A Novel Family of Chitin-binding Proteins from Insect Type 2 Peritrophic Matrix

cDNA SEQUENCES, CHITIN BINDING ACTIVITY, AND CELLULAR LOCALIZATION*

Gene WijffelsDagger §, Craig EisemannDagger , George RidingDagger , Roger PearsonDagger , Alun Jones, Peter WilladsenDagger , and Ross TellamDagger

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -70 °C.

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 approx  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.

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 approx  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.

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 lambda 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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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.


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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.

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.


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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).

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).

                              
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Table I
Comparisons of predicted mass, pI, and percent identity of the mature peritrophin-15 proteins from three species of fly
Lc15, L. cuprina peritrophin-15; Dm15a, D. melanogaster peritrophin-15a; Dm15b, D. melanogaster peritrophin-15b.

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 beta -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 beta -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).

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.


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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.

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).


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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).

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.


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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.

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.


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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.

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.


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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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  and beta  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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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