From the Commonwealth Scientific and Industrial
Research Organization Livestock Industries, Molecular Animal
Genetics Centre and the ¶ Institute of Molecular Biosciences,
Level 8, Gehrmann Laboratories, The University of Queensland,
St. Lucia, Queensland, 4072, Australia
Received for publication, October 16, 2000, and in revised form, January 2, 2001
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ABSTRACT |
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The peritrophic matrix is a prominent feature of
the digestive tract of most insects, but its function, formation, and
even its composition remain contentious. This matrix is a molecular sieve whose toughness and elasticity are generated by glycoproteins, proteoglycans, and chitin fibrils. We now describe a small, highly conserved protein, peritrophin-15, which is an abundant component of
the larval peritrophic matrices of the Old World screwworm fly,
Chrysomya bezziana, and sheep blowfly, Lucilia
cuprina. Their deduced amino acid sequences code for a 8-kDa
secreted protein characterized by a highly conserved and novel register
of six cysteines. Two Drosophila homologues have also been
identified from unannotated genomic sequences. Recombinant
peritrophin-15 binds strongly and specifically to chitin; however, the
stoichiometry of binding is low (1:10,000 N-acetyl
glucosamine). We propose that peritrophin-15 caps the ends of the
chitin polymer. Immunogold studies localized peritrophin-15 to the
peritrophic matrix and specific vesicles in cells of the cardia, the
small organ of the foregut responsible for peritrophic matrix
synthesis. The vesicular contents are disgorged at the base of
microvilli underlying the newly formed peritrophic matrix. This is the
first time that the process of synthesis and integration of a
peritrophic matrix protein into the nascent peritrophic matrix has been observed.
The cellular constituents of the intestinal systems of higher
organisms are protected from deleterious endogenous and ingested exogenous substances by mucus secretions. In insects and several other
phyla, a more elaborate secretion, the peritrophic matrix (PM),1 assumes this
protective role. It is also likely that the PM has other tasks: as a
molecular sieve of partially digested protein and carbohydrate, as a
scaffold for proteases, peptidases, and glycosidases, as a sink for
toxic substances, and as a barrier to ingested pathogens. There are two
quite different forms of PM: type 1, which is synthesized and secreted
by the midgut cells, usually in response to ingestion of a meal; and
type 2, which is constitutively synthesized, secreted, and extruded
into the gut by a small specialized organ known as the cardia typically located at the junction of the foregut and midgut (1). PM can be
present in both the larval and adult stages of most insects, although
different stages may produce a different class of PM.
The PM consists of a chitin fibril mesh embedded with glycoproteins and
proteoglycans. The assembly of these components imbue the PM with its
physical properties of semi-permeability, strength, and elasticity.
Although the architecture of the chitin meshwork is evident from
electron microscopy studies, few of the proteins have been studied in
any detail. Recent molecular studies on this unique structure have been
conducted on mosquitoes and the fly, Lucilia cuprina. The PM
is a potential target for vaccines or insecticides and may play various
roles in the transmission of disease (2-4).
The peritrophins are a group of proteins tightly associated with the
PM, either binding to chitin fibrils or possibly to other peritrophins
(5). Generally, the peritrophins can only be solubilized from the PM
with strong denaturants. Typically, these proteins are heavily
glycosylated and cysteine-rich, the latter characteristic reflecting
extensive intramolecular disulfide bonding. The sizes of these proteins
vary greatly. Although the sequences of five peritrophins from a number
of insect species have been published, very little is known of their
function within the PM. Three peritrophins, peritrophin-44 (L. cuprina larvae (6)), Ag-Aper-1 (Anopheles gambiae (7)),
and IIM (Trichoplusia ni (8)) have been shown to bind chitin
in vitro. These proteins contain multiple nonidentical copies of an approximate 65-amino acid domain characterized by a
specific register of 6 cysteines called the peritrophin-A domain (5)
but are otherwise unrelated and vary in size from 15.2 to 400 kDa.
We now report the identification and functional characterization of a
novel chitin-binding protein, peritrophin-15, that is a fundamental
component of the core complex of protein present in type 2 PM. A model
is presented for the biological role of this protein within PM. In
addition, the mode of transport and secretion of this protein out of
the cells of the cardia, where it is synthesized, is also demonstrated.
Insect Cultures and Collection of Larval PMs--
Larval and
adult L. cuprina were collected from an in
vitro culture maintained at the Long Pocket Laboratories,
Indooroopilly, Queensland, Australia. Adult and larval Chrysomya
bezziana were collected from an in vitro culture
maintained at the Research Institute of Veterinary Science, Bogor,
Indonesia. Larval PMs for both species of fly were harvested from
overnight cultures of third instar larvae (9-10). Briefly, third
instar larvae are obtained from a 72-h culture of larvae in PBS
supplemented with 40% newborn calf serum (Commonwealth Serum
Laboratories, Melbourne, Australia), 2% yeast extract (Sigma
Y-0373), and 0.2 mg/ml gentamycin sulfate (Sigma) and inoculated with
surface-sterilized freshly laid eggs. Third instar larvae collected
from this culture were placed into 150 ml of PBS containing 2.5 mM benzamidine and 5 mM EDTA. After 18-24 h at
37 °C (with ventilation), the larvae were removed, and the culture
medium was decanted and clarified (6,000 × g, 20 min,
4 °C). The pellets containing fragments of excreted PM were washed
once in PBS containing 2.5 mM benzamidine and stored at
Isolation of Cb-peritrophin-15--
The extraction of proteins
from PM was facilitated by disruption and progressive extraction by
homogenization and washing with various surfactants and chaotropic
agents of increasing solubilization capacity, as previously described
for the extraction of proteins from the larval PM of L. cuprina (5, 6, 11). Briefly, 8.5 g (wet weight) of C. bezziana larval PM was sequentially extracted by homogenization
(4 °C) in 40 ml (final volume) of the following solutions: water (5 min); 100 mM Tris-HCl, pH 7.2, containing 140 mM NaCl and protease inhibitors (4 mM EDTA, 50 µM aminoethylbenzylsulfonyl fluoride, and Complete®
protease inhibitor (Roche Molecular Biochemicals)) (30 min); 2%
Zwittergent 3-14 in 20 mM Tris-HCl, pH 7.2, 140 mM NaCl (TBS) containing protease inhibitors (2 h); 6 M urea in TBS containing protease inhibitors (16 h); and
finally, 6 M guanidine-HCl in TBS containing protease
inhibitors (16 h). After each extraction, homogenates were clarified
(48,000 × g, 30 min, 4 °C), and the pellet was
resuspended in the next extraction solution. The final pellet after
this extensive extraction was retained. Half of the guanidine-HCl-insoluble pellet was washed twice with MilliQ water and
twice in 50 mM Tris-HCl, pH 7.5. After each wash, the
suspension was pelleted by centrifugation (14,000 × g,
15 min, room temperature (RT)). The washed pellet was finally
resuspended in 2 ml 50 mM Tris-HCl, pH 7.5, containing 10 mM dithiothreitol and 5% SDS, heated for 30 min at
95 °C, and then centrifuged (14,000 × g, 15 min,
RT). The supernatant (1.3 ml), termed the SDS-soluble extract,
contained Cb-peritrophin-15.
Isolation of Lc-peritrophin-15--
After the same extraction
strategy described above, L. cuprina larval PM (1 g dry
weight) was homogenized and progressively washed by resuspension and
centrifugation (50,000 × g, 30 min, 4 °C) with 40 ml each of the following solutions: water; 100 mM Tris-HCl,
pH 7.5, containing 150 mM NaCl, 5 mM EDTA, 5 mM benzamidine, and 100 µM
phenylmethylsulfonyl fluoride (PMSF); 2% Zwittergent 3-14 in TBS
containing 100 µM PMSF; and TBS containing 6 M urea and 100 µM PMSF. The remaining residue
was then extracted with 40 ml of TBS containing 6 M
guanidine-HCl and 100 µM PMSF. This extract was
concentrated, dialyzed against TBS containing 8 M urea and
100 µM PMSF, and subjected to gel permeation
chromatography (0.5 ml/min) on a Superose 12 column (1.6 cm × 50 cm; Amersham Pharmacia Biotech) equilibrated with the same buffer.
Collected fractions were examined by reducing SDS-PAGE with Coomassie
Blue staining. A distinct peak of protein was eluted near the bed
volume of the column, and this contained a single major protein
(Lc-peritrophin-15) of Mr Peptide Purification and Amino Acid Sequencing--
A 400-µl
aliquot of the SDS-soluble extract of C. bezziana larval PM
was precipitated with methanol and then resuspended in SDS-PAGE-reducing sample buffer. The protein sample was resolved by
SDS-PAGE on a 12% analytical gel and visualized by staining for 1 h in 0.2% Coomassie Brilliant Blue R-250 (Bio-Rad) in 25% methanol,
10% acetic acid in water. The gel was twice destained overnight in
25% methanol, 10% acetic acid in water. A highly abundant protein of
Mr Cloning and Sequencing of Cb-peritrophin-15--
Redundant
primers were designed from the amino-terminal sequence of
Cb-peritrophin-15 and the amino acid sequence of peptide SDS1517.
The DNA sequences of the forward and reverse primers were
5'-GA(T/C)CCNGA(T/C)GGNAA(T/C)AA(T/C)CA(A/G)CC-3' and
5'-C(T/C)TCNGG(A/G)CANGG (T/C)TCNGTCCA-3', respectively.
cDNA was produced from a pool of C. bezziana larvae
at various instar stages as previously described (14). Polymerase chain
reaction was conducted in 100-µl volumes containing 3 mM
MgCl2, 0.25 mM of each dNTP, 100 pmol of each primer, 2.5 units of Taq DNA polymerase (Promega), and 5 ng
of C. bezziana larval cDNA through 40 cycles: 94 °C,
2 min; 58 °C, 1 min; 72 °C, 4 min. A dominant fragment of 213 bp
was purified and ligated into pGEM-T (Promega) for sequencing. The
translated sequence contained an open reading frame that included the
sequence of another peptide, SDS1506, as well as appropriate extensions of the amino-terminal sequence and peptide SDS1517. The 160-bp product
was digoxigenin-labeled (Roche Molecular Biochemicals) and used
to probe a first instar C. bezziana larval cDNA library constructed in Sequence of Lc-peritrophin-15--
A polymerase chain reaction
was conducted on third instar L. cuprina larval cDNA
(16) using the redundant forward and reverse primers described above
through 40 thermocycles: 94 °C, 1 min; 60 °C, 1 min; 72 °C, 1 min. A major product of 210 bp was gel-purified, ligated into pGEMT
(Promega), and sequenced on both strands. The sequence contained a
single open reading frame that contained the amino-terminal sequence
and all the peptide sequences derived from native
Lc-peritrophin-15.
Expression and Purification of Recombinant
Hexahis-Cb-peritrophin-15--
The recombinant protein,
hexa-His-Cb-peritrophin-15 (hhCb15), was expressed using the pQE9
vector (Qiagen) according to the manufacturer's instructions. Only the
DNA encoding the mature protein was used. HhCb15 was expressed as a
soluble protein and purified by nickel nitrilotriacetic acid affinity
chromatography according to the manufacturer's instructions (Qiagen).
The fractions containing hhCb15 were pooled and concentrated using a
Centricon 10 device (Amicon). Typical yields were 3-4 mg/liter of
culture. The amino terminus of the purified recombinant protein was
confirmed by amino-terminal amino acid sequencing.
Production of Antiserum to hhCb15--
HhCb15 (70 µg) was
homogenized in Montanide ISA70 (Seppic, Paris, France) and
injected subcutaneously over two sites along the spine of a female New
Zealand White rabbit (6 months). The rabbit received two further
injections 4 and 6 weeks after the primary immunization. Serum was
collected 2 weeks after the final immunization. The Australian code of
practice for the care and use of animals for scientific purposes,
National Health and Medical Research Council, 1997, was followed in the
maintenance of and procedures on all animals. The antibody titer of the
rabbit serum was measured by ELISA, and the antibody specificity was
examined by immunoblot analysis.
SDS-PAGE and Immunoblot Analyses--
SDS-PAGE was run using 12 or 15% resolving gels under reducing conditions. Gels were stained
with silver (17), and immunoblot analyses were conducted as previously
described (15). The rabbit serum was diluted at 1:1000 in TBS
containing 0.05% Tween 20 (TBST). Insect tissues were dissected from
whole larvae submerged in water (4 °C) containing a mix of protease
inhibitors (Roche Molecular Biochemicals). Dissected tissues were
transferred and collected into 200 µl of water with protease
inhibitors (on ice), then pelleted (15,000 × g, 5 min,
4 °C) and washed once with 500 µl of cold water. The supernatants
were discarded, and the pellets were resuspended by vortexing in
various volumes of reducing sample buffer. The suspensions were then
treated at 100 °C (5 min), homogenized by a motorized pestle, and
treated at 100 °C for a further 5 min before centrifugation
(15,000 × g, 5 min, 4 °C). Gel loading in terms of
tissue equivalents of each preparation is indicated in the figure
legends. Prevaccination serum (diluted 1:1000), used as control for the
immunoblots, did not produce signal (data not shown).
Intrinsic Fluorescence--
The intrinsic fluorescence of hhCb15
(dialyzed against TBS) was measured in a PerkinElmer Life Sciences
LS50B luminescence spectrometer at 25 °C in the presence or absence
of a range of GlcNAc-containing oligosaccharides and a soluble form of
chitin, glycol chitosan. GlcNAc, chitobiose, chitotriose,
chitotetraose, and glycol chitosan were purchased from Sigma. The
solutions were filtered (0.22-µm filter) before use. Excitation was
at 280 nm, and both slit widths were set at 5 nm. Emission spectra were
corrected for the small background signal from the appropriate solvent
(TBS) but remained uncorrected for the variation in detector efficiency with wavelength. The final concentrations of hhCb15 and glycol chitosan
were 4 and 180 µg/ml, respectively. Two irrelevant proteins acted as
controls, bovine serum albumin and a hexa-His-tagged bovine leptin
that, like hhCb15, is a small disulfide-bonded protein.
Chitin Binding Assay--
The ability of hhCb15 to bind chitin
was determined by titrating reacetylated chitosan against a fixed
concentration of hhCb15. After incubation, the suspension was separated
by centrifugation, and the concentration of unbound hhCb15 in the
supernatant was measured using a hhCb15-specific quantitative ELISA.
The hhCb15 bound to the reacetylated chitosan was qualitatively
examined by treating the chitosan pellets with SDS-PAGE-reducing sample buffer (100 °C, 10 min) followed by centrifugation. The supernatants were subjected to SDS-PAGE and silver staining. There was an inverse correlation between unbound and bound hhCb15 (data not shown).
In detail, reacetylated chitosan was prepared from horseshoe crab
chitosan (Sigma) as previously described (18). Various amounts of
reacetylated chitosan suspended in TBS were dispensed into preweighed
tubes and pelleted (30 s, 10,000 × g), and all liquid
was removed. HhCb15 (20 µg) in 200 µl of TBS was mixed with the
reacetylated chitosan for 5 h at RT. The supernatants containing
unbound hhCb15 were collected after brief centrifugation (10,000 × g,
3 min, RT). The pelleted chitin samples containing bound hhCb15 were
washed twice with absolute ethanol and then vacuum-dried to determine
the dry weight. The unbound hhCb15 was quantified by ELISA. Briefly,
samples of the supernatants were diluted 1:100 in 0.2 M
Na2CO3, pH 9.0, and dispensed as 100-µl duplicates into a microtiter plate (Falcon). A series of standards of
hhCb15 was also prepared on the same tray. After overnight incubation
(4 °C), the tray was washed in TBST and blocked with 5% skim milk
in TBST (150 µl/well) for 30 min at RT. After another wash step, the
tray was treated with rabbit antiserum to hhCb15 diluted 1:1000 in TBST
(100 µl/well) for 30 min at RT. The plate was washed in TBST, and the
wells were incubated with 100 µl of sheep anti-rabbit Ig horseradish
peroxidase conjugate (Silenus Laboratories, Melbourne) diluted
1:1000 in TBST for 30 min (RT). The plate was washed in TBST and 200 µl of 1 mM
2'-2'-azino-3-bisethylbenzthiazolone-6-sulfonic acid in 100 mM citric acid, pH 4.0, dispensed per well. Color development was measured at 405 nm using a Titretek Multiskan Plus
(Flow Laboratories, Rickmansworth, UK). A standard curve generated with
the hhCb15 standards over the concentration range of 1.56-100 µg/ml
(data not shown) allowed quantitation of the unbound hhCb15 in the supernatants.
Gel Permeation Chromatography--
A SEC3000 gel permeation
column (Phenomenex) was equilibrated with TBS delivered at 0.3 ml/min
by HPLC (Shimadzu). The column was calibrated with gel filtration
standards (Amersham Pharmacia Biotech): ribonuclease A (13.7 kDa, 220 µg), chymotrypsinogen (25.0 kDa, 100 µg), ovalbumin (43 kDa, 220 µg), and bovine serum albumin (67 kDa, 290 µg). Elution of the
standards and hhCb15 (25 µg) in TBS was monitored at 280 nm.
Mass Spectrometry--
HhCb15 was subjected to reverse phase
HPLC followed by mass spectrometry. HhCb15 (10 µg in water) was
injected onto a Zorbax 300SB (C3) cartridge (2.1 × 150 mm, 5 µm) equilibrated in 0.05% trifluoroacetic acid in water. After 5 min
in buffer A, a gradient to 60% buffer B (0.045% trifluoroacetic acid
in 90% acetonitrile) was delivered in 60 min at 150 µl/min by a
Perkin Elmer Biosystems 140B dual syringe pump. The protein
eluted as a single peak that was directly introduced into the mass
spectrometer. The mass spectrum was acquired on a Mariner
electrospray/time-of-flight spectrometer (PerSeptive Biosystems).
Sample droplets were ionized to a positive potential of 4 kV and
entered the analyzer at a nozzle potential of 160 kV. Full-scan spectra
were acquired over the range m/z 700-3000 in
2 s.
Tissue Processing for Immunogold
Localization--
L. cuprina larvae were used for
immunogold localization because live C. bezziana larvae
cannot be imported into Australia due to quarantine restrictions.
L. cuprina larvae were reared to third instar (still
feeding) on a diet containing 10% w/v skim milk powder and 2% w/v
brewers' yeast in 1% w/v agar gel. Cardiae were dissected from second
instar larvae in PBS and fixed in 4% paraformaldehyde (TAAB,
Reading, Berkshire, UK) and 0.3% glutaraldehyde (ProSciTech,
Thuringowa, Queensland, Australia) in 0.1 M PBS for 1 h at RT. The cardiae were then washed in PBS, dehydrated through an
ethanol series (30, 50, 70, 90%), and embedded in medium grade LR
White embedding resin (London Resin Co, Reading, Berkshire, UK), which
was then polymerized in airtight gelatin capsules at 50 °C
overnight. Ultra-thin sections were cut longitudinally through the
cardiae on an Amersham Pharmacia Biotech Ultrotome Nova
ultramicrotome, and the sections were taken up on Butvar-coated copper
grids. Sections on grids were processed by transferring to drops of
buffer (PBS with 0.5% ovalbumin (Sigma) and 0.1% Tween 20) containing (a) 10% normal goat serum, (b) pre-vaccination
or post-vaccination rabbit serum to hhCb15-diluted 1:500, and
(c) goat anti-rabbit Ig antibody conjugated to 10-nm
diameter colloidal gold particles (British Biocell International,
Cardiff, UK). Sections were washed on drops of diluting buffer after
steps b and c and finally on distilled water.
They were then stained (5 min each) in 2% aqueous uranyl acetate and
0.1 M lead citrate and examined in a JEOL 1010 transmission
electron microscope. Serum raised to hhCb15 cross-reacted strongly with
Lc-peritrophin-15 in both ELISAs and immunoblots. This was expected
since there was 82% amino acid sequence identity between the mature
sequences of the two proteins.
Sequence Analyses--
Database searches, alignments,
sequence analyses, molecular weight, and pI determinations were
performed on the WebANGIS computer system (University of Sydney,
Sydney, Australia) and the ExPASy computer system (Swiss Institute of
Bioinformatics, Switzerland). The former system also contains the EGCG
suite of computer programs. In addition, the gene analysis tools
accompanying the Berkeley Drosophila Genome Project were
used for the prediction of promoters and exons. Global protein sequence
alignments were computed using the program GAP (EGCG package, ANGIS),
which gives a measure of the overall percentages of amino acid sequence
identity and similarity. The latter used the PAM 250 similarity matrix.
The "Quality" score for the GAP alignment was compared with the
"Average Quality" score for 100 random alignments using the same
protein composition. Significant differences between these scores
provides evidence of significant sequence similarity.
Isolation and Sequences of Cb-peritrophin-15 and
Lc-peritrophin-15--
The sequential extraction of proteins from
dipteran PMs has proven an effective means of elucidating the protein
complexity of this structure (5). The insoluble residue left after
extraction of larval PM with high concentrations of urea or
guanidine-HCl still contains a significant protein content.
Subsequently, extraction of the guanidine-HCl-insoluble residue of
C. bezziana larval PM with 5% SDS and 10 mM
dithiothreitol solubilized a single dominant 15-kDa protein and minor
species at 18-20 kDa and 48-65 kDa, as judged by SDS-PAGE (Fig.
1, lane 2). The 15-kDa protein
was termed Cb-peritrophin-15. Biotinylated lectin blots (wheat germ
agglutinin, concanavalin A, and lentil lectin) indicated that
Cb-peritrophin-15 was not glycosylated. The amino-terminal sequence and
the two internal peptide sequences from Cb-peritrophin-15 were
determined. The amino-terminal sequence and the sequence of the longer
peptide (SDS1517) were used to design redundant polymerase chain
reaction primers to expedite cloning of a cDNA encoding
Cb-peritrophin-15 from a larval C. bezziana cDNA
library.
The DNA and predicted protein sequences of the insert from clone S3.4
are given in Fig. 2. The DNA sequence
codes for a 91-amino acid protein consisting of a 19-amino acid leader
sequence and a mature polypeptide of 72 amino acids. The predicted
molecular mass of the mature protein is 7894 daltons. Given the
migration of Cb-peritrophin-15 on reducing SDS-PAGE (~15 kDa) and
that no smaller forms of the protein were detected in higher percentage polyacrylamide gels, the small size of the predicted protein was surprising. The Cb-peritrophin-15 sequence contains a relatively high
number of prolines (~14%), mostly clustered near cysteines. Proline-rich proteins often have anomalous migration on SDS-PAGE (19-21). Consistent with the lack of reactivity with biotinylated lectins, there are no potential N-linked glycosylation sites
(i.e. NXS/T where X is not proline)
and no obvious potential O-linked glycosylation sites.
Lc-peritrophin-15 was isolated in a similar manner as Cb-peritrophin-15
and further purified by gel permeation chromatography in 6 M urea. Fig. 1 (lane 1) shows a silver-stained
SDS-PAGE gel of purified Lc-peritrophin-15. Lc-pertrophin-15 was
slightly larger in size than Cb-peritrophin-15 (Fig. 1, lane
2) and also migrated as a diffuse band, typical of
Cb-peritrophin-15 in the electrophoresis conditions used. A combination
of amino-terminal sequencing and internal peptide sequences generated a
total of seven peptide sequences from Lc-peritrophin-15. These were
highly homologous to regions within the Cb-peritrophin-15 primary
sequence. By alignment with the Cb-peritophin-15 sequence, the peptide
sequences covered 87.5% of the Lc-peritrophin-15 polypeptide. The
mature deduced protein sequence for Lc-peritrophin-15 was obtained by
sequencing a polymerase chain reaction fragment derived from L. cuprina larval cDNA using the Cb-peritrophin-15 degenerate
primer set.
Identification of D. melanogaster Peritrophin-15a and
Peritrophin-15b (Dm-peritrophin-15a and Dm-peritrophin-15b)--
The
deduced amino acid sequence of Cb-peritrophin-15 was used to search a
nonredundant protein sequence data base using the computer program
BLAST-P (22). No significant matches were obtained. However, searches
of the anonymous Drosophila melanogaster genomic sequence
and EST data bases using the computer program tBLASTn (22) retrieved
the EST LP08046 (GenBankTM accession number AI294630) and
the genomic clone DS02110.1 g10. The EST was from a larval/pupal
cDNA library. The sequence of the genomic clone contained two
contiguous hypothetical genes coding for proteins with highly
significant similarity to Cb-peritrophin-15 (Fig.
3). The putative protein sequences were
called Dm-peritrophin-15a and Dm-peritrophin-15b. The first
corresponded to the EST LP08046. Both D. melanogaster-deduced amino acid sequences contained putative amino-terminal signal sequences. The computer program SIGCLEAVE (23)
predicted signal sequence cleavage sites between residues 22-23 and
21-22 for Dm-peritrophin-15a and Dm-peritrophin-15b, respectively.
The percentage identities between any two of the four mature
deduced amino acid sequences ranged between 45.2 and 82.2% (Table I). The Cb-peritrophin-15 and
Lc-peritrophin-15 sequences show virtually the same percentage identity
to both sequences from D. melanogaster (i.e.
47.2-48.6% identities). However, there is much greater identity
between Cb-peritrophin-15 and Lc-peritrophin-15 (82.2%). Thus far,
only one peritrophin-15 has been identified in each of L. cuprina and C. bezziana at both the protein and mRNA level. Table I also summarizes the predicted isoelectric points and mature sizes of all four proteins. The pIs of these proteins
are acidic, which is a typical feature of peritrophic matrix proteins
(5).
A specific register of six absolutely conserved cysteine residues
characterizes all four protein sequences. This register consists of the
following consensus sequence:
CX9CX19-21CX10-11CX12 CX11C (where X is any amino acid
except cysteine). The predicted secondary structures of all four
sequences reveal the presence of several Gene Organization of Dm-peritrophin-15a and
Dm-peritrophin-15b--
The organization of the D. melanogaster genomic sequence containing two genes encoding
Cb-peritrophin-15 homologs is shown diagrammatically in Fig.
4. This segment of DNA, corresponding to
the reverse complement of nucleotides 16,736-13,184, was extracted from the sequence of the P1 genomic clone DS02110
(GenBankTM Accession AC004423) and was interpreted
with the aid of the EST sequence LP08046 (which corresponds to
Dm-peritrophin-15a), the sequence of Cb-peritrophin-15, and the
computer program DGRAIL. This gene-rich segment comprising 4 genes
encoding Dm-peritrophin-15a, Dm-peritrophin-15b, Acp29AB-I, and
Acp29AB-II, spans ~3.5 kilobases. The latter two genes are potential
unrelated C-type lectins.
Overall, the organizations of both peritriophin-15 genes are very
similar, suggesting a gene duplication event. The coding sequences
contributed by exon 1 for the two peritrophin-15 genes are both only 9 bp long. This is followed by a small intron (~66-70 bp) and then the
remainder of the coding sequence on a second exon. The putative
exon-intron and intron-exon boundaries conform to the appropriate
consensus sequences for these splice sites (24). Appropriate DNA
regulatory elements can be discerned in the D. melanogaster
genomic sequences upstream from the start sites. For each gene these
elements include a putative TATA box incorporated into a predicted
promoter region and an appropriately positioned arthropod
transcription initiator consensus sequence (25). The prediction
scores for the two promoters are highly significant (0.96 and 0.99),
respectively. The 5'-untranslated region and 3'-untranslated region
regions of the Dm-peritrophin-15 sequences are probably relatively
short, as is emphasized by the density of genes in this region. Only
one peritrophin-15 was expressed in C. bezziana and L. cuprina larvae as defined by protein and cDNA sequence
information. Furthermore, the Drosophila EST data base only
contained an EST representative of Dm-peritrophin-15a.
The Size of hhCb15--
To gain insight into the function of the
peritrophin-15 proteins, Cb-peritrophin-15 was expressed in
Escherichia coli as a soluble hexa-His fusion protein
(hhCb15). It was purified in a single affinity chromatography step
using nickel nitrilotriacetic acid-Sepharose. Like its native
counterpart, reduced hhCb15 migrated at ~15 kDa in 15 and 20%
SDS-PAGE gels rather than the 10.4 kDa predicted from the amino acid
sequence of the hexa-His-tagged recombinant protein (Fig.
5a). When hhCb15 was subjected
to gel permeation chromatography in nondenaturing conditions, only a single well defined peak was detected (Fig. 5b). The
apparent size of hhCb15 was measured by gel permeation chromatography
and compared with standards. The elution of hhCb15 was consistent with
a 24.5-kDa globular protein (Fig. 5b).
Mass determination of hhCb15 by mass spectrometry indicated that hhCb15
has a molecular mass of 10,395 Da, which is in agreement with the
expected molecular mass of 10,396 Da for the disulfide-bonded protein
(Fig. 5c). The mass spectrum revealed multiply charged ions
with charge states from +4 to +10. The reconstructed zero charge state
spectrum indicates 2 major components: the fully disulfide-bonded
species (10,395.2 Da) and a second species of 10,406.2 Da (Fig.
5c, inset), which represented about one-third of
the hhCb15 population. Both components were consistently detected (±1
Da) in three analyses. The larger sized component does not represent a
fully reduced form of hhCb15 (10,402 Da) nor does it indicate the
presence of an oxidized methionine (addition of 16 Da). The mass
determination of hhCb15 suggests that the apparent larger size of
hhCb15 observed by SDS-PAGE (~15 kDa) may be artifactual, probably
reflecting amino acid composition bias and/or an irregular shape. The
gel permeation chromatography data indicated a size of 24.5 kDa. This
result may also reflect an unusual shape, possibly rod-like in
character, or alternatively, it could reflect the presence of a
noncovalently dimer or trimer of hhCb15.
Binding of hhCb15 to Chitin--
The noncovalent interaction
between native Cb-peritrophin-15 or Lc-peritrophin-15 and their
respective PMs is very strong because strong denaturing conditions are
required to solubilize these proteins from PM. The nature of this
interaction is unclear, particularly whether the proteins are binding
to other proteins or to chitin (a linear polymer of GlcNAc) present in
the PM. Monitoring mixtures of hhCb15 with the purified native larval
peritrophins from L. cuprina, Lc-peritrophin-30,
Lc-peritrophin-44, and Lc-peritrophin-95, by gel permeation
chromatography did not reveal any protein-protein associations (data
not shown). Since hhCb15 did not appear to interact with these
peritrophins, its ability to bind chitin was examined.
Fig. 6a shows the intrinsic
fluorescence spectra for hhCb15 in the presence and absence of glycol
chitosan. The wavelength of maximal fluorescence emission for the
protein in the absence of the glycol chitosan was 345 nm, reflecting
the dominance of the emission characteristics of tryptophan (26). The
protein is relatively rich in aromatic amino acids, having four
tryptophans and one tyrosine. The fluorescence of hhCb15 in the
presence of 180 µg/ml glycol chitosan was enhanced and shifted toward
shorter wavelengths. The wavelength of maximal emission in the presence of the glycol chitosan was 343 nm, and the intensity at this wavelength was significantly increased by 10%. The shift toward shorter
wavelengths and increase in intensity suggest one or more tryptophans
in the protein are becoming buried as the protein binds glycol
chitosan. This behavior is very similar to the binding of wheat germ
agglutinin to chitotriose (27, 28). Increased concentrations of glycol chitosan did not further increase these effects, indicating that the
protein binding capacity was saturated. The intrinsic fluorescence spectrum of hhCb15 was unaltered by GlcNAc (10 mM),
chitobiose (11 mM), chitotriose (10 mM),
chitotetraose (5 mM), or a wide range of mono- and
disaccharide moieties (e.g.
N-acetylgalactosamine, methylmannopyranoside, fucose,
fructose, xylose, galacturonic acid, galactose, glucopyranosylglucose,
inositol, arabinose, all at 100 mM) in the presence
or absence of divalent cations (results not shown). Furthermore, glycol
chitosan (180 µg/ml) had no effect on the intrinsic fluorescence of
bovine serum albumin or hexa-His-tagged leptin at similar
concentrations (result not shown). These experiments indicate that
there is an interaction between hhCb15 and glycol chitosan that is
specific. If all the hhCb15 was bound, then a minimum ratio of 1 hhCb15
molecule to 2100 molecules of polymerized GlcNAc can be inferred from
this data.
The binding of hhCb15 to reacetylated chitosan was directly determined.
Increasing concentrations of reacetylated chitosan were titrated
against a constant amount of hhCb15 (20 µg). Bound and unbound hhCb15
were separated by centrifugation. The unbound hhCb15 was quantified by
ELISA (Fig. 6b). A minimum of 5 mg of reacetylated chitosan
was required to completely bind 20 µg of hhCb15, i.e. a
ratio of 1 molecule of hhCb15 to 10000 molecules of polymerized GlcNAc.
Calculation of the yield of Lc-peritrophin-15 from L. cuprina larval PM, where its GlcNAc content has been determined by
acid hydrolysis and monosaccharide analysis, indicates a similar ratio.2 HhCb15, like native
Cb-peritrophin-15, could only be removed from reacetylated chitosan by
boiling in SDS. Consequently, no attempt was made to measure the
binding constant for this interaction, which must be relatively strong.
GlcNAc (100 mM), glycol chitosan (180 µg/ml), and 8 M urea could not remove hhCb15 from the reacetylated chitosan (data not shown). The different binding stoichiometries inferred from the fluorescence data and the direct binding assay may be
due to the nature of the assays because the former assay only measured
the minimum ratio. Alternatively, the results may be explained by
differences in the types of chitin used (glycol chitosan and
reacetylated chitosan, respectively) and may possibly reflect differing
GlcNAc polymer length distributions. The low stoichiometry of hhCb15
binding to both preparations of chitin raises the question of uptake of
hhCb15 by a contaminant of the chitin preparations rather than the
chitin polymer itself. However, this argument requires that the
putative contaminant would survive the harsh chemical treatments and
extensive washings need to prepare these two very different forms of
chitin. Furthermore, this contaminant or a very similar constituent
would also have to be present in the larval PM since peritrophin-15 can
only be removed from PM and these resins with harsh denaturants.
Expression of Peritrophin-15 in C. bezziana--
SDS extracts of
adult C. bezziana PM and cardia, the PM of the three larval
instars and the cardia from third larval instar, were examined for the
presence of Cb-peritrophin-15 by immunoblotting. As demonstrated in
Fig. 7a, the PM from all three
larval instars contained Cb-peritrophin-15, and the variable tissue
loading and subsequent signal correlates with the body sizes of the
three larval instars, i.e. PM from third instar larvae
contained much greater quantities of Cb-peritrophin-15 then a second or
first instar PM, a reflection of the larger sized PM in the larger
sized larvae. The cardia is the site of synthesis of PM and, therefore, the putative site of synthesis of Cb-peritrophin-15. The cardia of
third instar larvae clearly contains Cb-peritrophin-15, but at
significantly lower levels compared with the signal obtained from a
single third instar PM. In contrast, Cb-peritrophin-15 was not detected
in adult PM or adult cardiae.
A more detailed investigation of third instar larval tissues confirmed
that expression is restricted to the cardia and PM and does not occur
in any other component of the digestive system (Fig. 7b).
Single whole PM produced a strong signal in the absence of signal from
equivalent tissue loading of the anterior, central and posterior
midguts, and hindgut. Of the other gut-associated tissues, only cardiae
produced a signal. Non-gut-associated tissues such as the Malpighian
tubules, the respiratory tracheae, and the fat body were also negative.
The absence of reactivity of prevaccination serum with the PM or
cardiae samples (not shown) and the absence of reactivity of the
antibodies to hhCb15 with the other tissues indicate that the
antibodies are specific for Cb-peritrophin-15. The highly
tissue-specific and life stage-specific expression pattern for
Cb-peritrophin-15 is characteristic of other previously described
larval peritrophins (5).
Immunogold Localization of Peritrophin-15 in L. cuprina
Larvae--
L. cuprina larvae were used in this study
because of the ready availability of this insect. The antibody used was
raised to hhCb15. Immunoblots and ELISAs demonstrated strong reaction
of the antibody with Cb-peritrophin-15 and Lc-peritrophin-15, as would
be expected for two proteins that are 82% identical in their mature
amino acid sequences. Fig. 8 shows
immunogold localizations of Lc-peritrophin-15 in L. cuprina
second instar larval cardia, the organ from which type 2 PM is
synthesized (1). This is a relatively small but complex organ located
at the junction of the cuticle-lined foregut (esophagus) and midgut
(intestine), which is formed by the intussusception of both tissues.
Fig. 8a shows a view that encompasses the newly formed PM
and the underlying cardiae epithelium. The PM is substantially and
uniformly labeled with gold particles, indicating the presence of
Lc-peritrophin-15. Immediately adjacent to and above the PM is the
foregut cuticle layer, which is not labeled with gold. The cardia
epithelial cell has substantial microvilli, and these are shown in
cross-section directly underlying the PM. This section shows little
gold labeling between or within microvilli, but low density of labeling
is observed in other sections. At the base of the microvilli and within
the cell is a concentration of membrane-bound vesicles whose contents are heavily labeled with gold particles. A corresponding control using
a prevaccination serum in the same region of the cardiae is shown in
Fig. 8b. Virtually no gold particles were seen in this
section. This result and the distinctive labeling of the PM and
vesicles in Fig. 8a indicate that the antibody was
specific.
Fig. 8c shows a higher magnification photo of the nascent PM
present in the cardiae. The PM is strongly and evenly labeled, and in
addition, there are some gold particles (arrows) associated with the intermicrovillar space. Again no gold particles are associated with the foregut cuticle layer. Fig. 8d shows the specific
gold labeling of vesicles concentrated within the cell at the base of
the microvilli. In general, there is greater gold label within vesicles
closer to the microvilli compared with vesicles relatively distant from
the microvilli (result not shown). The vesicles adjacent to the
microvilli often contain 2-3 "nodes," possibly formed by the
fusion of smaller vesicles (inset, Fig. 8d). The
gold particles are not uniformly distributed within the vesicles but
rather are peripheral to the nodes. Not all vesicles are labeled, and
those that are often have different densities of gold label, suggesting that different vesicles may contain different cargoes. Close
examination of the vesicles reveals that some are closely associated
with microfilament bundles extending from the microvillar base into the
cell proper (Fig. 8e). Fig. 8, e and
f, indicate that the vesicles apparently fuse with the cell
membrane at the base of the microvilli, where the contents of the
vesicles are released.
The PM is a highly specialized extracellular matrix that
physically separates the insect midgut epithelium from the gut luminal contents in most insects. Despite being almost universally present in
insects, including medically and economically important insects, little
is known of its constituents at the molecular level or its functions.
Only five PM proteins from a range of insect species have been
characterized in any detail (5). In this study, we have described a
novel PM protein, peritrophin-15, present in three higher Diptera,
L. cuprina, C. bezziana, and D. melanogaster.
Peritrophin-15 is the smallest and most highly conserved peritrophin
isolated from PM to date. The protein sequences of peritrophin-15 from
L. cuprina, C. bezziana, and D. melanogaster are unique and bear little resemblance to the
previously identified peritrophin-A and peritrophin-B domains conserved
in other peritrophins (5). The only significant similarity is the
presence of six highly conserved cysteines. It is likely that these
form three intramolecular disulfide bonds, which dominate the
architecture of peritrophin-15. However, the cysteine register found in
peritrophin-15 is novel. Disulfide bonds in peritrophin-44 have been
shown to confer remarkable resistance to proteolysis (6). It is
probable that the disulfide bonds in peritrophin-15 serve a similar
function. Proteins present in the PM must be resistant to proteolysis
because digestive proteases such as trypsins and chymotrypsins must
traverse the PM to gain entry to the gut lumen. Unlike all other
characterized peritrophins, peritrophin-15 is not glycosylated. None of
the sequences contains consensus sequences for potential
N-linked glycosylation or predicted sites for
O-linked glycosylation, and a range of biotinylated lectins
did not react with peritrophin-15 purified from L. cuprina or C. bezziana (results not shown). The lack of
glycosylation, relatively strong sequence conservation, and strength of
interaction with the PM collectively indicate that this protein is a
core component of the PM.
HhCb15 was shown to tightly bind glycol chitosan and reacetylated
chitosan in vitro. Other irrelevant hexa-His-tagged
recombinant proteins such as hexa-His-tagged bovine leptin and a
hexa-His-tagged glycosylated Cb-peritrophin-48 did not show any
evidence of binding (results not shown). Thus, the chitin binding
activity must be contributed by peritrophin-15. The smallest chitin
binding "modules" are the 29-30 amino acid anti-microbial peptides
from Amaranthus caudatus and hevein, a 43 amino acid
polypeptide isolated from the rubber tree (Hevea
brasiliensis). Variations of these cysteine-rich modules are
repeatedly used in chitin-binding proteins, various chitin binding
plant lectins and chitinases (29, 30). The amino acid sequence of
peritrophin-15 is not related to any of these proteins. Unlike some
chitin-binding proteins, it is clear that peritrophin-15 is not the
product of proteolytic processing of a larger precursor protein.
Carbohydrate binding proteins tend to use both hydrogen bonds and Van
de Waals forces in binding their ligands (31-33). Asparagine and
glutamine are commonly implicated as hydrogen donors, whereas tryptophan, tyrosine, and less commonly, phenylalanine, are involved in
hydrophobic interactions with the sugar moiety. Both Cb-peritrophin-15 and Lc-peritrophin-15 contain five asparagines within the 20 amino-terminal residues, and the most conserved regions of the
peritrophin-15 sequences contain aromatic amino acids. It is
interesting to speculate that the two independently conserved regions
of aromatic amino acids in the peritrophin-15 sequences have roles in
binding GlcNAc within the chitin polymer. This hypothesis is consistent
with the relative sequence conservation of these residues and the
intrinsic fluorescence results. The latter clearly demonstrates that
aromatic residues in hhCb15 are perturbed upon binding chitin. The lack of any apparent binding of GlcNAc, chitobiose (GlcNAc2),
chitotriose (GlcNAc3), or chitotetraose
(GlcNAc4) to hhCb15 indicates that this relatively small
protein binds to chitin in a manner possibly dictated by the higher
order structure of this helical polymer.
The relatively small size of peritrophin-15 and its low
stoichiometry of binding to chitin (~1 molecule hhCb15:10,000
molecules of polymerized GlcNAc) rule out a mechanism involving
peritrophin-15-mediated cross-linking function of chitin polymers
within a fibril. Furthermore, if peritrophin-15 is a rod-like monomeric
protein, which could be consistent with the apparent size determination
by gel permeation chromatography, then the protein would probably be
monovalent in its binding characteristics and, therefore, unable to
cross-link chitin polymers. Peritrophin-15 may be involved in capping
the ends of individual chitin polymer chains within a chitin fibril or
within the chitin meshwork of the PM (Fig.
9). Such a capping function may have a
protective role preventing degradation by exochitinases. Alternatively,
the capping function may be part of a regulatory mechanism that
controls the length of the chitin polymer. It is interesting to note
that chitin polymers in insect PM can contain as many as 10,000 molecules of GlcNAc (34). Unfortunately, the average polymer length
of the chitin polymer chains within dipteran larval PM has not been
determined. Individual chitin chains consist of a 2-fold helical
symmetry of 10.34-Å repeats consisting of 2 GlcNAc residues. This
periodicity is true for
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
15,000. Occasionally the protein was further purified by anion exchange
chromatography. Protein concentrations were estimated with the Pierce
BCA protein determination kit using bovine serum albumin as a standard.
Samples were diluted with water before measurement to reduce
interference of urea in the assay. Protein standards were measured in a
comparable buffer.
15,000 (Cb-peritophin-15) was excised from the gel and digested in situ with trypsin replaced with
endoproteinase Lys-C (Roche Molecular Biochemicals) (12). After
enzymatic digestion, the peptides released into the digestion buffer
were isolated by reverse phase HPLC chromatography on a Brownlee RP-300
C8 column using, sequentially, chromatography in 0.1%
heptafluorobutyric acid in an acetonitrile gradient then 0.1%
trifluoroacetic acid in an acetonitrile gradient (13). Peptide
sequencing was performed on an Applied Biosystems 471A protein
sequencer. Two peptides, SDS1506 and SDS1517, were sequenced. The
amino-terminal sequence was determined by blotting onto Problott and
sequencing (ABI User Bulletin 42, 1990). Purified Lc-peritrophin-15 was
digested in solution by endoproteinase Lys-C, and the peptides were
purified by HPLC and sequenced, as described above.
ZAP express vector (Stratagene) (14, 15). After 3 rounds of screening, 16 clones were isolated. The inserts from three
clones were further analyzed; however, only one clone, S3.4, contained
a full-length coding sequence. The DNA insert from this clone was
completely sequenced on both strands.
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Fig. 1.
Isolation of Cb-peritrophin-15 and
Lc-peritrophin-15. Purified Lc-peritrophin-15 (lane 1,
2 µg) and the SDS extract of the insoluble guanidine-HCl residue of
C. bezziana larval PM (lane 2, 3 µg) were
subjected to SDS-PAGE and silver staining.
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Fig. 2.
cDNA sequence and predicted protein
sequence of Cb-peritrophin-15. The DNA sequence of the insert from
clone S3.4 and the predicted amino acid sequence of Cb-peritrophin-15
are presented. When translated from the putative initiating ATG, the
DNA sequence contains a single open reading frame of 273 bp. A poly(A)
signal sequence (boxed) begins 40 bp after the stop codon.
The sequence ends with a 51-bp poly(A) tail (underlined).
The 5'-untranslated region is at least 26 bp in length, and the
3'-untranslated region length is 128 bp. The amino-terminal and peptide
sequences obtained from the native protein are in
bold, and the signal sequence is
italicized. The cysteines within the mature protein are
circled.
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Fig. 3.
Alignment of deduced amino acid
sequences of peritrophin-15 from three insect species. Single
underlining denotes signal sequences, the dotted
underline denotes experimentally determined amino-terminal and
internal peptide sequences, and the cysteine residues in the mature
proteins are boxed. Gaps introduced to optimize the
alignment are shown as dots. Amino acids conserved in all
four sequences are denoted by the symbol *. Dm-peritrophin-15a (Dm15a)
and Dm-peritrophin-15b (Dm15b) are two sequences deduced from an
anonymous D. melanogaster genomic sequence
(GenBankTM accession number AC004423); Cb15,
Cb-peritrophin-15 (GenBankTM accession number AF327453);
Lc15, Lc-peritrophin-15 (GenBankTM accession number
AF327454).
Comparisons of predicted mass, pI, and percent identity of the mature
peritrophin-15 proteins from three species of fly
-turns, which are generally
positioned between the cysteine residues for each sequence. The
relative abundance of cysteines in this extracellular protein and the
presence of appropriate potential
-turns suggest that each protein
contains three intramolecular disulfide bonds. Both Cb-peritrophin-15
and Lc-peritrophin-15 bind very strongly to PM, but both can be
solubilized from the PM using harsh denaturants in the absence of
reducing agents (results not shown). This indicates that the cysteine
residues are not involved in intermolecular disulfide bonds. In
addition to the conserved cysteine residues, there are additional
regions of local conservation particularly centered on conserved
aromatic amino acids between cysteines 2 and 3 (two regions:
F42W43 and Y48W49),
cysteines 4 and 5 (F69), and cysteines 5 and 6 ((F/W)79XXW82XW84)
(Cb-peritrophin-15 numbering).
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Fig. 4.
Diagrammatic representation of the gene
organization on a segment of the D. melanogaster P1
clone DS02110 containing Dm-peritrophin-15a and
Dm-peritrophin-15b. The diagram shows the reverse complement of a
segment of the GenBankTM sequence AC004423 (nucleotides
16,736-13,184). The boxes denote the coding sequence only.
Filled boxes correspond to Dm-peritrophin-15a
(Dm15a) and Dm-peritrophin-15b (Dm15b). The
hatched boxes correspond to the C-type lectin genes
Acp29Ab-I (26) and Acp29Ab-II (homologous to Acp29Ab-I).
CDS, coding sequence.
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Fig. 5.
Purification and size determination of
recombinant hhCb15. a, HhCb15 was purified from the
Tris-buffered extract of E. coli by nickel nitrilotriacetic
acid affinity chromatography. Two samples of hhCb15 (each 2 µg) were
subjected to SDS-PAGE under reducing conditions and then stained with
silver. b, gel permeation chromatography of hhCb15 in
Tris-buffered saline. Amersham Pharmacia Biotech gel permeation
standards were used to calibrate the column: bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease A
(13.7 kDa). AUFs, absorbance units at full scale. c,
electrospray time-of-flight mass spectrum of hhCb15 indicating 7 multiply charged (+4 to +10) molecular ion species.
Inset, reconstructed mass spectrum of hhCb15 showing
the observed zero charge state molecular mass (Da).
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Fig. 6.
Binding of hhCb15 to chitin.
a, intrinsic fluorescence spectra of hhCb15 in the absence
and presence of glycol chitosan. HhCb15 (4 µg/ml in TBS) was excited
at 280 nm, and fluorescence was monitored over 300-400 nm in the
presence and absence of glycol chitosan (180 µg/ml). b,
binding of reacetylated chitosan with hhCb15. Varying quantities of
reacetylated chitosan were titrated with 20 µg of hhCb15 in TBS. The
unbound hhCb15 was quantified by ELISA.
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Fig. 7.
Expression of Cb-peritrophin-15 in the life
stages and tissues of third instar larvae of C. bezziana.
Immunoblots were used to determine the presence of Cb-peritrophin-15 in
SDS extracts of various C. bezziana tissues. a:
lane 1, 20 adult cardiae; lane 2, 10 adult PMs;
lane 3, a single PM from a third instar larvae; lane
4, 2 PMs from second instar larvae; lane 5, 10 PMs from
first instar larvae; lane 6, 8 cardiae from third instar
larvae. b, third instar larval tissues with the equivalent
tissue loading in parentheses. Lane 1,
crop (5); lane 2, salivary gland (10); lane 3,
cardiae (10); lane 4, anterior midgut (0.9); lane
5, central midgut (0.8); lane 6, posterior midgut
(0.9); lane 7, peritrophic matrix (1); lane 8,
hindgut (0.9); lane 9, Malpighian tubules (1.5); lane
10, fat body (0.2); lane 11, tracheae (0.7); lane
12, cardiae (8). The immunoblots were probed with rabbit serum
raised to hhCb15.
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Fig. 8.
Immunogold localizations of Lc-peritrophin-15
in L. cuprina second instar larval cardia.
Panels a and c-f show photos of immunogold
localizations using rabbit serum raised to hhCb15. Panel
b shows a control using prevaccination serum. The
inset in panel d shows an enlargement of a
vesicle. C, cuticle; V, vesicle; MV,
microvilli; MF, microfilaments; RER, endoplasmic
reticulum; M, mitochondria. The scale bars
represent 500 nm in a and b, 200 nm in
c, 333 nm in d, 133 nm in e, and 100 nm in f.
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and
chitins and chitosan (35, 36). It
is possible that Cb-peritrophin-15 also requires this helicity to bind
to the ends of chitin fibrils. One prediction of the capping function
hypothesis is that peritrophin-15 would only bind to one end of the
chitin polymer because of the chemical asymmetry associated with the ends of the polymer. Peritrophin-15 can only be isolated from PM using
strong denaturing conditions. This information and the chitin binding
studies indicate that the protein is probably an intrinsic structural
component of the PM secured into the PM by strong interactions with
chitin fibrils. The uniform localization of Lc-peritrophin-15 on
nascent PM produced by the larval cardia also supports this
conclusion.
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Fig. 9.
Model of the possible interaction of
peritrophin-15 with chitin polymer. Schematic diagram depicting a
model of the interaction of peritrophin-15 with the ends of
N-acetylglucosamine polymer chains within the chitin fibril
arrangement of parallel-antiparallel-parallel polymer chains.
The cardia is a small organ whose anatomy has been examined in detail in some Diptera (1, 37-39). These studies revealed that the adult cardia is complex, often structured into multiple and distinct regions and often synthesizing multiple PMs. By comparison, the larval cardia is less complex, with a single formation zone synthesizing a single PM composed of 1-3 layers. Although different cell types are obvious in the larval cardia, there is no indication which cell types are responsible for the synthesis of PM proteins, the predominant component of the PM. Consequently, it has been difficult to understand the spatial events involved in PM formation. It is now clear that one major component of the PM, peritrophin-15, is synthesized by the cardia epithelia located throughout the cardia. However, there appears to be a gradient of peritrophin-15 concentration within the cells from the anterior region to the posterior region of the cardia (results not shown). These cells package peritrophin-15 into membrane-bound vesicles, which move to the base of microvilli, where the contents of the vesicles are discharged. The movement of newly formed vesicles to the base of the microvilli could be assisted by microfilaments associated with these structures (Fig. 8). The cells have abundant rough endoplasmic reticula, consistent with a secretory function tightly linked to constitutive PM production in this insect. The process that transports Lc-peritrophin-15 from the extracellular microvillar base to the nascent PM is unclear. However, peritrophin-15, when present with the PM, probably binds rapidly to chitin within the PM.
At the position in the cardia where Lc-peritrophin-15 is secreted,
there is already a defined but immature PM. This observation reinforces
the view that the addition of peritrophin-15 to the PM occurs
relatively late in PM synthesis and is consistent with a role of
peritrophin-15 in capping chitin polymers or fibrils. The PM at this
stage is still immature, as evidenced by its relatively weak lamellar
appearance in cross-section, which is much more marked in the mature
PM.2 Despite the ability to follow the synthesis,
transportation, and addition of Lc-peritrophin-15 to PM, much is still
unknown in relation to PM synthesis. First, what is the molecular
nature of the nodes within the vesicles containing Lc-peritrophin-15, and do these vesicles have multiple PM protein cargoes? Second, what
defines the assembly stoichiometries of the different proteins and
chitin within the PM? Third, where and how is chitin synthesized for
the nascent PM? This will require definition of the location of chitin
synthase, which despite its importance in arthropods, remains
uncharacterized at this time. Fourth, is the PM a passive semi-permeable partition between the contents of the gut lumen and the
digestive epithelia, or does the PM have a more active role in
facilitating digestion and interacting with and directing the growth of
the digestive epithelia? What is clear is that the composition,
structure, and synthesis of PM are complex, possibly reflecting its
intimate role in the insect digestive processes and its role as a
barrier to the invasion of the insect by microorganisms.
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ACKNOWLEDGEMENTS |
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We are grateful for the assistance of David Watisbuhl and Joanne Gough in the purification of hhCb15. We also acknowledge the dedication of Ibu Sukarsih and Edi Satria in the maintenance of the screwworm fly colony (Balitvet, Bogor, Indonesia), the supply of fly materials, and assistance with many laborious dissections.
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FOOTNOTES |
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* This research was funded in part by the Australian Center for International Agricultural Research and the L. W. Bett Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF327453 and AF327454.
§ To whom correspondence should be addressed. Tel.: 61-07-3346 2510; Fax: 61-07-3346 2509; E-mail: Gene.Wijffels@li.csiro.au.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M009393200
2 G. Wijffels, C. Eisemann, G. Riding, R. Pearson, A. Jones, P. Willadsen, and R. Tellam, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PM, peritrophic matrix; Cb, C. bezziana; Lc, L. cuprina; Dm, D. melanogaster; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; RT, room temperature; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; bp, base pair(s); hhCb15, hexa-His-Cb-peritrophin-15; Cb15, Cb-peritrophin-15; ELISA, enzyme-linked immunosorbent assay; TBST, TBS Tween.
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REFERENCES |
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