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Insect Biochemistry and Molecular Biology 34 (2004) 215–227 www.elsevier.com/locate/ibmb

Identi?cation of two new peritrophic membrane proteins from larval Trichoplusia ni: structu

ral characteristics and their functions in the protease rich insect gut
Ping Wang a,b,?, Guoxun Li a, Robert R. Granados b
a

Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA b Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, USA Received 8 August 2003; received in revised form 2 October 2003; accepted 3 October 2003

Abstract Peritrophic membrane (PM) proteins are important determinants for the structural formation and function of the PM. We identi?ed two new chitin binding proteins, named CBP1 and CBP2, from the PM of Trichoplusia ni larvae by cDNA cloning. The proteins contain 12 and 10 tandem chitin binding domains in CBP1 and CBP2, respectively. Chitin binding studies demonstrated the chitin binding activity of CBP1 and CBP2, and con?rmed the chitin binding domain sequence predicted by sequence analysis. Both CBP1 and CBP2 were not mucin-like glycoproteins, however, they were highly resistant to proteolytic degradation by trypsin. We found that in CBP1 and CBP2, potential trypsin and chymotrypsin cleavage sites reside primarily within the chitin binding domain sequences, limiting exposure of the potential cleavage sites to the digestive proteinases. This ?nding suggests a proteinase-resistance mechanism for non-mucin PM proteins to function in the proteinase rich gut environment. Immunohistochemical analysis showed that CBP1 and CBP2 are speci?cally localized in the PM. However, intact CBP1 and CBP2 proteins were not present in the PM, indicating that their partially degraded fragments were assembled into the PM. This observation suggests that the presence of a large number of chitin binding domains in PM proteins allows the proteins to tolerate limited proteolytic degradation in the midgut without loss of their chitin binding activity with multiple chitin binding domains. Alignment of the chitin binding sequences suggested that CBP1 and CBP2 evolved by gene duplication and the tandem chitin binding domains in the proteins arose from domain duplications. # 2003 Elsevier Ltd. All rights reserved.
Keywords: Peritrophic membrane; Peritrophic matrix; Chitin binding protein; Midgut; Trichoplusia ni

1. Introduction The gut epithelia of animals and humans are lined by a proteinaceous covering which performs important functions in assisting food digestion and protecting the gut epithelium. An intestinal mucus layer, for example, lines the gastrointestine in mammals and humans. In contrast, in arthropods and some other invertebrate animals, the gut is normally covered by the peritrophic membrane (PM), which is also known as the peritrophic matrix (Peters, 1992). The digestive tract is
? Corresponding author. Tel.: +1-315-787-2348; fax: +1-315-7872326. E-mail address: pw15@cornell.edu (P. Wang).

extremely rich in proteolytic enzymes, so protection against digestive enzymes is a prerequisite for the proteinaceous constituents of the gut lining in order to maintain their biological functions. In mammals and humans, it is well known that the gastrointestinal mucus layers are primarily made of hydrated intestinal mucins. Intestinal mucins are highly O-glycosylated proteins and the massive carbohydrate moieties of the mucins facilitate the formation of the viscous mucus gel and, importantly, protect the proteins from proteolytic degradation (Forstner and Forstner, 1994; Bansil et al., 1995). The invertebrate PMs are a thin gut lining with the thickness ranging from submicron to several microns (Spence, 1991; Lehane, 1997), in contrast to the 50–300 lm-thick mammalian mucus (Lichtenberger,

0965-1748/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2003.10.001

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1995). The PM structure and formation are much less understood at present. PMs are produced along the midgut epithelium, which are de?ned as Type 1 PMs, or are produced in a restricted structure at the anterior midgut, the cardia, which are de?ned as Type 2 PMs (Richards and Richards, 1977; Peters, 1992). The limited thickness of PMs imposes a signi?cant challenge on the PM to perform protective functions against mechanical stress, chemical damage and biological invasions. PMs are known to be composed of chitin ?brils and proteins. The incorporation of chitin ?brils in the PM by chitin– protein binding is believed to signi?cantly strengthen the physical property of the PM. To date, a total number of 10 PM proteins have been identi?ed from ?ve insect species that represent insects having Type 1 and Type 2 PMs (Elvin et al., 1996; Wang and Granados, 1997b; Casu et al., 1997; Shen and Jacobs-Lorena, 1998; Schorderet et al., 1998; Wij?els et al., 2001; Vuocolo et al., 2001; Tellam et al., 2003; Sarauer et al., 2003). These PM proteins have been biochemically characterized and their primary sequences analyzed by cDNA cloning. All the characterized PM proteins showed chitin binding activities and have also been collectively named ‘‘peritrophins’’ (Tellam et al., 1999). With the exception of peritrophin-15 from Chrysomya bezziana and Lucilia cuprina (Wij?els et al., 2001), these PM proteins contain two to six putative chitin binding domains, which are composed of 65–70 amino acid residues and are characterized by a conserved register of six cysteine residues and a few aromatic amino acid residues (Tellam et al., 1999). The conserved cysteine residues are suggested to form intradomain disul?de bonds which confer the stability of the protein in the protease rich gut environment (Wang and Granados, 1997b; Shen and Jacobs-Lorena, 1999; Schorderet et al., 1998; Wang and Granados, 2001). The importance of disul?de bonds for the stability of PM proteins has been demonstrated in Trichoplusia ni PMs (Wang and Granados, 1997b). An insect intestinal mucin (IIM) has also been identi?ed from the PM of T. ni (Wang and Granados, 1997b). This invertebrate intestinal mucin has similar molecular and biochemical characteristics to mammalian and human intestinal mucins (Wang and Granados, 1997b). Recently, a similar IIM was identi?ed from Plutella xylostella (Sarauer et al., 2003). With the known biochemical and protein sequence characteristics of the identi?ed PM proteins, it has been proposed that the presence of multiple chitin binding domains in PM proteins result in cross-linking of chitin ?brils by binding of the proteins to form the PM (Wang and Granados, 1997b; Shen and JacobsLorena, 1999; Tellam et al., 1999; Wang and Granados, 2001). PM proteins are necessary for the PM structural integrity (Zimmerman and Peters, 1987) and any disruption of this delicate chitin–protein interaction may

result in detrimental physiological e?ects to insects (Wang and Granados, 2001; Bolognesi et al., 2001; Okuno et al., 2003). Currently, identi?cation of PM proteins is limited to 10 PM proteins from ?ve di?erent insect species. The number of proteins in PMs has been reported from 2 to 30 in Type 1 PMs by SDS-PAGE analysis (Wang and Granados, 2001). The majority of the PM proteins in the lepidopterous insect, T. ni, may be solubilized by a chitin binding reagent (Wang and Granados, 2000). It remains unclear how all the PM proteins are involved in the PM formation and function. Notably, the PM functions in the extremely protease rich milieu, so the mechanisms for non-mucin proteins to survive and function under such conditions need to be understood. In this study, we identi?ed two new PM proteins from T. ni larvae by cDNA cloning. These two nonmucin PM proteins possess unique sequence features which allow the proteins to play their roles in PM formation.

2. Materials and methods 2.1. Insect larvae and PM preparation A laboratory colony of T. ni was maintained on a wheat germ-based arti?cial diet. The PMs from mid5th instar larvae were isolated and cleaned as described earlier by Wang and Granados (1997a) and were stored v at ?70 C before use. 2.2. Preparation of PM chitin binding proteins and generation of an antiserum against the proteins PM chitin binding proteins were solubilized from the PMs by a chitin binding reagent, Calco?uor, as previously described (Wang and Granados, 2000). Brie?y, 1.2 g (wet weight) of T. ni PMs were incubated with 2.5 ml of 1% Calco?uor (Calco?uor White M2R, Sigma, St. Louis, MO) containing 4 lg/ml aprotinin, 1 lg/ml leupeptin, 1 mM EDTA and 2 mM phenylmethylsulfonyl ?uoride (PMSF) for 2.5 h at room temperature with gentle homogenization, followed by centrifugation at 1000 g for 5 min to clarify the supernatant. The supernatant was collected and the remaining PM pellet was extracted again with 3 ml of 1% Calco?uor as described above. The supernatants collected from the two extractions were combined and further clari?ed by centrifugation at 1000 g for 5 min. The resulting supernatant was collected and subjected to two additional centrifugations at 15,000 g for 5 min to eliminate residual PM fragments from the solubilized protein preparation. The Calco?uor in the protein preparation was removed by gel ?ltration chromatography using the Econo-Pac 10DG column from Bio-Rad (Hercules, CA).

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The solubilized PM proteins were incubated with 800 mg regenerated chitin (wet weight) with constant mixv ing at 4 C overnight. The regenerated chitin bound with PM chitin binding proteins were collected by centrifugation and were thoroughly washed with 10 ml of PBS four times. The PM chitin binding proteins bound with the regenerated chitin were released from the chitin in 1.5 ml 2% SDS and 5% b-mercaptoethanol in PBS by boiling for 5 min, followed by centrifugation at 15,000 g for 5 min. The supernatant was collected and the regenerated chitin pellet was re-extracted with 1 ml of 2% SDS and 5% b-mercaptoethanol. The supernatants containing the extracted PM chitin binding proteins were combined and the SDS and b-mercaptoethanol were removed from the protein preparation by an Econo-Pac 10DG column as described above. The quantity of PM CBPs was determined using the BCA protein assay kit from Pierce (Rockford, IL) with bovine serum albumin (BSA) as standards. An antiserum against the PM chitin binding proteins was generated by immunization of a New Zealand rabbit with an initial injection and two subsequent boost injections of 150 lg proteins. This antiserum production procedure was performed in the animal facility at the School of Veterinary Medicine of Cornell University. 2.3. Cloning and sequencing of cDNAs for PM chitin binding proteins To identify cDNA clones coding for PM chitin binding proteins, a T. ni midgut cDNA expression library (Wang and Granados, 1997b) was screened with the antiserum generated against the PM chitin binding proteins. Since the PM chitin binding proteins include the IIM, to exclude cDNA clones for the IIM, which was cloned previously (Wang and Granados, 1997b), an antiserum against T. ni IIM (Wang and Granados, 1997a) was also used to identify IIM clones among the cDNA clones that were recognized by the antibodies to chitin binding proteins. Therefore, the same phage plates from the cDNA library were blotted with two ?lters and the ?lters were probed with antibodies to the chitin binding proteins and to the IIM, respectively. cDNA clones that showed a positive reaction to the antibodies against chitin binding proteins, but were negative to the antibodies against the IIM, were considered to be the clones coding for the new PM chitin binding proteins and were subsequently puri?ed by an additional one or two rounds of plating and screening. These new PM protein cDNA clones were ?nally processed for in vivo excision to rescue the pBluescript phagemids following the instructions provided by Stratagene (La Jolla, CA). DNA sequencing was conducted by the thermal cycle sequencing method, using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit

from Applied Biosystems (Foster City, CA) and sequences were determined with an ABI PRISM 310 Genetic Analyzer. Initially, 43 positive clones were selected and the 50 and 30 termini of their cDNA inserts were sequenced using the T3 and T7 primers, respectively, which are located in the pBluescript vector ?anking the cDNA inserts. Based on the sequences of the 50 and 30 terminal sequences of the cDNA inserts, two representative full length cDNA clones, designated as cbp1 and cbp2, were identi?ed. The plasmids with cbp1 and cbp2 were named pBluescript/CBP1 and pBluescript/CBP2, respectively. The full length sequences of cbp1 and cbp2 were determined by generation of nested deletion constructs from both 50 and 30 ends of the inserts using the Erase-A-Base system from Promega (Madison, WI), followed by sequencing of the deletion constructs using the T3 primer for the strand with forward orientation and T7 primer for the strand with reverse orientation of the inserts.

2.4. Immunolocalization of the chitin binding proteins, CBP1 and CBP2, in T. ni larvae To localize CBP1 and CBP2, which are encoded by the cDNAs cbp1 and cbp2, respectively, in T. ni larvae, cross-sections of T. ni larvae were prepared as described earlier (Wang and Granados, 2000). Fifth instar T. ni larvae were ?xed in 4% paraformaldehyde v solution at 4 C overnight and dehydrated with a series of concentrations of ethanol from 50% to 100%. The larval samples were ?nally embedded in LR White resin (London Resin Company, Berkshire, England) and 1 lm-thick cross-sections in the larval midgut region were prepared. For staining of the sections with antibodies, the sections were treated with 3% BSA in PBS for 1 h to block non-speci?c antibody binding, followed by incubation with antibodies to CBP1 or antibodies to CBP2, which were puri?ed by a?nity puri?cation method using E. coli expressed CBP1 or CBP2 proteins, at room temperature for 2 h. After washing the sections with PBS for three times, the sections were incubated with a goat anti-rabbit IgG antibody conjugated with Rhodamine at room temperature for 1 h. The sections were ?nally mounted with Gel/ Mount medium (Biomeda Corp., Foster City, CA) after washing with PBS for three times and observed under a ?uorescence microscope. 2.5. Construction of recombinant baculoviruses for expression of the CBP1, CBP2 and a fragment of CBP1 containing a single chitin binding domain The Bac-to-Bac baculovirus expression system (Invitrogen, Carlsbad, CA) was used to produce the recombinant CBP1, CBP2 and a fragment of CBP1 that

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contains a single chitin binding domain, CBP1–CBD1. To construct donor plasmids for the generation of recombinant baculoviruses, the cDNA for CBP1 was excised from pBluescript/CBP1 by digestion with EcoRI and XhoI and inserted into the multiple cloning site in the donor plasmid pFASTbac1 between EcoRI and XhoI. Similarly, the cDNA for CBP2 from pBluescript/CBP2 was also excised by digestion with EcoRI and XhoI and cloned into pFASTbac1 between EcoRI and XhoI sites. To construct a recombinant baculovirus to express the secreted CBP1–CBD1, a fragment between the positions ?99 bp upstream of the cbp1 cDNA insert in pBluescript vector region and +393 bp in cbp1 was excised by polymerase chain reaction (PCR). The PCR primers were designed as such that a stop codon and a HindIII site were introduced into the 30 end of the ampli?ed fragment for subsequent cloning: AATTAACCCTCACTAAAGGG (forward primer) and TTCACTAAGCTTTCAGTTGCAGGTGGCCGGACTA (reverse primer). The PCR fragment ampli?ed from pBluescript/CBP1 was digested with BamHI and HindIII and cloned into pFASTbac1 between the BamHI and HindIII sites. The procedures to generate the recombinant baculoviruses were the same as that provided by the manufacturer. The recombinant baculoviruses obtained were designated as vTnCBP1, vTnCBP2 and vTnCBP1–CBD1 for the inserted genes cbp1, cbp2 and cbp1–cbd1, respectively. 2.6. Isolation of recombinant PM chitin binding proteins and analysis of their binding to regenerated chitin Recombinant CBP1, CBP2 and CBP1–CBD1 were produced in the insect cell line BTI-Tn-5B1 (HighFive) (Granados et al., 1994) maintained in TNM-FH medium supplemented with 10% fetal bovine serum. The insect cells were infected with vCBP1, vCBP2 and vCBP1–CBD1, respectively, and the cell culture media were collected at 72 h post-infection. To isolate the recombinant CBP1 and CBP2 from the recombinant virus infected cell culture media, 12 ml of medium from an infected culture was incubated with v 800 mg (wet weight) of regenerated chitin at 4 C in suspension for overnight in the presence of 0.2 mM Pefabloc (Roche Applied Science, Indianapolis, IN). The regenerated chitin bound with chitin binding proteins was washed ?ve times with 15 ml of PBS to remove non-chitin binding proteins and the regenerated chitin was collected by centrifugation. The recombinant CBP1 or CBP2 bound to chitin were released from the chitin by incubation with 1.5 ml of 1% Calco?uor for 1 h at room temperature and the supernatant was collected after centrifugation at 15,000 g for 5 min. The regenerated chitin pellet was extracted again with 1.5 ml of 1% Calco?uor. The Calco?uor in the protein preparations was removed by permeation gel chromato-

graphy using a 10-DG column from Bio-Rad as described above. The isolated recombinant CBP preparations in PBS were quanti?ed by Bradford method (Bradford, 1976) with BSA as standards. Isolation of the recombinant CBP1–CBD1 from the cell culture medium was similar to the isolation of recombinant CBP1 and CBP2, with some modi?cations. One hundred milliliters of vCBP1–CBD1 infected cell culture medium was incubated with 10 g (wet weight) of regenerated chitin, followed by washing the chitin with 20 mM Tris–HCl bu?er (pH 8.0). The chitin was then incubated with 2% SDS to further remove non-CBP1–CBD1 proteins. After thorough washing of the chitin with 20 mM Tris–HCl bu?er (pH 8.0), the chitin bound recombinant CBP1–CBD1 was eluted from the chitin with 1% Calco?uor. The Calco?uor was removed using a 10-DG desalting column with deionized water for elusion. Analysis of binding of chitin binding proteins to chitin was conducted as described by Wang and Granados (2000). Brie?y, recombinant chitin binding proteins were incubated with regenerated chitin to allow the proteins to bind to the chitin, followed by a thorough washing with PBS. Aliquots of the resulting chitin bound with proteins were incubated with PBS, 6 M urea, 1% Calco?uor, 2% SDS, 50 mM acetic acid, 2% SDS ? 5% b-mercaptoethanol and 1 M NaCl, respectively. After a 1 h incubation, the supernatants containing proteins released from the chitin were collected and analyzed by SDS-PAGE. 2.7. Analysis of CBP1, CBP2 and PM proteins for their glycosylation with lectins A total of 0.2 lg of isolated recombinant CBP1 and CBP2 proteins were separated by a 7.5% SDS-PAGE gel and transferred onto Immobilon-P membrane (Millipore, Billerica, MA). The membrane blot was probed with digoxigenin-labeled peanut agglutinin (PNA) and galanthus nivalis agglutinin (GNA) to examine whether the proteins have glycosylations similar to the IIM (Wang and Granados, 1997a) by following the manual provided by the manufacturer (Roche Applied Science, Indianapolis, IN). T. ni PM total proteins (proteins extracted from the PM by boiling in the SDS-PAGE sample bu?er) were also analyzed for their glycosylation using the lectins, PNA and GNA, as described above. 2.8. Trypsinization of recombinant CBP1 and CBP2 and subsequent chitin binding analysis Trypsinization of recombinant CBP1 and CBP2 was conducted by incubating 4 lg of recombinant CBP1 or CBP2 with 20 lg of recombinant trypsin (Roche Applied Science, Indianapolis, IN) in 400 ll of 0.1 M Tris–HCl

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bu?er (pH 8.8) at 37 C for 22 h. To analyze the chitin binding activity of the protein fragments of the CBP1 and CBP2, the trypsinized fragments were incubated v with 80 mg of regenerated chitin (wet weight) at 4 C for 6 h. After washing with 1.5 ml of PBS for three times, the chitin bound protein fragments were solubilized from the chitin by boiling in 50 ll of SDS-PAGE sample bu?er for 5 min and a 15 ll aliquot of the supernatant was analyzed by SDS-PAGE analysis. 2.9. Western blot analysis Western blot analysis of chitin binding proteins from T. ni PMs was performed by separating the proteins with a 7.5% SDS-PAGE gel and then transferring onto Immobilon membrane. The blots were probed with a?nity puri?ed antibodies to CBP1 or antibodies to CBP2 after incubation with 3% BSA and positive reactions were detected with a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase followed by the colorimetric reaction with bromochloroindolyl phosphate/nitro blue tetrazolium as the substrate. 2.10. N-terminal protein sequencing of CBP1–CBD1 The N-terminal sequence of the secreted recombinant protein CBP1–CBD1 was determined by a microsequencing method (Ausubel et al., 2003). Brie?y, a sample of recombinant CBP1–CBD1 was separated by 15% SDSPAGE and the protein was transferred onto Immobilon membrane. The CBP1–CBD1 protein band was excised after a brief staining of the blot with 0.1% Coomassie blue in 50% methanol and destaining with 10% acetic acid and 50% methanol. Microsequencing of the protein was performed at the protein analysis facility at the BioResource Center of Cornell University. 3. Results 3.1. Identi?cation and sequencing of cDNAs for PM chitin binding proteins Screening of the T. ni midgut cDNA expression library with antibodies against chitin binding proteins resulted in positive staining of 0.55% of the clones. Of these positive clones, 18% were also positively stained by antibodies to the IIM, suggesting that 0.45% of the total cDNA clones in the library expressed the nonIIM PM chitin binding proteins. The cDNA clones that were positive by staining with the antibodies to the chitin binding proteins, but were negative by staining with antibodies to the IIM, were considered to code for new PM chitin binding proteins and were randomly selected for sequencing. Sequencing of both the 50 and 30 ends of the cDNA inserts of 43 randomly selected positive clones showed

v

that the majority of the selected clones were identical except for the di?erence in the cDNA length between some clones. From these selected cDNA clones, two representative cDNAs in full length were identi?ed and designated as cbp1 (GenBank accession no. AY345124) coding for chitin binding protein 1 (CBP1) and cbp2 (GenBank accession no. AY345125) coding for chitin binding protein 2 (CBP2), respectively. cbp1 is 3679 bp in length containing an open reading frame (ORF) of 3516 bp, followed by an AT rich 30 end region and a predicted polyadenylation signaling site ATTAAA located at 12 bp upstream of the polyA tail (Fig. 1). cbp2 is 3439 bp in length and contains an ORF of 3231 bp, followed by an AT rich region with two polyA signals AATAAA at 9 and 30 bp upstream of the polyA tail (Fig. 2). 3.2. Sequence features of CBP1 and CBP2 The deduced protein sequences from cbp1 and cbp2 indicate that CBP1 and CBP2 have 1171 amino acid residues with a predicted molecular weight of 124 kDa and 1076 amino acid residues with a predicted molecular weight of 114 kDa, respectively. Both CBP1 and CBP2 are predicted to have a 17 amino acid long signal peptide at their amino termini by the SignalP V1.1 signal peptide prediction software (Figs. 1 and 2) (Nielsen et al., 1997). Prediction of potential O-glycosylation sites using the NetOglyc 2.0 server (Hansen et al., 1998) showed that both of the proteins only have one potential O-glycosylation site, which is the threonine at position 1005 in CBP1 and the serine at position 935 in CBP2, indicating that these two PM proteins are not mucin-type PM proteins. Both of the proteins contain six potential N-glycosylation sites by Prosite protein pattern search (Falquet et al., 2002). These sequence features suggest that CBP1 and CBP2 might be N-glycosylated but are not highly O-glycosylated as the IIM (Wang and Granados, 1997b). Experimentally, staining of the recombinant CBP1 and CBP2 on the membrane blot with digoxigenin-labeled PNA and GNA showed negative results (data not shown), indicating that the recombinant CBP1 and CBP2 were neither O-glycosylated with galactose b(1–3) Nacetylgalactosamine as in the IIM nor were N-glycosylated with a terminal mannose residue. Probing of total PM proteins with the lectins showed that both PNA and GNA primarily reacted to the IIM. The pro?les of the lectin binding analysis with PNA and GNA were identical to the Western blot analysis using antibodies to the IIM, except PNA reacted to an additional protein band at 170 kDa (Fig. 3), indicating that the majority of the PM proteins are not O-glycosylated with galactose b(1–3) N-acetylgalactosamine and not N-glycosylated with a terminal mannose.

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Fig. 1. Nucleotide and deduced amino acid sequences of the PM protein cDNA cbp1 (GenBank2 accession number AY345124). The translation initiation codon ATG and stop codon TAA are double underlined. The predicted signal peptide cleavage site is indicated by a vertical arrow. The potential polyadenylation signal sequence is marked in box. Twelve chitin binding domains, peritrophin-A domains, are underlined and numbered from 1 to 12 from N- to C-terminus of the protein.

CBP1 and CBP2 are rich in asparigine, aspartic acid, cysteine and proline, accounting for 12.4%, 11.0%, 8.2% and 8.7% of total amino acid residues, respectively, in CBP1 and 12.1%, 16.0%, 7.5% and 8.7% in CBP2. Both CBP1 and CBP2 are composed of tandem putative chitin binding domains (Figs. 1 and 2) with conserved sequence motifs CX14–15CX5CX9CX12CX7, which are similar to the predicted chitin binding sequences from PM proteins of T. ni and other species

(Wang and Granados, 1997b; Tellam et al., 1999) and belong to the peritrophin-A domains (Tellam et al., 1999). The schematic structures of CBP1 and CBP2 are diagramed in Fig. 4. In these two proteins, the trypsin and chymotrypsin cleavage sites are primarily located within or in the vicinity of the predicted chitin binding sequences (Fig. 4), suggesting that the trypsin and chymotrypsin cleavage sites are primarily embedded within the chitin binding domains.

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Fig. 2. Nucleotide and deduced amino acid sequences of the PM protein cDNA cbp2 (GenBank2 accession number AY345125). The translation initiation codon ATG and stop codon TAA are double underlined. The predicted signal peptide cleavage site is indicated by a vertical arrow. The potential polyadenylation signal sequence is marked in box. Ten chitin binding domains, peritrophin-A domains, are underlined and numbered from 1 to 10 from N- to C-terminus of the protein.

3.3. Localization of CBP1 and CBP2 in T. ni larvae CBP1 and CBP2 were speci?cally localized in the PM of T. ni larvae by immuno?uorescence microscopy with antibodies against CBP1 and CBP2, respectively (Fig. 5). The control antibody did not show any speci?c ?uorescent staining on the section, con?rming the positive localization of CBP1 and CBP2 in the PM. 3.4. Binding of recombinant CBP1 and CBP2 to chitin Recombinant CBP1 and CBP2 were successfully expressed in insect cells (Tn-5B1-4) as secreted proteins

using recombinant baculoviruses (Fig. 6, lanes 1 and 3). The apparent molecular weights for both recombinant CBP1 and CBP2 were more than 200 kDa, signi?cantly higher than their predicted molecular weights of 124 and 114 kDa, respectively. This phenomenon is similar to PM proteins identi?ed from other species (Tellam et al., 1999) and to the PM protein from the same species, the IIM, which shows a signi?cantly higher apparent molecular weight even after deglycosylation (Wang and Granados, 1997a). Subsequent chitin binding assays demonstrated that both of the recombinant CBP1 and CBP2 had chitin binding a?nity (Fig. 6,

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lanes 2 and 4). The CBP1 and CBP2 tightly bound to chitin and did not dissociate from the chitin following treatment with PBS, 50 mM acetic acid and 1 M NaCl. The proteins were only partially dissociated with 6 M urea and 2% SDS. However, they were solubilized from the bound chitin by 2% SDS in the presence of 5% b-mercaptoethanol or by 1% Calco?uor (Fig. 7), which is similar to the characteristics of solubilizing proteins from T. ni PMs (Wang and Granados, 2000). 3.5. Con?rmation of the chitin binding activity of the putative chitin binding domain A fragment of the CBP1 containing a single chitin binding domain, CBP1–CBD1, was expressed in insect cells with the recombinant baculovirus, vTnCBP1– CBD1. The recombinant CBP1–CBD1 protein was secreted into the cell culture medium and showed chitin binding a?nity to regenerated chitin (Fig. 8). The CBP1–CBD1 appeared as a 22 kDa protein on the 15% SDS-PAGE gel, which was also signi?cantly higher than its predicted molecular weight of 11,246 Da. N-terminal amino acid sequencing of the protein isolated from the 22 kDa band showed that the N-terminal sequence of the recombinant CBP1–CBD1 is VDLDLKRQQ, con?rming that it was the CBP1–CBD1 with the 17-amino acid signal peptide removed as predicted in Fig. 1. 3.6. CBP1 and CBP2 are highly resistant to trypsin and their trypsinized fragments retain chitin binding activities Although both CBP1 and CBP2 are not heavily glycosylated mucins, they appeared to be highly resistant to degradation by trypsin, which represents the major digestive proteinase activities in the digestive tract of lepidopterous insect larvae. Partial degradation of the recombinant CBP1 and CBP2 by trypsin required a high protein:trypsin ratio of 1:5 and a prolonged incuv bation time period (22 h) at 37 C. The resulting

Fig. 3. Analysis of T. ni PM proteins with antibodies to the IIM and the lectins peanut agglutinin (PNA) and Galanthus nivali agglutinin (GNA). Lane 1: Western blot analysis of the PM proteins with antibodies speci?c to the IIM to localize the IIM on the blot. Lane 2: analysis of the PM proteins with PNA to detect the presence of Olinked galactose b(1–3) N-acetylgalactosamine on PM proteins. Lane 3: analysis of the PM proteins with GNA to detect the presence of terminal mannose residues of N-linked carbohydrate moieties on PM proteins. Thin arrows indicate the protein bands recognized by antibodies to the IIM and the thick arrow indicates the non-IIM protein band reacted to PNA.

Fig. 4. Schematic structures of the CBP1 (A) and CBP2 (B) proteins, showing their chitin binding domains (CBDs) and the distributions of trypsin and chymotrypsin cleavage sites in the proteins. The trypsin and chymotrypsin cleavage sites were identi?ed using the sequence analysis software Lasergen (DNAStar). The trypsin cleavage sites were de?ned as R^X and K^X, excluding R^P and K^P (^ indicates cleavage sites; X indicates any amino acid residues). The chymotrypsin cleavage sites were de?ned as W^X, F^X, Y^X, M^X, L^X and H^X, excluding Y^P.

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Fig. 5. Immunolocalization of CBP1 and CBP2 in T. ni larvae. Cross-sections of ?fth instar larvae were stained with anti-CBP1, anti-CBP2 or control antibodies. Phase contrast microscopy (images in the upper panel) was employed to identify larval tissues for localization of ?uorescent antibody staining (images in the lower panel).

trypsinized fragments retained their chitin binding activities (Fig. 9). 3.7. Antibodies to CBP1 and CBP2 reacted to multiple PM proteins with molecular weights lower than the intact CBP1 and CBP2 Western blot analysis of chitin binding proteins isolated from T. ni PMs using antibodies to CBP1 and

CBP2, respectively, showed that the antibodies recognized multiple PM protein bands. However, all the proteins detected by the antibodies showed their apparent molecular weights to be no more than 200 kDa (Fig. 10), which were lower than the apparent molecular weights of the recombinant CBP1 and CBP2 (Fig. 6). This result showed that intact CBP1 and CBP2 were not present in the PM, although CBP1 and CBP2 are localized in the PM (Fig. 5), indicating that fragmented CBP1 and CBP2 were present in the PM.

4. Discussion Proteins are the primary components of the PM and the formation of PMs has been suggested to involve binding of PM proteins to chitin ?brils (Wang and Granados, 1997b; Shen and Jacobs-Lorena, 1999; Tellam et al., 1999; Wang and Granados, 2001). It was previously demonstrated that the major PM proteins in T. ni larvae were chitin binding proteins that include the IIM (Wang and Granados, 2000). In this study, we identi?ed two new PM chitin binding proteins, CBP1 and CBP2, from T. ni and found that cDNA clones for these two proteins were abundantly present in the nonnormalized midgut cDNA expression library, which is in agreement with the previous observation that the majority of the PM proteins were chitin binding proteins. cDNA clones recognized by antibodies to PM chitin binding proteins but not by antibodies to the IIM represented an abundance of 0.45%, which is 3.5 times more abundant than those for the IIM. Sequence features of the CBP1 and CBP2 suggest that these two proteins are not heavily glycosylated and lectin binding analysis of the recombinant CBP1

Fig. 6. SDS-PAGE analysis of recombinant CBP1 and CBP2 expressed in insect cells using recombinant baculoviruses. Lanes 1 and 3: culture media from HighFive cells infected with vCBP1 (Lane 1) or vCBP2 (Lane 3), showing that the recombinant CBP1 and CBP2 were secreted into the medium. Lanes 2 and 4: chitin bound proteins from the media of vCBP1 (Lane 2) and vCBP2 (Lane 4) infected HighFive cells.

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Fig. 7. SDS-PAGE analysis of recombinant CBP1 (panel A) and CBP2 (panel B) dissociated from the CBP1/regenerated chitin or CBP2/regenerated chitin complex by PBS, 6 M urea, 1% Calco?uor, 2% SDS, 50 mM acetic acid, 2% SDS ? 5% b-mercaptoethanol and 1 M NaCl, respectively.

and CBP2 further indicated that they were not glycosylated proteins as the IIM. Moreover, lectin binding studies of total extractable PM proteins also showed that the majority of the PM proteins were not glycosylated as the IIM (Fig. 3). Although the CBP1 and CBP2 are not highly glycosylated proteins, these two proteins are highly resistant to trypsin degradation (Fig. 9). In lepidopteran larvae, trypsins and chymotrypsins are the predominant digestive proteinases in the midgut. Pri-

mary sequence analysis of CBP1 and CBP2 showed that the potential cleavage sites for trypsin and chymotrypsin reside primarily in the chitin binding domains (Fig. 4). Therefore, most of the trypsin and chymotrypsin cleavage sites are protected against midgut digestive proteinases by being buried in the chitin bind-

Fig. 8. SDS-PAGE analysis of chitin binding proteins isolated from cell culture media of uninfected HighFive cells (lane 1) and HighFive cells infected with the wild-type baculovirus (AcNPV) (lane 2) and with recombinant virus vCBP1–CBD1 (lane 3). The CBP1–CBD1 protein band was shown as a 22 kDa protein.

Fig. 9. SDS-PAGE analysis of trypsinized recombinant CBP1 and CBP2 fragments that were bound with regenerated chitin. Lanes 1 and 3: puri?ed recombinant CBP1 and CBP2, respectively. Lanes 2 and 4: chitin bound trypsinized protein fragments of recombinant CBP1 and CBP2, respectively.

P. Wang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 215–227

225

Fig. 10. Western blot analysis of T. ni PM chitin binding proteins with antibodies to the total PM chitin binding proteins, CBP1 and CBP2, respectively. A Western blot using the pre-immune serum (1:5000 dilution) was also included to con?rm the speci?c antibody staining of the PM proteins.

ing domains, which is critically important since PM proteins must function in an environment very rich in proteinases. This observation is consistent with the early reports that the intradomain disul?de bonds, which stabilize the CBD structure, in PM proteins play a signi?cant role for the resistance of the proteins to digestive proteinases (Elvin et al., 1996; Wang and Granados, 1997b). Therefore, the non-glycosylated PM proteins or the non-glycosylated regions of PM proteins become resistant to digestive proteinases as the result of: (1) the limited presence of proteinase cleavage sites outside the CBDs; (2) the restriction of the exposure of proteinase cleavage sites within the CBDs to the gut proteinases by the intradomain disul?de bonds; and (3) further restriction of exposure of the proteins to gut proteinases by the binding of the proteins to the chitin. To date, PM proteins identi?ed from several insect species are all chitin binding proteins with or without mucin domains and may contain up to six putative chitin binding domains. The presence of multiple chitin binding domains in PM proteins has been suggested to be a mechanism for PM formation (Wang and Granados, 2001). In this study, the chitin binding activity of a single putative PM protein chitin binding domain was experimentally con?rmed (Fig. 8), validating the previous predictions of chitin binding domains in PM proteins (Elvin et al., 1996; Wang and Granados, 1997b; Shen and Jacobs-Lorena, 1998). The CBP1 and CBP2 identi?ed from T. ni larval PMs are composed of 12 and 10 chitin binding domains, respectively, and are speci?cally localized in the PM (Fig. 5).

However, their intact forms appeared to be absent in the PM (Fig. 10). Instead, PM chitin binding proteins identi?ed with antibodies to CBP1 and CBP2 all showed molecular weights lower than the intact recombinant CBP1 and CBP2 (Fig. 10). These ?ndings suggest that CBP1 and CBP2 are associated with the PM as partially degraded fragments. The presence of 10–12 chitin binding domains in the proteins allows the partially degraded proteins to contain multiple chitin binding domains in the fragments which retain their functions in the PM formation. The observations that (1) the trypsin and chymotrypsin cleavage sites in CBP1 and CBP2 are mostly located within the chitin binding domains and (2) the proteins are composed of numerous chitin binding domains, indicated a mechanism for these non-mucin PM proteins to adapt and function in the proteinase rich gut environment. Analysis of PM chitin binding proteins with antibodies to CBP1 and CBP2 suggested that the PM proteins resolved by SDS-PAGE might be fragments of a few original high molecular weight proteins that are partially degraded by digestive proteinases. The PM proteins could be degraded before and/or after being assembled into the PM structure (Fig. 11). Therefore, a signi?cant number of T. ni PM proteins identi?ed by SDS-PAGE could be fragments of their original high molecular weight PM proteins, CBP1 and CBP2. In T. ni, the pro?le of PM proteins is rather complex based on SDS-PAGE analysis, and the majority of these proteins are chitin binding proteins (Wang and Granados, 2000). The results from this study suggest that the complex pro?le of PM proteins may originate from a smaller number of high molecular weight PM proteins, such as the CBP1 and CBP2. Therefore, it is possible that the permeability of the PM may be a?ected by the extent of degradation of the PM proteins, or the size of the PM protein fragments. Further studies will be needed to determine if the PM permeability is regulated by the extent of PM chitin binding protein degradation. The chitin binding sequences in CBP1 and CBP2 contain the same sequence motifs found in the chitin binding domains in the IIM identi?ed from the same insect (Wang and Granados, 1997b). Alignment of the chitin binding sequences from the three T. ni PM proteins with the Jotun Hein algorithm (Hein, 1990) using the DNAStar software package (DNASTAR, Madison, WI) indicates that the chitin binding domains in CBP1 and CBP2 have a higher degree of homology between the two proteins than to those in the IIM (Fig. 12). CBD1, 2, 3, 9, 10, 11 and 12 in CBP1 are highly similar to CBD1, 2, 3, 7, 8, 9 and 10 in CBP2, respectively. CBD4, 5, 6 and 7 in CBP1 are closely related. Similarly, CBD4, 5 and 6 in CBP2 are highly similar. These sequence similarities between the chitin binding domains suggest that these two proteins evolved from

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Fig. 11. A proposed model showing that high molecular weight multi-chitin binding domain PM proteins are assembled into the PM as partially degraded fragments.

References
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Stuhl, K., 2003. Current Protocols in Molecular Biology. John Wiley and Sons, New York. Bansil, R., Stanley, E., LaMont, J.T., 1995. Mucin biophysics. Ann. Rev. Physiol. 57, 635–657. Bolognesi, R., Ribeiro, A.F., Terra, W.R., Ferreira, C., 2001. The peritrophic membrane of Spodoptera frugiperda: secretion of peritrophins and role in immobilization and recycling digestive enzymes. Arch. Insect Biochem. Physiol. 47, 62–75. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Casu, R., Eisemann, C., Pearson, R., Riding, G., East, I., Donaldson, A., Cadogan, L., Tellam, R.L., 1997. Antibody-mediated inhibition of the growth of larvae from an insect causing cutaneous myiasis in a mammalian host. Proc. Natl. Acad. Sci. USA 94, 8939–8944. Elvin, C., Vuocolo, T., Pearson, R., East, I.J., Riding, G., Eisemann, C., Tellam, R.L., 1996. Characterization of a major peritrophic membrane protein, peritrophin-44, from the larvae of Lucilia cuprina: cDNA and deduced amino acid sequences. J. Biol. Chem. 271, 8925–8935. Falquet, L., Pagni, M., Bucher, P., Hulo, N., Sigrist, C.J., Hofmann, K., Bairoch, A., 2002. The PROSITE database, its status in 2002. Nucleic Acids Res. 30, 235–238. Forstner, J.F., Forstner, G.G., 1994. Gastrointestinal mucus. In: Johnson, L.R. (Ed.), Physiology of the Gastrointestinal Tract. Raven Press, New York, pp. 1255–1283. Granados, R.R., Li, G., Derksen, A.C.G., McKenna, K.A., 1994. A new insect cell line from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis virus. J. Invertebr. Pathol. 64, 260–266. Hansen, J.E., Lund, O., Tolstrup, N., Gooley, A.A., Williams, K.L., Brunak, S., 1998. NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility. Glycoconjugate J. 15, 115–130. Hein, J.J., 1990. Uni?ed approach to alignment and phylogenies. Methods Enzymol. 183, 626–645. Lehane, M.J., 1997. Peritrophic matrix structure and function. Ann. Rev. Entomol. 42, 525–550. Lichtenberger, L.M., 1995. The hydrophobic barrier properties of gastrointestinal mucus. Ann. Rev. Physiol. 57, 565–583.

Fig. 12. Dendrogram generated from Jotun Hein alignment of chitin binding domains from T. ni PM proteins.

the same origin by gene duplication and the presence of multiple chitin binding domains in these proteins resulted from domain duplication. Acknowledgements This work was supported in part by the Cooperative State Research, Education, and Extension Service, US Department of Agriculture, under agreement 99-353028083 and by the Cornell University Agricultural Experiment Station federal formula funds, project no. NYG-621510 received from Cooperative State Research, Education, and Extension Service, US Department of Agriculture.

P. Wang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 215–227 Nielsen, H., Engelbrecht, J., Brunak, S., von Heijne, G., 1997. Identi?cation of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6. Okuno, S., Takatsuka, J., Nakai, M., Ototake, S., Masui, A., Kunimi, Y., 2003. Viral-enhancing activity of various stilbenederived brighteners for a Spodoptera litura (Lepidoptera: Noctuidae) nucleopolyhedrovirus. Biol. Control 26, 146–152. Peters, W., 1992. Peritrophic Membranes. Springer, New York. Richards, A.G., Richards, P.A., 1977. The peritrophic membranes of insects. Ann. Rev. Entomol. 22, 219–240. Sarauer, B.L., Gillott, C., Hegedus, D., 2003. Characterization of an intestinal mucin from the peritrophic matrix of the diamondback moth, Plutella xylostella. Insect Mol. Biol. 12, 333–343. Schorderet, S., Pearson, R.D., Vuocolo, T., Eisemann, C., Riding, G.A., Tellam, R.L., 1998. cDNA and deduced amino acid sequences of a peritrophic membrane glycoprotein, ‘peritrophin-48’, from the larvae of Lucilia cuprina. Insect Biochem. Mol. Biol. 28, 99–111. Shen, Z., Jacobs-Lorena, M., 1998. A type I peritrophic matrix protein from the malaria vector Anopheles gambiae binds to chitin. Cloning, expression, and characterization. J. Biol. Chem. 273, 17665–17670. Shen, Z., Jacobs-Lorena, M., 1999. Evolution of chitin-binding proteins in invertebrates. J. Mol. Evol. 48, 341–347. Spence, K.D., 1991. Structure and physiology of the peritrophic membrane. In: Binnington, K., Retnakaran, A. (Eds.), Physiology of the insect epidermis. CSIRO Publications, Victoria, Austria, pp. 77–93. Tellam, R.L., Wij?els, G., Willadsen, P., 1999. Peritrophic matrix proteins. Insect Biochem. Mol. Biol. 29, 87–101.

227

Tellam, R.L., Vuocolo, T., Eisemann, C., Briscoe, S., Riding, G., Elvin, C., Pearson, R., 2003. Identi?cation of an immuno-protective mucin-like protein, peritrophin-55, from the peritrophic matrix of Lucilia cuprina larvae. Insect Biochem. Mol. Biol. 33, 239–252. Vuocolo, T., Eisemann, C.H., Pearson, R.D., Willadsen, P., Tellam, R.L., 2001. Identi?cation and molecular characterisation of a peritrophin gene, peritrophin-48, from the myiasis ?y Chrysomya bezziana. Insect Biochem. Mol. Biol. 31, 919–932. Wang, P., Granados, R.R., 1997a. An intestinal mucin is the target substrate for a baculovirus enhancin. Proc. Natl. Acad. Sci. USA 94, 6977–6982. Wang, P., Granados, R.R., 1997b. Molecular cloning and sequencing of a novel invertebrate intestinal mucin cDNA. J. Biol. Chem. 272, 16663–16669. Wang, P., Granados, R.R., 2000. Calco?uor disrupts the midgut defense system in insects. Insect Biochem. Mol. Biol. 30, 135–143. Wang, P., Granados, R.R., 2001. Molecular structure of the peritrophic membrane (PM): identi?cation of potential PM target sites for insect control. Arch. Insect Biochem. Physiol. 47, 110–118. Wij?els, G., Eisemann, C., Riding, G., Pearson, R., Jones, A., Willadsen, P., Tellam, R., 2001. A novel family of chitin-binding proteins from insect type 2 peritrophic matrix. cDNA sequences, chitin binding activity, and cellular localization. J. Biol. Chem. 276, 15527–15536. Zimmerman, D., Peters, W., 1987. Fine structure and permeability of peritrophic membranes of Calliphora erythrocephala (Meigen) (Insecta: Diptera) after inhibition of chitin and protein synthesis. Comp. Biochem. Physiol. 86b, 353–360.


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