of the liberated phenylisothiohyd-antoin amino acids were performed as described by the supplier.
2.6. Nomenclature
The Locusta and Blaberus proteins will be given a prefix, Lm and Bc, respectively, followed by the letters NCP for nymphal cuticular protein, and a number
denot-ing the molecular weight in kDa as calculated from the amino acid sequence.
3. Results
Urea-soluble proteins were extracted from fifth instar nymphs of B. craniifer andL.migratoria. Some of the extracted proteins were purified, and their amino acid sequences were determined by the combined use of mass spectrometry and Edman degradation. The proteins were chosen among the more abundant components in the two extracts and both small and medium size proteins were represented.
The first step in the procedure for sequence determi-nation was to obtain the mass of the individual proteins; plasma desorption mass spectrometry (PDMS) was used for the low-molecular weight proteins, and MALDI-MS for proteins with masses above 10 kDa. Table 1 com-pares the mass values obtained by mass spectrometry with the values obtained by sequence determination. The N-terminal sequences of the proteins, ranging from 35 to 50 residues, were obtained for most of the proteins by Edman degradation, but two of theBlaberusproteins (BcNCP14.9 and BcNCP21.1) appeared to be N-ter-minally blocked. Treatment of the proteins with pyroglu-tamine aminopeptidase removed the blocking group, and Edman degradation could be carried out, indicating that pyroglutamine is the N-terminal residue in these two proteins.
Samples of the proteins were digested by trypsin, and the tryptic peptides separated by RP-HPLC and sub-jected to mass determination by PDMS and Edman degradation. The sum of the masses of the tryptic pep-tides could in all cases account for the mass of the indi-vidual proteins, and good agreement was observed between the peptide masses determined by mass spec-trometry and those calculated from the sequences, indi-cating that no glycosylations or other secondary modifi-cations were present. A few tryptic peptides were too long to be sequenced completely, and it was necessary to subject them to further digestion by other proteolytic enzymes (chymotrypsin or endoproteinase Glu-C), and the new set of peptides could be sequenced completely. The intact proteins were also digested by chymotryp-sin or endoproteinase Glu-C to obtain peptides to estab-lish the sequential order of the tryptic peptides. The masses of the chymotryptic and Glu-C peptides were determined, and compared with the masses expected for peptides obtained by digestion of the tryptic peptides. By means of the sequences of the peptides, which according to their masses could represent overlaps between tryptic peptides, the complete amino acid sequences of the proteins were established.
Table 1
The molecular masses of nymphal, post-ecdysial cuticular proteins from B. craniiferand L. migratoriadetermined by mass spectrometry and calculated from the amino acid sequences together with the calculated isoelectric points
Protein Molecular mass according to: pI
Mass spectrometry Sequence
BcNCP3.8 3754.2 3753.3 9.3
BcNCP14.6 14592.9 14596.7 5.4
BcNCP14.9 14932.8 14930.5 5.3
BcNCP15.0 15043.8 15049.8 6.2
BcNCP21.1 21093.6 21085.5 8.7
LmNCP4.9 4921.3 4922.5 9.2
LmNCP5.1 5136.4 5142.7 9.2
LmNCP6.4 6446.0 6438.0 8.8
LmNCP9.5 9498.3 9505.6 9.1
LmNCP18.7 18733.0 18721.3 6.2
LmNCP19.8 19816.3 19815.3 6.1
LmNCP21.3 21329.8 21323.1 9.8
could only be determined unambiguously from residue 1 to residue 97, the remaining part of the sequence gave six closely related tryptic peptides, which contained neither glutamic acid nor aspartic acid, preventing the use of endoproteases Glu-C and Asp-N for establishing overlaps, and the content of tyrosine was so high that insufficient information could be obtained from chymo-tryptic digests. The problem was solved by incubating the protein with trypsin for 5 min at 0°C, resulting in partial digestion of the protein. The peptides from the partial digest were separated by RP-HPLC and their masses were determined by PDMS. Several of the pep-tides had masses corresponding to the sum of the masses of two or three of the peptides from the complete tryptic digest, and N-terminal sequence determination was used for deciding the order of tryptic peptides in the products from the partial digest.
The complete amino acid sequences were thus obtained for five proteins from post-ecdysial nymphal cuticle of B. craniifer (Fig. 1) and seven proteins from post-ecdysial nymphal cuticle of L. migratoria(Fig. 2). The cockroach proteins range in mass from 3.8 kDa to 21.1 kDa, and the locust proteins range from 4.9 kDa to 21.3 kDa. The masses and isoelectric points of the pro-teins as calculated by use of the GPMAW program (Lighthouse Data, Odense, Denmark) are shown in Table 1.
4. Discussion
Polyacrylamide gel electrophoresis and electrofocus-ing have shown that after ecdysis nymphs of both L.
migratoriaandB.craniiferdeposit a set of cuticular pro-teins different to the propro-teins deposited in the pharate cuticle before ecdysis (Nøhr and Andersen, 1993; Jensen et al., 1997). A few sequences of nymphal post-ecdysial
Fig. 1. Amino acid sequences for five proteins purified from extracts of abdominal cuticle from fifth instar nymphs ofBlaberus craniifer. N-terminal pyroglutamate is indicated by pQ. An 18-residue consensus sequence present in several cuticular proteins is indicated by double underlining.
cuticular proteins have been reported previously (Nøhr et al., 1992; Jensen et al., 1997), and a total of nine such proteins from locust nymphs and eight from cockroach nymphs have now been sequenced. According to electro-phoresis, none of the sequenced proteins are present in pharate cuticle from the same instar, indicating that they are not pre-ecdysial proteins which have escaped scler-otisation.
Fig. 2. Amino acid sequences for seven proteins purified from extracts of cuticle from fifth instar nymphs ofLocusta migratoria. An 18-residue consensus sequence present in several cuticular proteins is indicated by double underlining.
and Andersen, 1993; Jensen et al., 1997). The sequenced proteins appear to belong to the group of proteins which, according to two-dimensional electrophoresis, are com-mon to several regions (abdomen, pronotal shield, hind-leg femur, wing pads). Some of the analysed regions were completely free of arthrodial membranes, indicat-ing that the sequenced proteins are derived from the post-ecdysial layer of sclerotised cuticle. The sequenced proteins were purified from extracts of total cuticle in the case of locust nymphs, and from abdominal cuticle in the case of cockroach nymphs. Proteins from arthrodial membranes will be present in such extracts, but presum-ably in small amounts and they will not belong to the major components.
Comparing the nymphal sequences with each other shows common features in some of the sequences from the two species. Proteins BcNCP21.1 and LmNCP19.8 both contain a 68-residue sequence motif of the RR-2 type, known from a number of sequences of other cuticu-lar proteins (Andersen, 1998). The motif is characterised by containing a variant of the Rebers–Riddiford consen-sus sequence (Rebers and Riddiford, 1988) in its C-ter-minal region. The motif occurs in proteins derived from solid cuticle, such as pharate cuticle destined to be scler-otised. A related motif, the RR-1 motif, containing a per-fect Rebers–Riddiford consensus sequence, occurs in
proteins derived from soft cuticles (Andersen, 1998). The RR-2 motifs in BcNCP21.1 and LmNCP19.8 have identical residues in 41 positions out of 68, and most of the differences in the remaining positions represent conservative substitutions. Comparison of the RR-2 motifs in BcNCP21.1 and LmNCP19.8 with the corre-sponding regions in cuticular proteins from other insect species representing several orders (Orthoptera, Diptera, Coleoptera, and Lepidoptera), shows degrees of identity varying between 51% and 84% (Fig. 3). The proteins included in the comparison comprise only a small subset of the large group of cuticular proteins in which the RR-2 motif has been found.
Two of the Blaberus proteins, BcNCP14.9 and BcNCP15.0, contain a 75-residue region with significant sequence similarities (Fig. 4); a similar region has been described for a post-ecdysial cuticular protein, TmLPCP29, fromTenebriolarvae and pupae (Mathelin et al., 1998), for a protein, HaCP18.8, from the the exo-skeleton of the American lobster, Homarus americanus
(Nousiainen et al., 1998), and for a protein, AdACP15.5, from the spider, Araneus diadematus (Norup et al., 1996). This region also contains in its C-terminal end a variant of the Rebers–Riddiford consensus sequence, which differs in some respects from the variants present in the RR-1 and RR-2 proteins, but the differences between the three groups of proteins are most pro-nounced in the N-terminal parts of the conserved regions. I suggest naming the cuticular proteins with the 75-residue motif the RR-3 proteins, to indicate their relatedness to the RR-1 and RR-2 proteins. All five pro-teins belonging to the RR-3 group are derived from post-ecdysial cuticle.
Several of the post-ecdysial proteins fromLocustaand
Blaberusnymphs contain one or more copies of an 18-residue consensus motif, previously reported from a
Bombyx mori pupal cuticle protein, Bm-PCP (Nakato et al., 1990). TheBombyx protein contains three copies of the motif, one of the locust proteins (LmNCP18.7) con-tains four copies, two copies are present in BcNCP14.6, and a single copy is present in BcNCP14.9 and in BcNCP15.0. Two copies of the motif were reported present in BcNCP9.9 (formerly called Bc-NCP2) (Jensen et al., 1997), and a single copy is present in the post-ecdysial cuticular protein, TmLPCP29, from larvae and pupae of Tenebrio molitor(Mathelin et al., 1998). The motif has also been observed in two proteins (HaCP18.8 and HaCP20.2) extracted from the exoskeleton of the lobster, H.americanus (Nousiainen et al., 1998) and in one protein (CpCP18.76) from the crab,Cancer pagurus
(Andersen, 1999). The 18-residue motifs are aligned in Fig. 5, and it appears that their consensus sequence is: (P/V)xDTPEVAAA(K/R)AA(H/F)xAA(H/Y).
Fig. 3. Alignment of sequence regions in BcNCP21.1 and LmNCP19.8 to corresponding regions in six other proteins from insect cuticles. A (*) beneath the alignments indicates that the position is identical in all eight sequences. Residues identical in five or more of the sequences are indicated by double underlining. The Rebers–Riddiford consensus sequence is indicated above the alignments. The sequences are from: LmACP21 (Jensen et al., 1998); DmEDG84 (Apple and Fristrom, 1991); TmLCP-A1a (Andersen et al., 1997); TmACP20 (Charles et al., 1992); HcCP66 (Lampe and Willis, 1994); AgCP1 (della Torre et al., 1996). The numbers to the left and right of the sequences refer to the position of the aligned segments in the intact proteins.
Fig. 4. Alignment of sequence regions in BcNCP14.9 and BcNCP15.0 to corresponding regions in cuticular proteins fromTenebrio molitor
(Mathelin et al., 1998),Homarus americanus(Nousiainen et al., 1998), andAraneus diadematus(Norup et al., 1996). A (*) beneath the alignments indicates that the position is identical in all five sequences. Residues identical in three or more of the sequences are indicated by double underlining. The Rebers–Riddiford consensus sequence is indicated above the alignments. Gaps (–) have been introduced in some of the sequences to optimize the alignments. The numbers to the left and right of the sequences refer to the position of the aligned segments in the intact proteins.
Fig. 5. Alignment of 18-residue motifs in various arthropod cuticular proteins. A (*) beneath the alignments indicates that the position is identical in all 16 motifs, while underlining indicates identity in six or more of the segments. The sequences are from: BcNCP9.9 (Jensen et al., 1997); Bm-PCP (Nakato et al., 1990); TmLPCP29 (Mathelin et al., 1998); HaCP18.8 and HaCP20.2 (Nousiainen et al., 1998); CpCP18.76 (Andersen, 1999). The numbers to the left and right of the sequences refer to the position of the aligned segments in the intact proteins.
suggested that it might be involved in protein–protein or protein–chitin interactions.
Several of the post-ecdysial proteins from the nym-phal cuticles are also characterised by long stretches of alanine, valine and proline, a feature they have in com-mon with pharate cuticular proteins from some insect species. The short motif, AAPA/V, abundant in pharate proteins from adult locusts and Tenebrio larvae and pupae (Andersen et al. 1995a, 1997) occurs several times in LmNCP19.8, and a single copy is present in LmNCP4.9 and LmNCP5.1, whereas it is not found in any of the cockroach proteins.
LmNCP6.4 is remarkable for its high content of gly-cine and tyrosine, which together account for more than 60% of the sequence. The complete sequence resembles an enlarged version of the N-terminal region of many of the pharate cuticular proteins from adult locusts (Andersen et al., 1993). A glycine–tyrosine rich protein has also been described from Drosophila pupal cuticle (Apple and Fristrom, 1991). A rather unusual compo-sition is found in LmNCP9.5, where the dominating amino acids are valine (22.2%), glutamine (14.4%), ala-nine and proline (each 12.2%), and tyrosine (11.1%).
So far, only a relatively small number of proteins have been sequenced from post-ecdysial, solid cuticles, and generalisations can only be tentative. The post-ecdysial proteins from nymphs of Blaberusand Locustaas well as those from pupae of Tenebrio (Bærnholdt and And-ersen, 1998; Mathelin et al., 1998) resemble the proteins from pre-ecdysial cuticle from adult locusts and Teneb-rio larvae/pupae in being mainly hydrophobic; some of them contain the RR-2 motif and others are rich in ala-nine, valine and proline. In these respects they differ from the post-ecdysial proteins from adult locusts, which resemble the proteins from soft, flexible cuticles in being hydrophilic, containing the RR-1 motif and not being enriched in alanine and valine (Talbo et al., 1991; And-ersen, 1998).
A model has recently been suggested for the molecu-lar organisation of arthropod cuticles (Andersen, 1999). According to the model, cuticular proteins containing RR-1 or RR-2 regions are bound to the chitin filament system via the consensus motifs, and the N- and C-ter-minal regions are folded into the space between the chi-tin filaments. Iconomidou et al. (1999) have recently pro-vided evidence indicating that both RR-1 and RR-2 regions will tend to form ß-pleated sheets which presum-ably will have a pronounced tendency to bind to chitin, in agreement with the above mentioned model. It appears that only proteins containing the RR-1 region are present in soft cuticles, such as dipteran and lepidopteran larval cuticles, adult locust post-ecdysial cuticle, and crustacean arthrodial membranes, and the interfilament space is accordingly occupied by the terminal regions of the chitin-bound proteins plus some water.
Solid cuticles contain a number of proteins which lack an RR-2 region. These proteins are assumed not to be bound directly to chitin, but to be located in the inter-filament space, where they constitute the main part of the matrix bulk phase (Fig. 6). The proteins interact hydrophobically with each other and with the terminal regions of the RR-2 proteins. Some, but not much water will also be present in the interfilament space, allowing a certain mobility and flexibility of the protein chains. It is envisaged that interactions between neighbouring proteins are as strong or stronger than interactions between chain segments in the same protein molecule, so that when external forces are applied to the cuticles
Fig. 6. Model proposed for the molecular arrangement of chitin and proteins in arthropod solid cuticles. Chitin filaments are indicated as broad, grey bands, and the proteins are shown as irregularly kinked chains, some of which are attached to the chitin filaments, while others are free in the interfilament space. Water molecules are indicated by small circles. The figure is a redrawn version of the model shown in Andersen (1999).
the protein chains will be stretched and unfolded rather than sliding past each other and along the chitin frame-work.
A post-ecdysial cuticular layer constructed according to this model would be expected to be relatively stiff, due to a low water content, but not as stiff as the overly-ing sclerotised cuticle. Duroverly-ing bendoverly-ing the inner post-ecdysial layer will become compressed, while the outer, sclerotised pre-ecdysial layer will resist stretching. The pronounced differences between the post-ecdysial cuticular proteins from nymphs ofLocustaandBlaberus, resembling the pharate proteins from solid cuticle, and the post-ecdysial proteins from cuticle of adult locusts, which resemble the proteins from soft cuticles, indicate fundamental functional differences between these two types of post-ecdysial cuticles. One difference which might be of importance is that the mechanical demands on cuticle of adult insects during flight are much more severe than the demands upon cuticle of the non-flying stages, and the thorax cuticle, in particular, is exposed during flight to very strong deforming forces. Only a small amount of protein can be extracted from the thorax and leg cuticle of adult locusts, due to a significant degree of sclerotisation of both pre- and post-ecdysial proteins. This suggests that the properties of a post-ecdysial cuticle containing hydrophilic and sclerotised proteins may be better suited for withstanding strong deforming forces, than is a material held together mainly by hydrophobic interactions.
post-ecdysial cuticle can be allowed to be indigestible, as it has to last for the rest of the life of the animal. To be easily degradable, the nymphal post-ecdysial cuticle should not be sclerotised, and it has been reported that sclerotisation of locust nymphal cuticle occurs only dur-ing the first day after ecdysis, the proteins deposited thereafter are readily extracted (Andersen, 1973). It therefore makes sense that proteins in the nymphal post-ecdysial cuticle have to resemble pharate cuticular pro-teins in being hydrophobic and rich in alanine, valine and proline, to give the material sufficient stiffness with-out sclerotisation. More detailed characterisation of both pre- and post-ecdysial cuticular proteins will show whether these suggestions are valid for other insect spec-ies.
The sequence data reported in this paper have been deposited in the SWISS-PROT protein sequence datab-ase with the following accession numbers: BcNCP14.6: P82118; BcNCP14.9: P82119; BcNCP15.0: P82120; BcNCP21.1: P82121; BcNCP3.8: P82122; LmNCP18.7: P82165; LmNCP19.8: P82166; LmNCP21.3: P82167; LmNCP4.9: P82168; LmNCP5.1: P82169; LmNCP6.4: P82170; LmNCP9.5: P82171.
Acknowledgements
Thanks are due to Lene Skou for obtaining MALDI mass spectra and to Dorthe Nielsen for excellent techni-cal assistance. Economic support from the Danish Natu-ral Science Foundation, the Novo Nordisk Foundation and the Carlsberg Foundation is gratefully acknowl-edged.
References
Andersen, J.S., Andersen, S.O., Højrup, P., Roepstorff, P., 1993. Pri-mary structure of a 14 kDa basic structural protein (Lm-76) from the cuticle of the migratory locust, Locusta migratoria. Insect Biochem. Mol. Biol. 23, 391–402.
Andersen, S.O., 1973. Comparison between the sclerotization of adult and larval cuticle inSchistocerca gregaria. J. Insect Physiol. 19, 1603–1614.
Andersen, S.O., 1988. Comparison between cuticular proteins from two locust species,Locusta migratoriaandSchistocerca gregaria. Insect Biochem. 18, 751–760.
Andersen, S.O., 1998. Amino acid sequence studies on endocuticular proteins from the desert locust, Schistocerca gregaria. Insect Biochem. Mol. Biol. 28, 421–434.
Andersen, S.O., 1999. Exoskeletal proteins from the crab,Cancer pag-urus. Comp. Biochem. Physiol. 123A, 203–211.
Andersen, S.O., Højrup, P., 1987. Extractable proteins from abdominal cuticle of sexually mature locusts, Locusta migratoria. Insect Biochem. 17, 45–51.
Andersen, S.O., Højrup, P., Roepstorff, P., 1995a. Insect cuticular pro-teins. Insect Biochem. Mol. Biol. 25, 153–176.
Andersen, S.O., Rafn, K., Krogh, T.N., Højrup, P., Roepstorff, P., 1995b. Comparison of larval and pupal cuticular proteins in Teneb-rio molitor. Insect Biochem. Mol. Biol. 25, 177–187.
Andersen, S.O., Rafn, K., Roepstorff, P., 1997. Sequence studies of proteins from larval and pupal cuticle of the yellow meal worm,
Tenebrio molitor. Insect Biochem. Mol. Biol. 27, 121–131. Apple, R.T., Fristrom, J.W., 1991. 20-hydroxyecdysone is required for,
and negatively regulates, transcription ofDrosophilapupal cuticle protein genes. Dev. Biol. 146, 569–582.
Bærnholdt, D., Andersen, S.O., 1998. Sequence studies on post-ecdys-ial cuticular proteins from pupae of the yellow mealworm,Tenebrio molitor. Insect Biochem. Mol. Biol. 28, 517–526.
Charles, J.-P., Bouhin, H., Quennedey, B., Courrent, A., Delachambre, J., 1992. cDNA cloning and deduced amino acid sequence of a major, glycine-rich cuticular protein from the coleopteranTenebrio molitor. Temporal amd spatial distribution of the transcript during metamorphosis. Eur. J. Biochem. 206, 813–819.
Chihara, C.J., Silvert, D.J., Fristrom, J.W., 1982. The cuticle proteins of Drosophila melanogaster: stage specificity. Dev. Biol. 89, 379–388.
Della Torre, A., Favia, G., Mariooti, G., Coluzzi, M., Mathiopoulos, K., 1996. Physical map of the malaria vector,Anopheles gambiae. Genetics 143, 1307–1311.
Haebel, S., Jensen, C., Andersen, S.O., Roepstorff, P., 1995. Isoforms of a cuticular protein from larvae of a meal beetle,Tenebrio moli-tor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electro-phoresis. Protein Sci. 4, 394–404.
Iconomidou, V.A., Willis, J.H., Hamodrakas, S.J., 1999. Is ß-pleated sheet the molecular conformation which dictates formation of heli-coidal cuticle? Insect Biochem. Mol. Biol. 29, 285–292. Jensen, C., Andersen, S.O., Roepstorff, P., 1998. Primary structure of
two major cuticular proteins from the migratory locust, Locusta migratoria, and their identification in polyacrylamide gels by mass spectrometry. Biochim. Biophys. Acta 1429, 151–162.
Jensen, U.G., Rothmann, A., Skou, L., Andersen, S.O., Roepstorff, P., Højrup, P., 1997. Cuticular proteins from the giant cockroach,
Blaberus craniifer. Insect Biochem. Mol. Biol. 27, 109–120. Jespersen, S., Højrup, P., Andersen, S.O., Roepstorff, P., 1994. The
primary structure of an endocuticle protein from two locust species
Locusta migratoria and Schistocerca gregaria determined by a combination of mass spectrometry and automatic Edman degra-dation. Comp. Biochem. Physiol. 109B, 125–138.
Kollberg, U., Obermaier, B., Hirsch, H., Kelber, G., Wolbert, P., 1995. Expression cloning and characterization of a pupal cuticle protein cDNA ofGalleria mellonellaL. (1995). Insect Biochem Mol. Biol. 25, 355–363.
Lampe, D.J., Willis, J.H., 1994. Characterization of a cDNA and gene encoding a cuticular protein from rigid cuticles of the giant silk-moth, Hyalophora cecropia. Insect Biochem. Mol. Biol. 24, 419–435.
Lemoine, A., Millot, C., Curie, G., Delachambre, J., 1990. Spatial and temporal variations in cuticle proteins as revealed by monoclonal antibodies, immunoblotting analysis and ultrastructural immunolo-calization in a beetle,Tenebrio molitor. Tissue and Cell 22, 177– 189.
Mathelin, J., Bouhin, H., Quennedey, B., Courrent, A., Delachambre, J., 1995. Identification, sequence and mRNA expression pattern during metamorphosis of a cDNA encoding a glycine-rich cuticular protein inTenebrio molitor. Gene 156, 259–264.
Mathelin, J., Quennedey, B., Bouhin, H., Delachambre, J., 1998. Characterization of two new cuticular genes specifically expressed during the post-ecdysial molting period inTenebrio molitor. Gene 211, 351–359.
Nakato, H., Toriyama, M., Izumi, S., Tomino, S., 1990. Structure and expression of a mRNA for a pupal cuticle protein of the silkworm,
Bombyx mori. Insect Biochem. 20, 667–678.
Neville, A.C., 1975. Biology of the Arthropod Cuticle. Springer, Berlin.
Purification and characterization of five cuticular proteins from the spiderAraneus diadematus. Insect Biochem. Mol. Biol. 26, 907– 915.
Nousiainen, M., Rafn, K., Skou, L., Roepstorff, P., Andersen, S.O., 1998. Characterization of exoskeletal proteins from the American lobster, Homarus americanus. Comp. Biochem. Physiol. 119, 189–199.
Nøhr, C., Højrup, P., Andersen, S.O., 1992. Primary structure of two low molecular weight proteins isolated from cuticle of fifth instar nymphs of the migratory locust, Locusta migratoria. Insect Biochem. Mol. Biol. 22, 19–24.
Nøhr, C., Andersen, S.O., 1993. Cuticular proteins from fifth instar nymphs of the migratory locust, Locusta migratoria. Insect Biochem. Mol. Biol. 23, 521–531.
Rebers, J.E., Riddiford, L.M., 1988. Structure and expression of a
Manduca sextalarval cuticle gene homologous toDrosophila cuti-cle genes. J. Mol. Biol. 203, 411–423.
Roberts, P.E., Willis, J.H., 1980. The cuticular proteins ofTenebrio molitor. I. Electrophoretic banding patterns during postembryonic development. Dev. Biol. 75, 59–69.
Roepstorff, P., 1993. 252-Californium plasma desorption time-of-flight mass spectrometry of peptides and proteins. Methods Mol. Biol. 17, 229–235.
Rondot, I., Quennedey, B., Courrent, A., Lemoine, A., Delachambre, J., 1996. Cloning and sequencing of a cDNA encoding a larval– pupal-specific cuticular protein in Tenebrio molitor (Insecta, Coleoptera). Developmental expression and effect of a juvenile hor-mone analogue. Eur. J. Biochem. 235, 138–143.
Skelly, P.J., Howells, A.J., 1987. Larval cuticular proteins ofLucilia cuprina. Electrophoretic separation, quantification and develop-mental changes. Insect Biochem. 17, 625–633.
Talbo, G., Højrup, P., Rahbek-Nielsen, H., Andersen, S.O., Roepstorff, P., 1991. Determination of the covalent structure of an N- and C-terminally blocked glycoprotein from endocuticle of Locusta migratoria. Combined use of plasma desorption mass spectrometry and Edman degradation to study post-translationally modified pro-teins. Eur. J. Biochem. 195, 495–504.
Wolfgang, W.J., Fristrom, D., Fristrom, J.W., 1986. The pupal cuticle of Drosophila: differential ultrastructural immunolocalization of cuticle proteins. J. Cell Biol. 102, 306–311.
