CA2331921A1 - A novel invertebrate intestinal mucin cdna and related products and methods - Google Patents

Abstract

The invention represents the disclosure of a novel insect intestinal mucin.
The IIM protein was been identified and cloned using T. ni larva. The cDNA and amino acid sequences have been determined and are disclosed. The novel protein has an approximate molecular mass of 400 kDa. These sequences are useful for the production of transgenic or recombinant vectors including viral, microorganism, cell, plant, or animals, wherein the virus, microorganism, cell, plant, or animal is the product of an insertion of a gene expression vector including a DNA that encodes an IIM protein sequence. Thereafter the engineered host of the IIM DNA sequence is capable of expressing said IIM
protein in a functional form. Also useful is a purified and isolated recombinant DNA sequence comprising a DNA sequence that codes for an IIM
protein. The recombinant DNA sequence used can be a cDNA sequence for either IIM14 or IIM22, SEQ. ID.'s No. 1; and 2 respectively. The current invention also provides for the use of the purified amino acid sequences of IIM14 or IIM22, SEQ. ID.'s 3 or 4 respectively. With this knowledge of the proteinaceous components of the PM, and particularly the mucin-like proteins it will be possible to enhance the effectiveness of bio-engineered pesticides, recombinant viral vectors, enhance the defenses of transgenic plants, or protect insect vectors susceptible to attack by organisms utilizing enhancin or enhancin-like enzymes.

Description

A NOVEL INVERTEBRATE INTESTINAL MUCIN cDNA
AND RELATED PRODUCTS AND METHODS
FIELD OF THE INVENTION
The invention pertains to the field of proteins associated with the peritrophic membranes of insects. More particularly, the invention pertains to a novel invertebrate intestinal mucin cDNA and related products and methods.
BACKGROUND OF THE INVENTION
Vertebrate epithelial organs are covered, throughout the body, with a mucus lining, which serves as a selective physical barrier between extracellular contents and the epithelial cell surface. The mucus lining, especially in the gastrointestinal tract, is highly resistant to various digestive enzymes and provides protection and lubrication for the underlying cells. The protective functions of the mucosal layer are largely dependent upon heavily glycosylated proteins known as mucins. Mucins play an active role in preventing bacterial, viral, and other pathogens from interacting with vertebrate intestinal epithelia.
Mucins are highly p-glycosylated proteins. Carbohydrate moieties on mucins commonly account for more than 50% of the protein by weight. The biochemistry and molecular biology of mucins from vertebrates has been broadly investigated, with ~SUBST11'L)TE SHEET (Rule 26) human epithelial mucins being the most extensively studied. Several mucins from humans and other vertebrates gave been completely or partially sequenced, and this has contributed to a greater understanding of their structwe and function. Full cDNA
sequences for human mucin MUC 1, MUC2, and MUC7, have been obtained. In addition, mucins from other vertebrates, including mouse MUC-1, rat ascites sialo-glycoprotein-1, canine tracheolbronchial mucin, bovine submaxillary mucin-like protein, and frog BM-A.1, have also been fully sequenced by cDNA cloning.
Studies on invertebrate mucins are very limited in comparison with vertebrate mucins. Drosophila melanogaster "glue proteins" from salivary glands have structural characteristics of mucin-like proteins. These "glue protein" have been sequenced but their function has not been fully determined. Mucin-like proteins have also been reported in protozoans. A secretory mucin involved in maintaining the cohesiveness of a clutch of a squid egg-mass formation was identified from that animal's nidamental gland. A glycoprotein from Drosophila melanogaster cultured cells was reported to be a mucin-like protein. Recently, a membrane-associated mucin from the hemocytes of Drosophila. melanogaster was identified, and a cDNA for the mucin was subsequently cloned. However, to date, there have been no reports on mucins identified from invertebrate digestive tracts.
Part of the reason for this may be that insects do not possess a mucus layer lining the digestive tract and/or other epithelial cells, as do vertebrates.
The digestive tract in insects is commonly li ed with an invertebrate-unique structure, the peritrophic membrane (PM). PMs are non-cellular matrices composed primarily of chitin, protein, and glycoproteins. PMs demonstrate a protective function similar to the mucus layer in vertebrates (e.g. a selective barrier protecting the digestive tract from physical damages and microbial infections).
Although there are few studies on the interaction between microbial pathogens and PMs, these structures are proposed to serve as a physical barrier to invasion or infection by pathogenic micra~organisms. The chitin component of PMs is normally present as a network of chitin fibrils in which proteins and glycoproteins are present.
The chitin can be a potential target substrate for intestinal pathogens. This was SUBSTIT><JTE SHEET (Rule 26) demonstrated through the degradation of chitin in the PM by a pathogen-encoded chitinase allowing an avian malaria parasite to overcome its mosquito vector intestinal PM barrier and infect the vector itself.
Proteins are the major 1'M component; however, their functions in the PM are unknown. Studies on the PM proteins are limited to analyses of the amino acid composition of total PM proteins and PM protein profiles as determined by electrophoresis. The only PM protein characterized to date, peritrophin-44, was isolated from Lucille cuprina larvae, but its biological function is not fully understood. To date, studies on the interaction of PM proteins with microbial pathogens are limited to the effect of a baculovirus enhancin on lepidopteran PM proteins.
Previous studies have demonstrated that a Trichoplusia ni ganulosis virus (TnGV) encodes an enhancin protein, a viral enhancing protein, that was identified as a metalloprotease. Enhancin degrades high molecular weight PM proteins in vivo and in vitro. In addition, the protein ~degadation initiated by these enhancins is correlated with the disruption of the stru<~ural integrity of the PM thereby "enhancing"
viral infection. It was recently demonstrated that enhancin could degade high molecular weight PM proteins from several lepidopterous species; however, the chemical nature and function of these proteins in baculovirus pathogenesis were previously unknown.
With a more complete knowledge of the proteinaceous components of the PM, and particularly the mucin-like proteins it will be possible to use that information to enhance the effectiveness of bio-engineered pesticides, recombinant viral vectors, enhance the defenses of transgenic plants, or protect insect vectors susceptible to attack by organisms utilizing enhancin or enhancin-like enzymes.
SUMMARY OF T$E INVEN1TON
Briefly stated the current invention represents the disclosure of a novel intestinal insect mucin compriising two nearly identical isoforrns, IIM14 and SUBSTITUTE SHEET (Rule 26) respectively. The proteins are identical except for slightly different peptide length in some repetitive regions, which is commoon in mucin proteins. This IIM protein has been identified and cloned frorn T. ni larva. Its cDNA and amino acid sequences have been determined and are disclosed. The IIHi protein has an approximate molecular mass of 400 kDa. These sequences are useful for the production of transgenic or recombinant vectors including viral, microorganism, cell, plant, or animals, wherein the virus, microorganism, cell, plant, or animal is the product of an insertion of a gene expression vector including a I)NA that encodes an IIM protein sequence.
Thereafter the engineered host of the IIM DNA sequence is capable of expressing said IIM
protein in a functional form. One easiliy used host is the bacteria is Escherichia coli.
Also useful is a purified and isolated recombinant DNA sequence comprising a DNA sequence that codes for an IIM protein. The recombinant DNA sequence used can be a cDNA sequence for eiither IIM14 or IIM22, SEQ. ID.'s No. 1; and 2 respectively. 7.'he current invention also provides for the use of the purified or recombinant proteins, IIM14 or IIM22, SEQ. ID.'s 3 or 4 respectively.
With the cloned IIM sequence it is possible to prepare an IINi protein or peptide by transforming a host cell with an expresssion vector comprising a promoter operatively linked to a nucleotiide sequence which codes for a fusion protein wherein said fusion protein comprises a~ first protein or peptide fused directly or indirectly with a transfer molecule said first protein or peptide being a predetermined protein or peptide of a T. ni IIll~i protein. Then culturing the host cell under conditions such that the fusion protein is expressed in recoverable quantity. When harvesting the protein or peptide the cells must be collected, isolated, lysed, and the fusion protein purified from the cytosol.
A gene expression vector containing a recombinant DNA sequence encoding a T. ni TINi protein sequence can also be constructed with this technology. This is accomplished through the use of a recombinant plasmid adapted for insertion into and transformation of bacteria or tz~ansgenic plants such that these hosts can express either the IIM protein or antibodies to disrupt pertrophic membrane function and formation in larval pests. The antibodies e~:pressed by the plant could bind to the mucin or its ligand SUBSTITUTE SHEET (Rule 26) or portions the IIM protein could be expressed by the plant to result in competive binding with the larvae's expressed mucin. As oppossed to transformation with the entire IIM sequence, important peptide fragments or functional domains of the IIM
protein can individually be transfected into expression vectors.
BRIEF DF;SCRIPTION OF THE DRAWING
FIG. 1 shows a schematic structure of the IIM protein.
FIG. 2 shows that greater amounts of FITC-dextran (3.2 nm dia) diffused across the peritrophic membrane of ligated T.ni alimentary canals taken from larvae fed on IgG containing diet for 2.5 hours.
FIG. 3 shows that the presence of IgG increased larval mortality due to AcMNPV
infection.
FIG. 4 shows the chitin binding regions of the IIM protein shown in SEQ. D7.
NO. 3 and SEQ. ID. NO. 4.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The following detailed description describes the methods used to discover and sequence a novel invertebrate iintestinal mucin (IIM), isolate cDNAs encoding this novel mucin, and determine the role of this mucin in the function of the peritrophic membrane during infection by a pathogenic viral organism.
Isolation and Anal~rsis of A Novel Invertebrate Intestinal Mucin PMs have long been proposed as selective physical barriers in invertebrate intestines. The primary components of PMs include chitin protein and glycoprotein, but SUBSTTTI1TE SHEET (Rule 26) WO 99/67373 PCTlUS99/14220 only one PM protein has been isolated and characterized thus far. PMs in invertebrates is analogous to vertebrate intestinal mucosal components that are secreted by epithelial cells. These vertebrate mucus secretions are composed primarily of one major constituent, intestinal mucin. bntestinal mucins from humans have been broadly studied, and the major human intestinal mucin (MUC2) was fully sequenced. Prior to the present invention, no intestinal. mucin had been identified from invertebrates.
The present invention shows a novel invertebrate intestinal mucin ~. The novel mucin was first isolated from an insect larvae, T. »i larvae from a laboratory colony reared on a high wheat-germ diet. Midgut PM was dissected from mid-fifth instar T. ni larvae, thoroughly rinsed with de-ionized water, and stored at -70°C. PM
proteins were solubilize by boiling PMs in SDS/PAGE sample buffer, and then separated by SDS/PAGE electrophoresis The IIM protein disclosed herein is a new type of mucin that represents the first intestinal mucin identified from an invertebrate.
To prepare IIM for antiserum production, protein bands can be first visualize by staining the gel with 0.05% Coomassie blue R-250 in 40% methanol follows by de-staining with de-ionized water; this procedure can be followed by excision of the ZIM band from the eletrophore;sis gel. After equilibration in a SDS/PAGE
running buffer, the IIM in the gel slice is electroeluted, and the preparation is purified and concentrated and re-suspended) in PBS by ultrafiltration using a centriprep-30 concentrator (Amicon).
For general biochemic~~l analyses, PM protein bands on the SDS/PAGE gel can be initially visualized by copper staining, which facilitates the excision of the iIM band.
UM from this gel slice is also electroelute after copper ions are removed by washing the gel slice several times in 0..2 M EDTA. Subsequently, the elute protein preparation is desalted by ultrafiltration.
To isolate and purify the IIM protein for amino acid composition analysis, the sodium phosphate-bui~ered SI)S/PAGE system is used. The gel is stained with copper chloride after equilibration of the gel in 0.375 M Tris-HCI (pH 8.8) with 0.1%
SDS.
The IIM band is excised and tlhe IIM is recovered by electroelution as described above;
SUBSTTTUTE SHEET (Rule 26) the preparation is further desalted by extensive dialysis against de-ionized water and then lyophilized.
TIM from T. ni PMs is a 400-kDa protein on 3.5% SDS/PAGE gels. The association of the IIM with PHis is stable over a wide range of pH, in the presence of non-ionic and ionic detergents., and in the presence of protein denaturing reagents.
Therefore, very little, or no IIl~ri was present in the supernatants from these treatments.
IIM, the predominant PM protein, could be released from the PM by a combination of 2% SDS plus S mM DTT, confirming that it was strongly associated with the chitin-containing PM matrix. The IIIV was not extracted from the PM by boiling in 2%
SDS
for 10 min unless a reducing agent was included, demonstrating the presence of intermolecular disulfide bonding in native IIM
Amino acid composition analysis of IIM, indicated that IINI was rich in threonine (18.'7%) proline (16.9%), and alanine (15.9%). These three amino acids accounted for 51.5% of the total amino acid residues in the protein, while aromatic amino acids accounted for les:~ than 5% of the amino acid residues in the protein, and may account for the ability of Invi to by strongly associated to the invertebrate PM
chitin fibrils. The IIM amino .acid composition profile resembles that of a typical vertebrate mucin that is commonly rich in threonine, serine, proline, alanine, and glycine, and rare in aromatic amino acids.
Quantification of the protein and carbohydrate content of IIM indicated that it was highly glycosylated. Carbohydrate content on IIM accounted for 56% of the total IIM mass, with protein accounting for 44%. Terminal mannose residues and galactose B(1-3) N-acetylgalactosamine were detected on TIM by the specific binding of peanut agglutinin and Galanthus nivalis agglutinin (GNA). The lectin binding assays using IIM samples pretreated with either O-glycosidase or N-glycosidase showed no binding or significantly reduced binding of the lectins, confirming the positive recognition of G.
nivalis agglutinin and peanut ;agglutinin to IIM. These results demonstrated that lIM
has both N-glycosylation and O-glycosylation, since terminal mannose is present in N-linked carbohydrate moieties .and galactose B(1-3)N-acetylgalactosamine is one type of O-linked carbohydrate moiety found in glycoproteins. In addition, removal of the ~SUBSTTTfJTE SHEET (Rule 26) disaccharide, galactose D(1-3) N-acetylgalactosamine by O-glycosidase treatment, resulted in significant reduction (approx. 100 kDa) in the molecular weight of the BM, further confirming the heavy O-glycosylation on IIM.
The experiments conducted demonstrated the highly protease-resistant nature of the isolated IIM. The stability of the IIM when exposed to degestive enzymes for long periods is aided by the O-linked carbohydrate moieties found in associated glycoproteins. The IIM was highly resistant to endogenous digestive even after a sixteen hour incubation, no degadation of IIM in PMs was observed. However, in the presence of O-glycosidase, IIr!i was quickly degaded. Control treatments using PMs with inactivated or inhibited endogenous midget proteases, confirmed that the degadation of IIM in the presence of O-glycosidase was a result of hydrolysis by endogenous digestive protease;s, following removal of the protective carbohydrate moiety, galactose 13(1-3) N-acetylgalactosamine.
The isolated and sequenced IIM from T. ni PM resembles mammalian secretory mucins in several characteristics, including high O-glycosylation, possible intermolecular cross-linking disulfide bonds, high concentrations of threonine alanine and proline, and resistance to proteases. Selective removal of galactose !3(I-3) N-acetylgalactosamine resulted in geatly increased susceptibility to proteolysis indicating that this O-Linked disaccharide plays an important role in protecting the IIM
protein from digestive degadation. Unike vertebrate mucins, insect PM proteins are embedded in a chitin fibril network. The iinability to extract the IIM from PMs with various detergents and extreme conditions in the absence of a reducing agent demonstrate that IIM is tightly associated with the chitin-rich PM matrix and that disulfide bonding is seemingly important for this a;3sociation.
Isolation and Seduencing of .A Novel Invertebrate Intestinal Mucin cDNA
The present invention leaches cloned and sequenced full-length cDNAs for IIM
from T. rri. IINi has a similar structural organization to human intestinal mucin, MUC2, SUBSTITUTE SHEET (Rule 26) and is expressed in midget tissue. Sequencx analysis indicates potential chitin binding domains that may interact with the chitin present within the PM.
A cDNA expression literary was constructed from T. ni midget mRNA. Midget epithelial tissues were dissected from early to mid-fifth instar T. ni larvae in cold Rinaldini's solution. PMs with food contents and other attached tissues (i.e.
fat bodies, trachea, and malphighian tubules) were quickly removed from the midget epithelium.
Isolated midget epithelia were rinsed with cold Rinaldini's solution, quickly frozen in liquid nitrogen, and stored at -70°C prior to use. Midget mRNA was isolated using the RNeasy total RNA isolation ki.t and the Oligotex mRNA isolation kit (Qiagen Inc., Chatsworth, CA), according to the manufacturer's specifications. The quality of mRNA
was confirmed by Northern blot analysis, which showed no detectable degradation of mRNA after probing with ~i-tu~bulin DNA. The cDNA library was constructed from T.
ni midget mRNA using the ZAP-cDNA Gigapack Cloning Kit (Stratagene, La Jolla, CA), following the manufacture's instructions. cDNA was unidirectionally ligated into the Uni-ZAP XR vector (Stratagene, La Jolla, CA) between the EcoRI and Xhol sites and packaged with the Gigapack II Gold package extract. The resultant cDNA
library was amplified once at 50,000 ~plaques/15-cm plate in XL1-Blue MRF E. coli host cells.
The library has a complexity of 2.35x106 plaques, of which over 99.5% were recombinants. Screening of the cDNA expression library for IIM cDNA clones was conducted using an IIM-specif c polyclonal antiserum in conjunction with the pico Blue Immunoscreening Kit (Stratagene, La Jolla, CA), according to the manufacturer's specifications. The first round of screening was performed at a high density (i.e. 50,000 plaques/15-cm plate). Positive plaques were selected and further purified by screening at a low plating density (i.e. 20-50 plaques/10-cm plate). From purified positive phages the pBluescript SK (-) phagemid (Stratagene, La Jolla, CA) was excised in vivo following the ZAP-cDNA Gip;apack cloning kit protocol.
Screening of the library with the antiserum specific to IIM indicated that the mRNA for the IIM was abundant; 50 positive plaques were obtained from 50,000 plaques. Since only one in thr~~ plaques will be in the correct reading frame for protein expression, the frequency of hQvt cDNA clones could be 1 in 333. From these 50 SUBSTTT'UTE SHEET (Rule 26) plaques, 20 positive plaques were further purified. From these 20 plaques, the pBluescript SK(-) phagemids vwere rescued by in vivo excision. Following restriction enzyme analysis to map the selected clones, two different full-length clones, plIHil4 and pI1Ht22, were chosen for sequencing.
Nested deletions from >;~oth orientations of the cDNA inserts were constructed using the Erase-a-Base System (Promega Corp., Madison, WI). Both strands of the cDNA were sequenced by automated cycle sequencing using T3 and T7 primers, complementary to the pBluescript SK(-) sequences flanking the cDNA inserts.
DNA
sequence analysis and a data b~~se search were conducted using the DNASTAR
software package (DNASTAR Inc., Madison, WD and BLAST data base search programs. Protein O-glycosylation sites were predicted following an O-GLYCBASE
search.
The cDNAs from both pBMl4 and pIIM22 were full-length clones, encoding a protein of 788 and 807 amino acid residues, respectively. The nucleotide sequence of each is shown in SEQ. ID. NO. 1 & 2, respectively. The open reading frame in the cDNA from IBvIl4, was 57 base pairs shorter than in IIM22; otherwise, the open reading fi~ames in these two clones were identical. IIM22 contains a putative polyadenylation signal consensus, AATAAA, located 331 base pairs downstream of the translation stop colon, TAA and 17 base pairs upstream of the poly(A). BM14 contains a putative polyadenyla~tion signal, AATTAA, located 15 base pairs upstream of the poly(A).
The deduced protein se~Iuences from IIM14 and IIM22 showed a hydrophilicity profile characteristic of a signal sequence at the N terminus of protein sequences. The N-terminal amino acid sequence determined from purified IIM indicated that the cDNA
clones encode a protein containing a signal peptide 25 amino acids long and confirmed that the cDNA clones code for the IIM. The amino acid composition of the deduced proteins from IIM14 and IIM22 were very similar to the composition of IBvi isolated from T. ni further confirming that the cDNA clones code for the IIM. Protein sequence data reveal that there are four potential N-glycosylation sites. This is in agreement with the biochemical analysis results which demonstrated that LINI has N-linked STJBSTIT(TTE SHEET (Rule 26) glycosylation. The amino acid sequence of IIM14 and IIM22 is shown in SEQ. ID.
NO. 3 & 4 respectively.
Referring to FIG. 1, thE; overall nM sequences can be divided into six distinct regions based upon their sexlue,nce features. Figure 1 shows a schematic structure of the IIM protein. The amino aciid composition of each region shows characteristics of a secreted epithelial mucin. Both the N-terminal and C-terminal domains, are rich in cysteine, which accounts for 8.2 and 7.8% of the total amino acid residues, respectively. Region III is rich in threonine, proline, and alanine (49.2, 16.2, and 21.5%, respectively) and contauns two types oftandem repeats, TTTQAPT and AATTP, which are typical features for a mucin (6, 32). Region IV is similar to regions II and VI and contains 9.0% cysteine residues. Region V is another threonine-, Proline, and alanine-rich section, containing a repetitive sequence, TAAP This region differed between 1IM14 and IIM22 in ;sequence length, but the sequence features ofthe IIM
protein isomers, and their respective cDNA clones were similar. T his region ('~, contains 25 TAAP repeats in IlTvI22.
Northern blot analysis .of T. ni midgut RNA with a probe made from I1M22 showed a single band with a molecular size of 3.1 kilobase pairs, indicating that there was no similar polydispersity i.n IIM transcription, as is found in mammalian mucin transcripts.
Biochemical analysis has shown that lTlvi from T. ni midget peritrophic membranes is a novel invertebrate intestinal mucin. The cDNA sequence presentexi here confirms the identity of this secreted invertebrate intestinal mucin. The overall structural organization of IIM is similar to human intestinal mucin, MUC2, which can be described as follows: (a) as a secreted mucin, the IINi contains a 25-amino acid signal peptide at the N terminus (region I); (b) relative to MUC2, which has two different tandem repeat domains interspersed by a cysteine-rich region that distinguishes MUC2 from other mucins, IIM also contains two threonine-rich tandem repeat regions (regions III and V) where potential O-glycosylation sites are located; and (c) the two tandem repeat regions are flanked by cysteine-rich regions (regions II, IV, and VI) (Fig. 1).
SUBSTTTL1TE SHEET (Rule 26) In comparison with M1:JC2, which contains more than 5100 amino acid residues, the apoprotein in IIM( is relatively small. The mature IIM contains either 763 or 782 amino acid residues. Prediction of O-glycosylation using the O-GLYCBASE
search program indicated that 127 of the 147 threonine residues and 5 of the 23 serine residues in IIMM22 (excluding the signal peptide) were potential O-glycosylation sites.
In regions III and V, all threonine residues, except the two at the boundaries of region III (at position 99) and region V (at position 486), were potential O-glycosylation sites.
There is only one threonine in the non-tandem repeat domains (at position 314) marginally predicted as a potential O-glycosylation site. A PROSITE data base search using DNASTAR demonstrated four tentative N-glycosylation sites. All four sites were located within region V.
Regions III and V contain high levels of threonine, alanine, and proline, and do not contain any aromatic or sulfur-containing amino acids, which is similar to the corresponding domains in MUC2. I1M contains multiple repeating units. These repeating units are short compared with those found in mammalian mucins.
Region III
contains two tandem repeating sequences, TTTQAPT and AATTP, throughout the whole region. Region V contains an even shorter repeating unit, TAAP. The repeating units in this region are dispersE:d at four potential N-glycosylation sites and several other locations. Sequences TT'VT(V/S)PP and TTAVPEI occur frequently in the disrupted locations in region V. The repeating sequences in IIM did not exhibit similarity to any known repeating sequences from other mucins.
The difference between cDNAs IIM14 and IIIvI22 is in region V. In this region, IIM14 contains 19 fewer amino acids than IIM22, which could be due to genetic polymorphism, as reported for human and other vertebrate mucin genes. Both IINI
cDNAs contain G + C-rich repeated sequence units in region III and V. These G
+ C-rich repeated sequences (with ;~-like sequence features), could be responsible for the evolution of genetic polymorphisms. This difference between IIM14 and Ilivi22 could also be the result of alternative. splicing during RNA processing. Such a phenomenon has been observed in mucin gene expression. The AG at position 2005 and 2006 in SUBSTITUTE SHEET (Rule 26) IllVVi22 could potentially serve as a 3'-splicing site, which would lead to a mRNA
corresponding to IIIViI4.
The protein sequence features of the IIM are in agreement with the data from the biochemical analysis of IIM. The presence of N-glycosylation motifs and mucin-characteristic threonine-rich tandem repeats in the IINi sequence confirmed the presence of N-giycosylation arvd extensive O-glycosylation of IIM, previously analyzed by carbohydrate-specific lectin binding and specific glycosidase analyses.
Cysteine-rich domains are common in mucins and have been demonstrated to cause oligomerization of mucins by disulfide bonding. These cysteine-rich regions might also contain globular structures with intramolecular disulfide bonds.
These protein regions could become exposed once the disulfide bonds are reduced.
Disulfide bonds in the non-heavily O-gl;ycosylated regions of IIM are involved in maintaining a digestive protease-resistant structure. However, protein sequence analysis did not show significant sequence similarity between the cysteine-rich regions in IIM and the cysteine;-rich regions from MLJC2, or other mammalian mucins. This is not surprising, since insects are phylogenetically very distant from mammals and since IIM is a constituent of a unique invertebrate chitin-containing structure.
IIM is tightly associated with the PM, and is a major structural constituent of the PM. These results indicate; that IIM may have a high affinity to the chitinous fibril network of PMs. By computer-assisted sequence analysis, a protein fragment in region IV was aligned to two chitin binding domains in chitinases from a yeast, Saccharomyces cerevisiae, and a fungus, Rhizopus oligosporus. In addition to region IV, sequences in regions II and VI also show a certain degree of similarity to the chitin binding domains described above; however, the levels of similarity were lower than that found in region IV. In a recent report, a non-mucin insect PM protein from Lucilia cuprina, peritrophin-44, showed binding capability to chitin, but it did not show significant sequence similarity to known chitin binding sequences. However, the cysteine-rich domains with pe:ritrophin-44 shared the same structural feature, a six-cysteine-containing sequence present in cysteine-rich domains in chitinases.
SUBST1T1JTE SHEET (Rule 26) Surprisingly, the sequE;nce features of IINI in the cysteine-rich regions are similar to what Elvin et al. proposed for peritrophin-44. Almost all sequences in regions II, IV, and VI are composed o~f such a six-cysteine consensus. This result supports the conclusion that IIQvvI may tightly bind to the chitin network of PM in the non-glycosylated cysteine-rich regions. The strong binding of BM to chitin could be a very important factor for the formation of PMs in invertebrates and aid in the stability of the chitin network. Based on the structural characteristics of IIHI and the strong binding associated with IIM and chitin, it is likely that the chitin fibrils in PMs are protected from enzymatic degadation by IIM. Considering the biochemical properties of IIM
and the putative chitin binding sequences in non-glycosylated regions in IIM, the IIHI
protein backbone is protected from degadation in the hydrolytic enzyme-rich midget environment by two different mechanisms: (a) the densely O-glycosylated regions (regions III and ~ are protected by oligosaccharide moieties; and (b) the cysteine-rich non-glycosylated or less glya~sylated regions (regions II, IV, and VI) are protected by disulfide covalent bonding forming a "buried" structure or by the protein binding to chitin in the PM. The mucin nature and chitin binding capability of IIM can explain the high resistance of IIM to midget digestive enzymes and the protective functions of PMs in invertebrates, especially in insects, Any reagents with the potential effect of damaging IIM, such as baculovirus enhancins or reducing agents, will result in the destruction or attenuation of tlhe protective role of the PM against parasites and other microorganisms.
Localization of Einr~ssion a~f the Mucin in the Peritronhic Membrane By immunolocalization in tissue sections, it was determined that IIIVVI is expressed in midget tissues.
The IIM from T. ni larvae was localized by immunocytochemistry with the antiserum to IIM. An antiserum to DM was generated by immunizing a Flemish Giant/Chinchilla Cross rabbit with purified IIM from T. »i PMs. Preimmune serum from the rabbit was collected .and used as a control for immuno-detection of IIM.
Fourth instar T. ni larvae were. fixed in 4% paraformaldehyde overnight at 4°C and SUBSTTT'~JTE SKEET (Rule 26) embedded in para~n. After tissue sectioning and de-waxing immunostaining was performed as follows: sections on glass slides were blocked for nonspecific staining with 3°/. bovine serum albumin in phosphate-buffered saline, followed by incubation with antiserum against IINI in lphosphate-buffered saline containing 3% bovine serum albumin. After incubation witlh the first antiserum, the sections were washed with phosphate-buffered saline and incubated with a secondary antibody against rabbit IgG
conjugated with colloidal gold (Sigma). Following secondary antibody incubation and subsequent washing, the sections were fixed with 2.5% glutaraldehyde.
Immunogold staining was intensified by silver enhancement using the Silver Enhancer kit (Sigma).
The immunostained sections were counterstained with hematozylin and eosin and examined by microscopy.
Microscopic observations showed that IIM was localized in the peritrophic membrane and in the area surrounding the midgut epithelial brush border.
Observation at a high magnification demonstrated that IIM could be secreted from goblet cells of the midgut epithelium. Immunostauning with preimmune serum from the same rabbit used to generate the anti-IIM antiserum did not show any positive reaction. In addition to the midgut, positive staining was occasionally observed in malpighian tubules on the lumen side. To verify whether this occasional positive staining in malpighian tubules was specific to IIM and to test whether IIM was present in other tissues, a Western blot analysis of extracts from various tissues of T. ni larvae using anti-IIM
antiserum was conducted.
Tissues were isolated firom fifth instar T. ni larvae and rinsed with phosphate-buffered saline. The tissues were then homogenized and boiled in 0.0625 M Tris-HCI
(pH 6.8) containing 2% SDS, :5% Beta-mercaptoethanol, and 10% glycerol.
Undissolved materials were removed by centrifugation. Protein concentrations in the supernatants were estimated using the Bradford protein assay. One microgam of protein from each tissue extract, except for the PM extract, for which 0.04 pg of protein was used, was loaded onto the gel. Proteins were separated by SDS-PAGE, followed by blotting onto Immobilon memlbrane (Millipore Corp., Bedford, MA), and probed with anti-IINi antiserum.
S'~UBSTITUTE SHEET (Rule 2b) The Western blot analysis showed that IIM was primarily present in the non-cellular PM. A broad band at 2,00 kDa could also be detected in the PM extract when this sample was overloaded. This band is considered a degradation product of IIM by active midget digestive enzymes, since the PM moved through the digestive tract. The midget was the only tissue in vvhich a significant amount of IIM was detected.
Besides the BM band, some lower molecular weight bands were also present in the midget extract. These bands possibly were the IIM protein in the process of glycosylation but not yet fully glycosylated. The extract from malpighian tubules did not show any positive staining at the gel position for IIM. Some weak positive staining was detected in the extract from hemolymph with a major broad band between 66 and 97 kDa.
Salivary gland, fat body, and epidermis extracts did not show any positive reaction to the anti-IIM antiserum. The bands detected in the malpighian tubules and hemolymph did not show the correct molecular weight corresponding to IM, and the reactivity to the anti-BM serum was very low. Therefore, the proteins represented by these bands do not indicate the presence of II1VI in tissues other than the PM.
Localization of IIM by immuno cytochemistry indicates that IIM is primarily expressed in the midget tissue and is likely to be secreted by goblet cells.
Interestingly, this is similar to the secretion of mucins by goblet cells in vertebrate intestinal epithelium.
Peritronhic Membrane Secretion Pat~grns of Invertebrate Intestinal Mucin T. ni PM first appears iin larvae before feeding starts and is present along the entire length of the mesentero:n. IIM plays a significant role in the formation and function of the peritrophic membrane. To ascertain the secretion patterns of I1M, PM
structure and secretion patterns were examined in the anterior, middle and posterior regions of the mesenteron.
Third instar larvae were allowed to fed on diet up to 24 hours. Prior to dissection, larvae were placed in wax-filled Petri dishes, stretched and pinned through the head capsule and telson, using pins held with forceps. The larvae were then flooded ;SUBSTTrIJTE SHEET (Rule 26) with cold fixative (3.2% formaldehyde, 5% glutaraldehyde in 0.1 M Sorensen's phosphate buffer, pH 7.2 containing 3% sucrose) and dissected to remove the cuticle.
The exposed alimentary canal was fixed for 2 hours at 4°C, washed in 0.1 M Sorensen's phosphate buffer containing 3°,io sucrose for 2 hours, post-fixed in 1%
osmium tetroxide in 0.1 M sodium cacodylate buffer, washed in double distilled water (ddw), stained en bloc for 4 hours with 2% aqua~us uranyl acetate (on ice), washed in cold ddw for 0.5 hour, and then dehydrated in an ascending ethanol series from 50 to 100'/0.
The specimens then were infiltrated with a 1:2 mixture of ethanol: Spuds resin for 1 hour, followed by a :l :1 mixture for 2 hours, and lastly placed in 100% Spurr's resin overnight. The specimens in resin were embedded in molds and cured for 60°C for 24 hours Other specimens also were embedded in LR White resin for immunocytochemical procedures. Dissections were performed as above except the fixative contained 4% paraforrnaldyde and 0.5% glutaraldehyde in 0.1 M
phosphate buffer saline (PBS), pH 7.2. Freshly dissected alimentary canals were fixed in this solution overnight, incubated in 0.1 M ammonium chloride in PBS for 1 hour, washed in PBS for 2 hours, and dehydrated in ascending ethanol series from 50 to 100%. The specimens were resin infiltrated with a 1:1 LR White: ethanol mixture for 2 hours, transferred to 100% resin with one change, and kept overnight to allow complete resin infiltration. The specimens in resin were loaded into gelatin capsules and allowed to polymerize at 50°C overnight. Thick sections (O.S~m) were cut using glass knives on Reichert Ultramicrotome. For transmission electron microscopy (TEM), thin sections were cut using a diamond knife and mounted on naked or formvar-coated nickel grids and observed on a Phillips EMC 201 transmission electron microscope.
For wheat germ agglutinin (WGA) staining, thin sections were incubated for 1 hours at room temperature in Mocking buffer [0.01 M PBS (pH 7.2) containing 1%
cold water fish gelatin, 0.075% Tween 20, and 0.075% Triton X-100] and subsequently incubated in a 1:100 dilution a~f 20 nm gold-labeled WGA (20 ~g/ml) (E-Y
Laboratories, San Mateo, CA) in blocking buffer for 1 hour. After incubation, grids were washed with PBS, ddw and stained with uranyl acetate (UA) and lead citrate SUBSTTrUTE SHEET (Rule 26) (PbC). Cytochemical controls consisted of addition of 1 part 10 mM chitotriose with 1 part WGA solution at twice the above concentration.
Invertebrate intestinal mucin (mvn was localized in thin sections which were first blocked in blocking buffer then incubated in a 1:300 dilution of anti-IIM
preparation for 1 hour. Sections were then washed in multiple changes of blocking buffer for 1 hour then incubated in 1:100 dilution of 20 nm gold conjugated goat anti-rabbit IgG (E-Y Laboratories, San Mateo, CA) for lhour. Sections were then washed with blocking buffer, PBS, ddw and stained with UA and PbC. Cytochemical controls were first incubated in a 1:300 dilution of rabbit preimmune serum for 1 h, washed in PBS for 1 hours and incubated in secondary antibody as described above.
Scanning electron microscopy (SEM) was performed on T. ni larvae. The midget and PM
were dissected and placed in Karnovsky's fixative for 2 hours. The specimens were then dehydrated in an ascending ethanol series from 70 to 100%, critical point dried, fixed to aluminum stubs with silver paste, sputter coated with gold-palladium, and viewed in an AMR-100A scanning electron microscope.
PM was present along the entire length of the mesenteron. In the most anterior midget region examined, PM appeared as a single thin structure located between the stomodeal valves and midget epithelium. Slightly posterior to this region (about 2 mm) PM appeared slightly thicker. This slight increase in thickness may be the result of the association of fine thread-like material to the delaminated PM. In the middle region of the mesenteron, the morphology of the PM changed to a more robust structure composed of compact layers. Similar in appearance to PMs located in the middle portion of the mesenteron, PM in the posterior mesenteron (just adjacent to the proctadeaum) can be seen at lower magnifications partitioning dietary plant cell walls and microbes from the underlying midget epithelium.
Observations taken from electron micrographs shows PM formation begins with the appearance of fine fibrous-like material within the brush border of the anterior mesenteron. These nascent PMs first appear in the upper third of the microvillar brush border as diffuse structures. Probing these regions with anti-IIM and WGA
gold, produce discrete lines of labeling confined to these fibrous-like structures.
These SUBSTITUTE SHEET (Rule 26) staining patterns indicate IIM and chitin (or N-acetyl-D-glucosamine containing structures) to be present in the nascent PM. This same binding pattern can be seen at the tips of the microvillar brush border demonstrating that nascent PM moves apically for delamination into midget lumen. These delaminated PMs have a fibrous appearance and bind both WGA-gold and anti-InVt. Scanning electron microscopy (SEM) of the anterior midget region revealed a microvillar brush border inundated with various amounts of material. Interestingly, SEM apparently captured individual secretion events where PM was resting above cells. At higher magnifications, these newly delaminated PMs possessed fibrous-like material, which is mostly obscured by smooth matrix material. Finally, these individual secretion events coalesce form a large smooth and continuos PM which now conceals the underlying midget epithelium.
To determine when PM first appears within the midget lumen, third instar and newly molted third instar larvae; were examined for the presence of PM.
Although PM
was not found in the pharate stage, there was localization of anti-IIM within the brush border (data not shown). Examination of newly ecdysed larvae (which have just passed their exuviae across the telson) showed a well-developed PM within the middle part of the midget. In these larvae, the anterior midget showed the presence of diffuse material packed between the interstices of microvilli. This material labeled extensively with anti-IIM and was present in the gut lumen above newly secreted PM.
Interestingly, there was an association of this diffuse material to delaminated PMs. Finally, the staining patterns of IIM were investigated through out the length of the mesenteron.
Cells located in the anterior midget possessed vesicles, which were extensively labeled with anti-IIM. In the posterior regions, anti-IIM localized to microvillar brush border to columnar epithelial cells adjacent to goblet cells. This same phenomenon was observed in the brush border of cells from the middle portion of the mesenteron.
At the entrance of the mesentron, the PM was observed as a thin structure sandwiched between the tips of"the microvillar brush border and intima of the stomodeal valves. This delicate-looking membrane increased in thickness as is it moved in a posterior direction toward the proctodaeum. The delamination of PM
from the microvillar brush border w;as only observed in the anterior mesenteron. No PM
SUBSTITUTE SHEET (Rule 26) delamination events were seen in the middle or posterior mesenteron.
Furthermore, sections representing the mid- and posterior mesenteron showed no discrete lines of labeling within the brush border when probed with anti-BM or WGA-gold. This observation demonstrates that chitin and IINI do not aggregate to form nascent PM in regions past the anterior meser~teron. Within the anterior mesenteron, PM
formation begins with the secretion of chitin and matrix material (IIMj. These PM
components appear to first aggregate within the upper part of the brush border to from a nascent PM. This is followed by delamination of PM into the midget lumen. Even though PM
delamination events appears to~ be restricted to the anterior mesenteron, there is secretion of IIM from cells located in the middle and posterior midget. In the middle and posterior mesenteron, the majority of anti-IIM localized to the brush border.
Secretion of IIM through out the entire length of the mesenteron may account for the observed increase in PM thickness. Interestingly, IIM secretion was often localized to columnar epithelial cells directly adjacent to goblet cells.
Our observations that PM formation is restricted to the anterior part of the midget is consistent with previous studies. In one study, the European corn borer (ECB, Ostrinia nubilalis) larvalL PM formation was found to be limited to the anterior mesenteron. In this region, ECB nascent PM was embedded within the brush border and stained with WGA-gold (indicating the presence of chitin containing structures).
Even though the authors were rtble to determine an anterior site of chitin substructure assembly and delamination, they were unable to directly determine where protein matryx was synthesized and secreted. The current disclosure demonstrates that the midget region is responsible for the secretion of protein matrix in T. ni larvae. By probing the midget for the major protein moiety IIM, it was determined that the chitin substructure and protein matrix (IIM) apparently are secreted together from cells located within the anterior part of the mesenteron. These results are consistent with the SEM observations which show fibrous linear structures (assumed to be chitin microfibrils) embedded in a proteinaceous matrix. Finally, another very interesting observation is the secretion of IIM through out the mesenteron. This whole midget secretion phenomenon may provide additional amounts of matrix material to damaged SUBSTITUTE SHEET (Rule 26) PMs. This may in turn preclude microbes and rough dietary components access to the midget epithelium.
The Role of tag Mucin in thg Function of the Peritronhic Membrane and Baculovirus Infection A baculoviros enhancing which is encoded and carried by specific baculoviruses, has mucin-degrading activity both in vitro and in vivo. The in vivo degradation of ZIM by enhancin was correlated with the enhancement of baculovirus infections in insects. These findings show that Viruses have evolved a novel strategy to overcome intestinal mucinous ibarriers against microorganisms by utilizing a mucin-degrading enzyme.
Incubation of IIM with Tn enhancin showed that the enhancin had activity against ZIM. To demonstrate proteolytic activity by TnGV enhancin against BM, purified IIM was incubated with 1.25 ~tg/ml TnGV enhancin in 0.05 M Tris-HCl buffer (pH 7.5) containing a cocktail of protease inhibitors minus the metalloprotease inhibitor, EDT'A at 37°C for 3 hours or overnight. The degradation of IIM was examined by SDS/PAGE anal~~sis. A parallel treatment of IIM without enhancin was included as a control. The degradation products of IIM displayed a banding pattern similar to that observed during; incubation of intact PMs with enhancin. To confirm the metalloprotease nature of enhamcin, IIM was incubated with TnGV enhancin in the presence of 10 mM EDTA. The addition of 10 mM EDTA to the incubation buffer blocked the digestion of the IfM and confirmed the metalloprotease nature of enhancin.
In vivo IIM degradation assays with T. ni neonate larvae demonstrated that enhancin degraded IIM in the midget of living insects and that the degree of degradation appeared to be dose-dependent. Two in vivo assays were developed to include neonate and fifth instar T. ni larvae, based on the methods employed to determine the efficacy of an enhancin on virus infections. The in vivo neonate IIM
assay and a concomitant virus bioassay were conducted by feeding T. ni neonate larvae SUBSTITUTE SHEET (Rule 26) with inoculum droplets containing 103 occlusion bodies/ml of AcMNPV, and varying doses of TnGV enhancin, as described by Wang et al. Following ingestion of the inoculum, 25 larvae from each treatment were transferred onto artificial diet, incubated at 28°C for 90 minutes, and collected for Western blot analysis using an antiserum specific to IIM. For Western blot analysis, the larvae were homogenized in 100 ul of SDS/PAGE sample buffer. Subsequently, 4 pl of each sample was electrophoresed through a 7.5% SDS/PAGE gE;l, blotted, and then probed with anti-IIlvI
antiserum.
To assess the correlation between the extent of IIM degradation in living insects and the degree of enhanced Ac;MNPV infection by TnGV enhancin, 60 neonate larvae from each feeding group were also collected and individually reared on artificial diet.
Viral infections were monitortxl and examined throughout the whole insect larval developmental stages, as described by Wang et al. The extent of degradation of IIM
was correlated with increased AcMNPV infection in larvae. This enhanced mortality was statistically significant and can be presented by the regression analysis:
Probit mortality = 4.72 + 0.256 X enlhancin dose (ng/larva) (RZ = 99.2; P = 0.004).
The in vivo I1M degradation assay was also conducted by feeding fifth instar T.
ni larvae with TnGV enhancin and analyzing the residual IIM in the fecal pellets. Early fifth instar T. ni larvae were feed 10 ul of inoculum containing 5% sucrose, 10 p,g/ml blue food coloring, and 5 ~g 7.'nGV enhancin in 25 mM sodium carbonate buffer (pH
10.5). Afterward, the larvae were transferred to individual rearing cups containing artificial diet and incubated at 28°C. During the incubation period, enhancin will digest the InVI present in the PM. Pl~is are secreted within the intestine and later excreted with fecal pellets, which are normally ensheathed within the remnants of a PM. The first three fecal pellets marked with blue food coloring therefore were collected and subjected to Western blot analysis using the IIM-specific antiserum. The in vivo ZIM-degradation assay using fifth instar larvae showed that IIM was present in the control fecal pellets and exhibited sonne minor degradation. However, no IIIvi was detected in the fecal pellets collected from the TnGV enhancin-fed larvae, confirming that enhancin completely degraded IIM in the digestive tract of living insects.
SUBSTT»'(JTE SHEET (Rule 26) The presence of an IIM protein and its degradation by enhancin is not restricted to the species, T. ni. Another mucin, similar to the IIM from T. ni PMs, was also isolated from Pseudaletia unipuncta PMs and biochemicaliy characterized. This mucin is also degraded by the TnGV enlhancin and degradation was correlated with enhanced baculovirus infections in P. a»ipuncta larvae.
The PDV that crossed enttancin treated T. ni PMs was infectious, as was demonstrated by increased mortality rates compared to control treatments {Table 1).
The effect of enhancin on PM permeability to infectious viruses was confirmed using a second insect species, P. unipuncta. Enhancin had a significant effect on PM
permeability, although the P. unipuncta PMs appeared to be more permeable to the virus (Table 1).
In lepidopterous larvae, the PM is a structure containing pores which may vary in size among different insect species. Low level permeability of untreated T.
ni PMs to blue dextran 2000 appears to confirm the presence of naturally occurring pores within the PM matrix. Although the purpose of this study was not to determine the approximate pore size of T. ni or P. unipuncta PMs, these studies did show that control T. ni PMs were permeable to blue dextran (diameter: 54nm) but were almost impermeable to AcMNPV PDV i;186nm diameter x 357nm length) over an 8-hour period. Insect bioassays also suggested that untreated P. unipuncta PMs probably had a larger pore size and allowed passage of more PDV particles than PMs from T. ni since control mortality values were higher for samples obtained from P. unipuncta PM
permeability experiments (1% vs, 38%, respectively; Table 1).
Table 1. T. ni eeonate bioassays showing increased permeability of T. ne and Pserrdoleiia nnipunda peritrophic membrane to AcMNPV PDV following treatment with enhancin.
T. nl Peritrophic Mfatrix' P. urripuncta Peritrophic Mahixb Total Avg. % t-Test Total Avg. % t-Test Treatment Insects Mortality f ( P ) Insects Mortality t ( p ) Tested SE Tested SE
SUBSTITUTE SHEET (Rule 26) PM' 90 1s.6 t z.9 e1s0 90.7 t 2.9 < 0.01 < 0.01 M 9p 1.O t 0.3 ~~1 1s0 38.0 t 8.2 V 9p 97.8 t 2..2 150 100 t 0 control a. Swnmary of 3 independent tests.
b. Summary of s independent tests.
c. PMs mounted in a Wi chamber permeability apparatus were treated with 3 mglml enhancin for 1 how and samples were collected 16 how~s post-treatment.
Our work showed that sephacryl-purified enhancin preparations contain traces of contaminating insect professes. In a subsequent study, Lepore et al. (1996) showed that extensive purification of enhancin by ion exchange chromatogaphy and immobilized a-macroglobulin removed the contaminating professes without diminishing the in vivo and in vitro activity of enhancin, thus providing evidence that these professes did not have a role in the enhancement of infections.
Furthermore, in that same study, Lepore, et al. (1996) also demonstrated that purified TnGV
enhancin, expressed by a recombinant AcIMNPV in insect cells, was active on insect PMs.
Addition of protease inhibitors provided evidence that potential contaminating professes did not have a role in increasing the PM permeability. The metalloprotease inhibitor EDTA was able to inhiibit the action of enhancin. Although there is no published evidence that ganulosis viruses encode a chitinase, it was recently reported that such a functional gene was present in the nuclear polyhedrosis virus, AcMNPV.
To rule out the effect of any possible chitinase contamination in our enhancin preparation a potent chitinase inhibitor was used and no effect on the ability of .
enhancin to increase PM permeability was found. Chitinase activity was not detected in our preparations using a chitinase activity assay.
Previous studies with enhancin suggested that the PM, though clearly not an impenetrable barrier, does reduce the exposure of susceptible midgut cells to SUBSTTI'IJ1'E SHEET (Rule 26) baculoviruses. It appears that some insect viruses may have evolved similar mechanisms to degrade the struc~ral integrity of the PM and facilitate the passage of infectious virus. Derksen and 1 iranados (1988) reported that an unidentified factor in the polyhedrin fraction of AcMNPV was able to affect the protein profile and structure of the PM. This observation was recently confirmed by Faulkner et al. (1997) who found that OBs from both a mostant and wild-type AcMNPV could degrade the PM
from T. »i larvae. Furthermore, the presence of an enhancin-type gene was recently reported from Ly»rantria dispurr nuclear polyhedrosis virus suggesting that other similar nuclear polyhedrosis viiruses (NPVs) may carry enhancin genes. Begon et al.
(1993) reported Plodia interpu~nctella GV (PiGV) OBs caused dramatic and significant effects of the PM structure from the same species and concluded that the PM
provided a barrier to PiGV infection at lower virus doses.
Although there have been many investigations concerning the mode of action of enhancin, prior to the work of inventors consensus has not been reached. It was previously reported that an enlancin from PuGV acted on the plasma membrane of midgut cells and cultured insect cells, facilitating the entry of virus particles into the cells by providing attachment cites or facilitating membrane fusion for the virus particles. Based upon the worlk described in this patent application, the inventors believe a major role of GV enhancins is to disrupt the structural integrity and increase the permeability of the PM to baculovirus particles. Our previous studies demonstrated that enhancin from TnGV digested a specific major PM protein, insect intestinal mucin.
The digestion of this PM mucin and the resulting degradation of the PM
structure was correlated with enhanced baculovirus infection of insect larvae. It is reasonable to conclude that the disruption of the PM structure resulted in the increased porosity of the PM, thereby facilitating the infection of the underlying epithelial cells.
Thus, these viral-encoded proteins appear to play an important role in baculovirus pathogenesis.
T. ni PMs are present in all larval instars and at all stages between molts.
Therefore,1ZM may play a protective role throughout the entire larval period.
No mucin degrading protease has been previously reported to be associated with a virus to assist the penetration of a pathogen through a mucinous protective barrier;
therefore, this SUBSTITUTE SHEET (Rule 26) study represents a novel concept in animal virus pathogenesis. The present invention enables further studies on the sopecific recognition sites and cleavage of mucins by baculovirus enhancins, and the biological properties of iZM and enhancins.
Furthermore, use of Invl degrading enzymes in recombinant plants or baculoviruses will decrease Larval growth and increase the pathogenesis of virus infections.
Having discovered the 1QM protein and its function, the inventors were able to develop applications for use of the novel cDNA sequences and the recombinant protein.
Diet Incornoration Experiments Using Anti IIM Serum Polyclonal antibodies against an insect peritrophic membrane (PM) prntein from the Australian blowfly, Lxrcilia cuprina inhibited growth and caused mortality of blowfly Larvae. It was reported that this biological response was caused by the PM
antibody, which blocked nutrient diffusion across the PM. The present invention includes the discovery that a po~lyclonal antibody against the T. ni PM mucin (IIM) has an adverse effect on T. ni growth and survival.
Mucin was prepared from T. rri fifth instar larval PM by preparative PAGE. The gel was stained by CuClz (0.3M~ for S min and the band containing mucin was isolated and destained in 0.2 M EDTA. Mucin was further eluted from the gel slices by electroelution, and used to immunized rabbit following a standard rabbit immunization protocol. 0.2 mg mucin was used per injection for a total of 3 injections.
Serum was collected at 6 weeks after the first injection and IgG was purified from the serum using caprylic acid and ammonium sulfate methods (Harlow, E. & Lane, D. 1988-Antibody, a laboratory manual. Cold Spring Harbor Laboratory). Control rabbit IgG was also purified from normal rabbit seru~.m (Gibco).
A laboratory colony of T: ni reared on high wheat germ diet was used in these experiments. Ta prepare diet incorporated with IgG, high wheat germ diet was prepared but with less water (10% less than the final diet volume). After mixing all the components, the diet was allowed to cool gradually to 45°C, and IgG
solution was SLBSTITVTE SHEET (Rule 26) added with vigorous stirring. lHeat inactivation experiments showed that the immunoreactivity of the anti-III serum was reduced above 60°C (data not shown).
Water was added when necessary to adjust the volume. The diet prepared in this way has exactly the same concentration of each component as normal high wheat germ diet, with the exception of the additiion of IgG. The final concentration of IgG in the diet was 20% of the original IgG concentration (V/V) in original anti-IIM serum.
The diet was aliquoted (2.5 mls/cup) into 1 oz cups which was sufficient diet to allow the larvae to develop into pupae.
T. ni neonates were placed individually into the cups with standard (no IgG) or IgG- incorporated diet. This timg point was designated as time zero. The larvae were incubated at 28°C and the larval growth was recorded every 8 hours. The larval weight was also recorded at the 3rd and 6th day. Pupal weight was measured when all the larvae had pupated. The experiment was conducted twice with 30 insects per treatment.
Incorporation of IgG into the diet had a significant effect on T. ni larval development (Table 2). Although control rabbit IgG containing diet had a strong effect on larval growth compared to larvae on standard wheat germ diet, the anti-IIM
IgG
treatment had an even stronger and statistically significant effect. The duration of growth from neonate to pupa was delayed in anti-IIM IgG fed larvae, and was significantly longer than control IgG containing diet fed larvae. Similarly, the anti-IIM
IgG fed larvae had the lowest v~reight at day 3 and day 6, and their weight was also significantly lower than larvae iEed on control IgG diet at day 6 in both experiment and at day 3 for experiment 2. No difference in pupal weight was found between all the treatments in bath experiments.
Table 2. Comparison of T. n! lwal and pupa Wv~gias and ,developmental oration from modte to pupa.
Treatment~1 Duration Larval Larval Pupa!
from Weight Weight Weight NoorWes at at to Pupae Day Day of ()rtSE) P' ~mB~E) P' ~m~E) P' (mBtSE) P' insect Exp. 30 201.4711.71 6.4510.49 142.5017.33 225.5713.43 )-Ca~trol Exp.l- 30 212.4812.78 3.9110.33 93.633b.93 221.1632.94 Normal 0.0:5 0.11 0.01 0.62 IgG

S1JBSTI1'UTE SHEET (Rule 26) Exp.l-Mti- 30 219.0711.79 3.2810.19 72.4714.23 223.2813.10 I<M IgCi F.xc.2- 30 193.611.46 5.78tQ.30 167.17tb.26 223.8313.21 Codroi Exp.2- 30 206.9012.91 3.9910.24 112.9319.39 224.2013.04 Normal IgC3 0.02 0.02 0.03 0.45 Exp.2-Aali- 30 216.3312.82 3.1710.24 88.941b.82 227.7013.29 BM IgU
' From t.te~t compering weigh a duration between larvse on uarnoal IgC3 diet and anti-IIM IgO diet.
An effect of anti-I1M IgG on T. ni larval development was observed. Compared with larvae fed on control rabbivt IgG containing diet, the larvae on anti-IIM
IgG
containing diet required a longer time to develop from a neonate to pupa, and had a lower larval weight at day 3 and day 6. In most cases, the differences were statistically significant. Since no difference; in pupal weight for the various treatments was observed, the differences in larval weight might be caused by a difference in speed of development. It is clear that the presence of anti-IIM IgG in the diet resulted in significantly slower growth of .'T: ni larvae. Anti-IIM IgG binds to the major protein on the insect peritrophic membrane, which could result in the blockage of nutrient flow through the peritrophic membrane.
The control rabbit IgG lhad a significant effect on larval development, compared with larvae growing on standard high wheat germ diet. Several different commercial rabbit sera were compared (two batches from Sigma, and one from Gibco), and they ali showed a similar effect on T ni development. The reason for this is not clear.
No major cross-reaction of normal. rabbit IgG to T. ni peritrophic membrane components was detected in western blot e:~cperiments. It is possible that IgG somehow interferes with the digestive physiology of the insect, or has some feeding deterrent effect. A
similar phenomena was also rc;ported by Casu et al. (1997) where the growth of the blood feeding insect, Lucilia c~uprina was inhibited in the presence of high concentration of normal control IgG.
The design of the experiments conducted were effected by the relatively low amount of serum that can be obtained from rabbits (i.e.,70 mls/rabbit) for~use in experiments. Using a PM permeability chamber it was also demonstrated that anti-IIM
SUBSTITUTE SHEET (Rule 26) serum could block the permeability of the PM to particles smaller than 5 nm.
This demonstrates that such a phenomenon, if it occurred in viva, might have a detrimental effect on the nutritional physia~logy of the insect. These data demonstrates that the delivery of anti-IIM antibodies through transgenic plants is a novel approach far affecting insect development a~r mortality.
Altered In Situ Peritrophic ll~Iembrane Permeabiliri The present invention includes the discovery that feeding larvae anti-IIM IgG
affects the permeability of the peritrophic membrane.
Fifth instar larvae reared on a high wheat germ diet were starved for 1 hours.
Starved larvae were then injected per os with 20 pl of anti-IIM IgG (2X
concentrated) swlution and placed on a high wheat germ diet containing an equivalent of 20%
anti-IlM IgG and 4% (dry wt) FITC-Dextran (3.2 nm diam.). Controls larvae were injected per os with either PBS or normal serum IgG and placed on their respective diets. After feeding for 2.~ hours at 28°C, larvae were chilled on ice and dissected under saline buffer to expose the alimentary canal. Once the esophagus and proctodeaum were ligated, a small hole (0.2 x 2 mm) was made to expose the PM. This hole was made in the middle portion of the midget just immediately anterior to the anastomosing malpighian tubules. These me:~enterons were then severed from the alimentary tract and placed in a small dish which G~ntained 15-ml buffer. To help remove any free FITC-dextran, the ligated midget was rinsed 3 times with 15-ml aliquots of buffer.
When the final rinse solution was removed, the ligated midget was re-suspended in 4 ml of saline buffer and incubated under gentle mixing. Aliquots of incubating solutions were removed every 0.5 hours and measured for the amount of fluorescence using a fluorescent plate reader set at a 485-nm excitation of 530-nm emission.
The permeability characteristics of PMs to passage of FITC-dextran is presented below. T. ni larvae fed on dieta containing IIM-IgG showed greater amounts of FITC-dextran in the incubating buffer as compared to those larvae fed on diets containing SUBST'IT'iJTE SHEET (Rule 26) normal serum and PBS (Fig. :;). Intact, lighted midget showed FITC-dextran is confined within the midget proper and that the midget wall acts as a barrier to the 3.2 nm FITC-dextran.
Figure 2 shows permeability characteristics of ligaxed midget from larvae fed diet containing either ZIM-IgC~, normal serum IgG, or PBS. An intact, ligated midget showed low passage of FITC-dextran across midget wall. There was more FITC-dextran present in the incubatiion buffer of IIM-IgG ligated midget. Each treatment and control are replicated.
In contrast, insect larvae that have fed on diets containing IIM-IgG have a greater PM permeability to FITC-dextran. The final amount of fluorescence in the incubating medium (at 3 hours) was greatest from IIM-IgG fed insects. One possible explanation for this is that ingested IgG may bind to newly secreted IINi thus altering the amounts of protein matrix available for normal PM synthesis. These results are contradictory when compared to the blocking ability of anti-IIM to passage of FITC-destran in the in vitro studies ~of peritrophic membrane permeability. In those in vitro studies, PMs were dissected and treated with serum. In the in vivo studies, insects are fed IIM-IgG for 2.5 hours. Tlnerefore, IgG may bind to delaminated PM
resulting in a "short term blockage" which could be followed by a subsequent "long term structural alteration" of PM. PM alterations could result from antibody competing for IIM
(especially during PM formation). These interactions could produce very porous PMs.
IIM-IgG induced PM structural abnormalities may be an appropriate explanation for the observed weight changes .and increased development time of larvae from the diet incorporation experiments.
Thus, the use of IIM anti-serum against larval pests would first block the insects ability to absorb nutrients and then dramatically increase the infection rate of ingested baculoviruses due to the increased permeability. Furthermore, this disruption effect can be caused by antibodies expressed by a transgenic plant binding to IIM or expression of portions of the IIM by the plant that competively bind to the peritrophic membrane.
SUBSTTTLJTE SHEET (Role 26) Effect of TIM IgG on AcMIVI'V Infection Based on the observations that IIM IgG may interfere with PM stnrcture, a viurs bioassay was conducted to detE;rmine if the ingestion of IIM IgG along with AcMNPV
would increase larval mortality due to viral infeciton. A neonate droplet bioassay was conducted as reported by Lepore et al. ( 199 except IgG replaced enhancin. T.
ni neonates consumed approximately 1 occlusion body and 10 nl of IgG solution profied from normal rabbit serum or anti-IIM serum. After droplet consumption, neonates were placed on high wheat germ diet and monitored for mortality due to AcMNPV
infection. In two preliminary experiments there was a trend in increased mortality (but not significant) of those neonates which consumed IIM IgG as compared to those fed normal serum IgG as shown in Figure 3. This shows that the administration of virus with IIM IgG will increase insect mortality and can be an important strategy in the suppression of insect damage.
Ubiauitv ntMucins In Insect S ecie T. ni mucin or I1M is an integral peritrophic membrane or matrix (PM) protein.
IINI with its cysteine rich domains, apparently binds chitin to farm a strong semipermeable structure which partitions ingested food and microbes from the midgut epithelium and may aid in digestion. The inventors examined the distribution of mucin (IIIVt) in different insect species.
Table 3.
Common nacre Genus species Fay C
Reactivity with anti IIM

Cabbage looper T. ni Nociuidae yes ~Yw'onn Pseudaletia untpunctaNoctuidae yes Tobacco budworm Herliothis virescensNoctuidae yes B~ ~o~ ~g~,o~s ipsilon Noctuidae yes Beet armyworm Spodoptera exigua Noctuidae yes SLBSTITZJTE SHEET (Rule 26) Fall webworm ,FlyphmrMa coma An~tiidae yes Banded wooUybear ,Pyrrharctia isabellaArctiidae yes Imported Cabbageworm.Pieris rapae Pieridse ?

Common white butterfly:Pieris napi Pieridae no Silkworm .8ombyx morl Hombycidae yes European oorn borer ~Dstrinia nubilalis Pyralidae yes Monarch butterfly .Danes plexzppus Danaidae yes Gypsy moth .Lyrnantria dispar Lymantriidae yes Potato tuberworm .l'Irthorimaea operculellaGelechiidae yes Diamondback.moth .l'lutella xylostellaPlutellidae yes House fly .Musca domesticcr Muscidae yes Tarnished plant bug L,~gus lineolaris Miridae yes Sweet potato whitetly8emisia tabaci Aleyrodidae yes English grain aphid Sitobion aveae Aphididae yes American cockroach Periplaneta americanaBlattidae yes German cockroach BlatteJla germanica Blattellidae yes Fruitfly Drosopi:ila melanogasterDrosophilidae?

Yellowfever Mosquito.Aedesae~pti Culicidae ?

Fungus gnat Bradysia ssp. Sciaridae no Colorado potato beetleLeptinotarsa decemlineataChrysomeGdae no Western spotted cucumberDiabrotica undecimpunetataChrysomelidaeno beetle Mealybug Planococcus citri Pseuclococcidaeno Insect midget was dissected to remove the PM. PM proteins were solubilized in SDS sample buffer containing; mercaptoethanol. Supernatants were subjected to SDS-PAGE, blotted onto nitrocellulose membranes, probed with a polyclonal anti-IIM
antibody preparation, washed, and incubated in a secondary antibody labeled with alkaline phosphatase. Bands were visualized by the addition of NBTBCIP
solution to the blots.
Seventy-six percent of the insect species tested (16/21) possess protein or protein moieties which cross reacted with anti-TIM antibody. Table 1 lists the insect species tested for the presencE; of mucin. PMs were examined in all insects except for mealy bugs and sweet potato whitefly where the whole insect was used. Only midgets of Lygus bugs were extracted and examined for the presence of IIM.
SUBSTITUTE SHEET (Rule 26) Examination of blots showed the presence of strong to weak signals.
Immunoreactive band development was strong in the tobacco budworm, fall armyworm, banded woollybe~~r, armyworm and cabbage looper. The remainder (listed below) gave moderate, weak or no cross reactivity to anti-mucin antibody.
Also, some insects had high molecular weight bands similar in size to T. ni IINi (denoted by asterix) Strong Band Dwelopment Weak Reactivity * Tobacco budworrn *European corn borer * Fall armyworm *Monarch butterfly * Banded Woollybear American cockroach * Armyworm Beet armyworm * Cabbage Looper Moderate Reactivity * Black cutworm No Reactivity * Gypsy moth Imported cabbageworm House fly Mealybug German cockroach Fungus gnat Tarnished plant bug Colorado potato beetle Diamondback moth Potato tuberworm White .fly * = possess bands which are around 400 kD

These studies have demonstrated that mucin (IIM) or mucin-like PM proteins are present in a wide variety of insect species in 5 orders. These insects and possibly many other species may share common mechanisms which involve mucin or mucin like proteins which bind chitin thus permitting the formation of PM. It is interesting to note that a Ho~rropteran and a Hemipteran possess discrete bands which cross-react with anti-II1VI antibody. This is interesting observation since these insects may not produce a PM as found in other insects. Some investigators feel these insects may produce extracetlular secretions that may be functional analogues to the chitinous PM.
Based on our observations, there may exits in Homopterans and Hemipterans a protecxive barrier present which contains mucin-like proteins.
Two potential relevant applications exist to this work. First, the insects which cross react with anti-II1VI may be sensitive to the PM degrading molecule enhancin.
SUBSTITUTE SHEET (Rule 26) Second, these same insect PMs rnay be susceptible to antibody binding which would reduce nutrient assimilation thus leading to a pre-reproductive growth or death.
chitin Binding and its Potential as an Insecticidat Target Plant lectins, which are carbohydrate binding proteins, have been tested for their insecticidal activity against many insect species and some show promise for use in transgenic plants. The mechanism for this anti-insect activity is not known but is believed to be mediated by lectin binding to chitin in the PM or by interacting with glycoproteins on the midgut epithelial cells. Wheat germ agglutinin (WGA) is a chitin specific lectin and others have shown that in the European corn borer, Ostrinia nubilalis, WGA could bind to tlae chitin in the midget and interfered with hM
formation. Such interference rE;sulted in an altered and discontinuous PM
structure, which allowed the food content: to penetrate through the PM protective barrier. Our recent ultrastructural studies on the PM formation in T ni larvae have shown that chitin is always co-localized with ilNl in the midget. These immunocytochernical studies showed that nascent PMs were initially delaminated as chitin containing fibrils from the anterior region of the midget and subsequently, the major protein (InVi) was added to the PM matrix.
Calcofluor is a fluorescent dye with high chitin binding affinity. It has been utilized in studies on the formation of fungi and algal cell walls which are protective structures containing chitin and proteins. Calcofluor interferes with the cell wall formation by binding to nascent chitin molecules during cell wall formation, thus blocking chitin fibril assembly. Similar investigations on insect midget chitin fibril formation using the chitin binding agent Calcofluor had not been approached until our recent studies were carried out. Our studies have shawn that Calcofluor can be used to extract and solubilize chitin binding proteins from dissected T ni PMs. These isolated proteins have high chitin binding properties and are normally not extractable from fully formed PMs by detergents or extreme pH conditions. Calcofluor fed to T ni larvae completely inhibited and/or disrupted PM formation. We believe that this phenomenon SUBSTTI'CTTE SHEET (Rule 26) is due to the disruption of chitin fibril formation by the binding of Calcofluor to nascent chitin molecules as observed in other organisms.
This PM disruption/inhibition phenomenon was further verified in Lymantria dispar, Pseudaletia unipuncta, Helicoverpa zea, and Hyphantria cones. Elegant studies with plant fungal systems whi<;h used dye compounds including Calcofluor showed that chitin biosynthesis and assembly was probably disrupted. We believe that binding of Calcofluor to the PM chitin blocked the interactions among chitin molecules and/or the binding between chitin and newly synthesized PM proteins, and severely interfered with PM formation. Feeding 7C ni larvae with an artificial diet containing 1%
Calcofluor (a concentration used by most investigators) resulted in insect mortality and significantly slowed the growth of the treated larvae. As expected the disruption of PM
formation by Calcofluor resulted in significantly increased baculovirus infections in the larvae.
This same phenomenon of increasing virus infection was first observed by others; however, the mechanism of action on the insect PM was not determined until now. Our studies on the effect: of Calcofluor on PM formation has uncovered a unique mode of action of this chitin bonding agent in the insect midget. These findings confirm our hypothesis that targeting the chitin in the insect midget by chitin binding peptides can affect PM formation or its properties, causing significant disruption of midget physiology and function. If these chitin targeting molecules are shown to have possible insecticidal properties, the genes for chitin binding peptides will serve as new genetic tools for use in recombinant microorganisms and transgenic plants.
Our current studies have demonstrated that PM proteins strongly bind to the chitinous PM matrix and such binding is critical for the PM formation and its function.
Sequence analyses of T ni IIM; and other PM proteins have shown that these midget proteins contain multiple putative chitin binding domains as follows:
Amino acid position See SECI. ID. NO. 3 & 41 IIM region II-- amino acid 26 to 98 IINI region IVs- amina~ acid 243 to 315 SUBSTITUTE SHEET (Rule 26) Iilvvi region IVb- amino acid 320 to 392 IIM region IVo- amina~ acid 408 to 478 IIM region VI
IINI 14--amino acid 695 to 757 Invl 22-amino acid 714 to 776 Nucleotide position(See SE(~. ID. NO. 1 & 2) I1HI region II
Invi 14--nucleotide 113 to 331 IIM 22--nuclea~tide 101 to 319 IINi region IVa IIM 14--nucleotide 767 to 982 IIM 22--nucleotide 755 to 970 IINi region IVb IIM 14--nucleotide 995 to 2013 IIM 22--nucleotide 983 to 2001 IIM region IVc IIM 14-- nucle~~tide 1258 to 1471 IIM 22-- nucleotide 1246 to 1459 IIM region VI
BM 14-- nucleotide 2120 to 2308 IIM 22-- nucleotide 2165 to 2353 To isolate these chitin binding domains, one can express T ni IIM in insect cells using a recombinant baculovirus. Construction of recombinant baculoviruses to express foreign proteins is a routine technique. To construct the recombinant baculovints, one clones the ITM cDNA into a baculovirus expression transfer vector which utilizes the polyhedrin gene promoter to express the IIM (e.g.
pBlueBac4.5 from Invitrogen). Recombinant bac~uloviruses cant be generated by cotransfection of insect cells with the constructed transfer vector and linearized Autographa californica nuclear polyhedrosis virus DNA (e.g. Bac-N Blue AcN/fNPV DNA from Invitrogen). The I1M
can be expressed in the high recombinant protein producing cell line, HighFiveTM
(Invitrogen). The suitability of the insect cell expression system for IIM
expression can SUBSTITUTE S~iEET (Rule 26) be confirmed by assaying the .chitin binding activity of the insect cell-expressed IINI to regenerated chitin. Briefly, insect cells infected with the recombinant AcN/fNPV are lyzed with a non-ionic detergent, such as Triton X-100 and sonication. The cell lysate is clarified by centrifugation and incubated with regenerated chitin to let 1TM
bind to the chitin. The chitin/protein complexes are thoroughly washed with buffer and isolated by centrifugation. Chitin bound IlM are released with a SDSB-mecaptoethanol sample buffer and subsequently analysed by SDS-PAGE and Western blot analysis with an anti-IIM antiserum. A similar approach was used for a mosquito PM protein which was over expressed in insect cells and demonstrated to have chitin binding activity.
The chitin binding regions can be confirmed by a biochemical approach. T ni IIM can be over expressed in insect cells using a baculovirus expression vector and bound to regenerated chitin follawing the procedures described above.
Following the binding reaction, the chitin/IIh!i complexes are washed with buffer to remove unbound IIM and contaminant proteins. Our current studies on isolated native T ni PM
proteins have shown that these proteins have chitin binding activities and the proteins/chitin complexes are strongly bound and resistant to washing with stringent buffers (e.g.
20mM acetic acid or I %SDS). Controlled proteolysis with protease K of the chitin bound IIM is performed to selectively degrade non-chitin-binding regions of the IIM.
Chitin bound fragments are isolated as protein/chitin complexes by centrifugation and subsequent washing with buffer and then solubilized with a SDS/0 mecaptoethanol-containing sample buffer. The chitin bound fragments with an expected low molecular weight (chitin binding domains) are analyzed by SDS-PAGE (15% to 20'/o gels) to separate individual fragments. 'These fragments are subsequently isolated and subjected to N terminal protein sequencing by microsequence analysis. Based on the amino acid sequences derived from the chitin bound fragments, it is possible to design and synthesize peptides to test their chitin binding activities. Competitive binding assays of IIM to regenerated chitin with synthetic peptides as competitors can be conducted to determine the chitin binding activities of these synthetic peptides.
In order to determine the conserved amino acid residues important for chitin binding, one can design synthetic peptides based on published conserved sequences in SLTBSTTT'UTE SHEET (Rule 26) addition to the identified chitin binding domains above. Putative chitin binding sequences have been identified by sequence analysis in several PM proteins from different species (Elvin et al., 1.996; Wang and Granados, 1997; Schorderet et al. 1998;
Shen and Jacoba-Lorena, 1998) and these sequences are similar. Based on these reported sequences one can design synthetic peptides with a mutation to these conserved amino acid residues to identify and establish the conserved amino acid residues responsible for the strong chitin binding activity.
The chitin binding domains in IIM can also be identified by making deletions and mutations of the IIM gene. One can express truncated and mutated IINI
proteins in insect cells using a baculovirus~ expression vector. The truncated I1M cDNA
fragments are prepared using polymerase chain reactions (PCR) using oligonucleotide primers flanking the desired cDNA fragments. These primers are designed to contain suitable restriction enzyme digestion sites so that the amplified cDNA fragments can be easily cloned in frame into the expression vector. Truncated proteins are transiently expressed by transfecting insect cells (e.p;. High FiveTM cells) with the expression vectors.
Chitin binding activities of the expressed proteins will be assayed by their incubation with regenerated chitin followed by analyses of the chitin bound proteins by SDS-PAGE and Western blotting using an anti-IIM antiserum.
IIM fragments can be expressed in insect cells as intracellular proteins and be released from cells by solubili;zation with a nonionic detergent such as Triton X-100 and by sonication. The chitin t>inding activities of the expressed peptides are assayed by incubation of the cell lysates v~rith regenerated chitin in an Eppendorf tube followed by washing of the chitin by centrifugation. Subsequently, the chitin bound peptides is analyzed by SDS-PAGE and Western blot anaiysis with an anti-IIM antiserum. If assays using whole cell lysate:> results in high cell protein background, one can construct an expression vector containing a secretion signal peptide at the N-termini of the peptides to be expressed. In such a way, one can obtain the expressed peptides from serum free cell culture mediunn, thereby minimizing contamination with cellular proteins. Alternatively, one can construct a vector containing a polyHistidine tag fused to the cDNA inserts. PolyHistidine fused peptides can be isolated using a metal-SUBSTITUTE SHEET (Rule 26) charged agarose resin (e.g. Probond Metal Binding Resin from Invitrogen) before chitin binding assays are conducted.
One can use the fragments to identify and evaluate amino acid residues necessary for chitin binding activity by substitution of these candidate residues. Amino acid residue substitution is accomplished following site directed mutagenesis of the cloned cDNA fragments for chitin binding domains. Oligonucleotides containing a mutated site are generated and nnutant clones are obtained using a site directed mutation kit (e.g. GeneEditor in vitro site-directed mutagenesis system from Promega).
Mutated chitin binding domains are expressed in insect cells and their chitin binding activities are assayed. Such assays will idlentify specific residues necessary for chitin binding.
Identified chitin binding domains can be over-expressed as chitin binding peptides in E coli using an E coli expression vector, such as PRSET expression vector series (Invitrogen), to determine. if E. coli expressed peptides have chitin-binding activities. The over expressed peptides carries a fused polyHistidine tag so that these chitin binding peptides can be easily isolated using nickel-charged agarose Fesin. Tests of chitin binding activities of E. coli expressed peptides are performed using the chitin binding assay described above. If the expressed peptides show chitin binding activities, this provide an effcient and economical system for production of these chitin binding peptides for use in biological st<idies.
Chitin binding peptides i.~,an also be over expressed in an eukaryotic system using insect cells and recombinant baculovirus vectors. cDNA fragments coding for chitin binding peptides are clon~xl into a baculovirus expression tranfer vector which utilizes the polyhedrin gene promoter to express polyHistidine fusion proteins (E.g.
pBlueBacHis2 series from Invitrogen). Recombinant baculoviruses are generated as described above. Expressed chitin binding peptides are isolated using a nickel-charged agarose resin.
SUBSTITLTT'E SHEET (Rule 26) Anti-IIM Antibody and Serum Production To isolate an Anti-I1M antibody serum ,11M is purified by solubilizing T.ni PM
in SDS buffer containing mercaptoethanol according to the extraction procedure described in the literature (Wang and Granados, Pros. Nail. Acad Sci U.S.A., 97, 6977-6982). The solubilized PM proteins are subjected to SDS-PAGE and bands are visualised by copper staining. ".fhe band containing IIIVI is cut from the gels, destained and electro-eluted. To help remiove SDS from proteins, elutant will be loaded on a column containing AG-I-X2 resin (Biorad). The elutant is lyophilized leaving the concentrated protein. Generally, 1000 PMs yields 30 pg of purified IIM.
To obtain large amounts; of serum goats are used and inoculated with Invi protein antigen. A similar technique has been used by Casu et al. (Pros. Natl.
AcaG
Sci. U.S.A., 94, 8939-8944) TelLlam and Eisemann's injection protocol is used (Int. J.
Parasitol , 28, 439-450) where IIM is first mixed in Freund's incomplete adjuvant and then equal portions are injectec intramuscular into each rear leg. A second injection is given 1 month later in the neck region. The goats are bled prior to each injection and 2 weeks after the first injection.
lIM can also be isolated from insect frass by collecting excreted PMs for the isolation ofPM protein. T.ni larvae are reared to the fifth instar on a high wheat germ diet and then placed on diet containing sucrose and agar. Feeding insects on this diet should clear their alimentary canals of ingested high wheat germ diet and produce PMs relatively clean of dietary proteiin. PMs are collected, IllVI solubilized and purified as described above.
To generate antibodies to chitin binding domains of PM proteins, chitin binding peptides are expressed using a traculovirus expression vector in High FiveTM
insect cells for optimum expression of peptides as described above. Polyclonal antibodies are produced in New Zealand White rabbits by injecting them with a total of 25-50 Ig of purified peptide. Preimmune serum is collected and used for control experiments. An antigen-capture ELISA is performed to determine the concentration of the total IgG in the original sera. To create a monoclonal antibody the antigen would be injected into a SUBSTITUTE SHEET {Rule 26) mouse and a hybridoma is created by well known methods. The gene encoding the antibody can then be isolated and used to transfect plants.
The antigen for any of the above can also be recombinant protein, which would be most useful if the desire was to target specific chitin binding sites.
There are five chitin binding sites in IIM and they are depicted in Figure 4. Anyone of these regions could be expressed in an appropriate vector, e.g. baculovirus expression system, to create antibodies that bind specifically to these regions.
Trans~enit Organisms Exp~ressinQ anti IIM-IQG
The present invention includes a transgenic plant that express IIM-IgG. Since the immunotherapeutic potential of antibodies produced in plants has been demonstrated in a number of cases, we believe that using peritrophic matrix InVI-specific Ab in plants could be used as immunocontrol strategy for control of insect pests. The concept of using lPM Ab to control insect pest has been established in the case of insects that are animal pests. Researchers in Australia have shown that PM
proteins injected into sheep produce antibodies that interfere with the growth or even kill the fly pest, Lucilia cuprina that causes cutaneous myiasis in the sheep, a conditions that causes over 200 million dollars in losses per year. These researcher provided evidence that the Ab were able to interfere with the porosity of the fly PM
and interfered with the normal digestive processes of the insect. They speculated that this type of approach could be used in plants to control insects, however, provided not guidance as to how to accomplish such and approach.
A gene encoding an antibody that binds IIM or a fragment thereof may be used to transfect a microbial host. Microorganism hosts may be selected which are known to occupy the environment that the insect larval pest occupies. Such microorganisms include bacteria, algae, and fungi. A number of ways-are known in the art for introducing a such a gene into the microorganism host under conditions which allow for stable maintenance and e:rcpression of the gene. For example, expression cassettes can be constructed which include the DNA constructs of interest operably linked with SUBSTTEI1TE SHEET (Rule 26) the transcriptional and translational regulatory signals for expression of the DNA
constructs, and a DNA sequence homologous with a sequence in the host organism, whereby integation will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.
A transgenic plant expressing IIM-IgG can be constructed using available techniques for inserstion of cIDNA encoding an antibody to IIM into a plant genome.
Referring figure 1, the regions designat~i II, IV and VI are chitin binding regions.
Antibodies that bind to any of the regions would block chitin binding and provide the desired effect.
Thus the preferred first step in developing a transgenic plant is to raise one or more antibodies to the chitin binding regions. However, it could be desirable to raise an antibody that bound to a non-chitin binding region of the protein so that the chitin binding function of the protein remained intact. The antibody could then block pores in the PM but not disrupt PM formation.
Technology for using transgenic plants to express such antibodies is known in the art. Specifically, U.S. Patent No 5,686,600 teaches the production of antibodies that bind to insect midgut tissue and the use of such antibodies. The teaching of this patent are incorporated herein by reference. The novel IIM protein discovered by the applicants is an excellent target protein for the antibody binding.
An antibody, monoclonal antibody, or fragment thereof is said to be capable of binding a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody, monoclonal antibody, or fragment thereof.
The term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as fragments or binding regions or domains thereof which are capable of binding to the regions described above. Such fragments are typically produced by proteolytic cleavage, such as papain or pepsin, but can be produced through the application of recombinant DNA technology or through synthetic chemistry.
Methods for the preparation of the antibodies of the present invention are generally known in the art. F'or example, see Antibodies, A Laboratory Manual, Ed SUBSTITUTE SHEET (Rule 26) Harlow and David Lane (eds.) Cold Spring Harbor Laboratory, N.Y. (1988), as well as the references cited therein. Standard reference works setting forth the general principles of immunology include: Klein, J. Immunology: The Science of Cell Noncell Discrimination, John Wiley &: Sons, N.Y. (1982); Dennett, R, et al. Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, N.Y.
(1980); and Campbell, A. "Monoclonal Antibody Technology," In Laboratory Techniques in Biochemistry au~d Molecular Biology, Vol. 13, Burdon et al.
(eds.), Elsevier, Amsterdam (1984). See also, U.S. Pat. Nos.: 4,609,893; 4,713,325;
4,714,681;
4,716,111; 4,716,117; and 4,720,459.
The antibodies which possess the desired binding specificity can be used as a source of messenger RNA for cloning of the cDNA for the particular monoclonal antibody. Antibody genes can be cloned from hybridoma cells using primers to conserved DNA sequences within the constant regions and the framework regions of the variable regions. This can be followed by amplification of the DNA for cloning using the polymerase chain r~;action (PCR). A database of mouse heavy chain and light chain sequences complied by Kabat et al. has been successfully used to generate both isotype specific and degenerate prim for cloning antibody genes (Kabat, E. A.
et al., 1987, U.S. Dept Health and Human Services, U.S. Government Printing Offices and Jones, S. T. and Bendig, M., 1991, Biotechnology 9:88=89). Additionally, there is a wealth of knowledge concerning the cloning of smaller fragments of antibodies which possess the binding properties of the original antibody.
The cloned DNA can then be sequenced by methods known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd.
Edition, Cold Spring Harbor Laboratory Press, N.Y. (1989) vol. 1-3, and the references cited therein. From the nucleic acid sequence, the protein sequence of the binding region from the selected MAb can be deduced.
The antibodies and monoclonal antibodies of the invention find use in the production of hybrid toxin molecules. By "hybrid toxin molecules" or "hybrid toxins"
is intended, fusion proteins or immunotoxins, which comprise a monoclonal antibody or antibody fragment operably linked to a toxin moiety and which is capable of binding ~SUBSTIT'iJTE SHEET (Rule 26) to the gut of an insect. That is, when linked, the monoclonal antibody or antibody fragment retains its binding properties and the toxin moiety retains its cytotoxic properties. DNA sequences encoding the toxin moiety of the hybrid toxins are known in the art. See, Lamb et al. (1985) Eur. J. Biochem. 148:275-170 (Ricin); Gray et al.
(1984) PNA.S 81:2645-2649 (I~seudomonas toxin DNA Sequence); Hindley and Berry (1988) Nuc. Acids Res. 16:4168 (B. sphaericus toxin gene); Bauman et al.
(1988) J.
Bacteriol 170:2045-2050, Bamman et al. 1987) J. Bacteriol 169:4061-4067, Berry and Hindley (1987) Nucleic Acids Res. 15:5891, Berry et al. (1989) Nucleic Acids Res.
17:7516 (B. sphaericus); WO 9309130-A (gelonin); EP 466222-A, U.S. Pat. No.

5,128,460 (ribosome-activating; protein); EP 412911-A (barnase); Heernstadt et al.
(1987) Gene 57:37-46 (cryIIIA.); Brizzard and Whiteley (1988) Nucleic Acids Res 16:2723-2724 (cryIB); and Ge:iser et al. (1986) Gene 48:109-118 (cryIA(b)).
See also Porter et al. (1993) Microbiolo~gical Reviews 57:838-861; Hofte and Whiteley (1989) Microbiological Reviews 53:242-255 The antibody genes can be cloned and expressed in plants in such a manner that functional antibodies are assembled. See, for example, Hiatt et al. (1989) Nature 342:76-78 During et al. ( 1990) J. Plant Molecular Biology 15:281-293 and PCT
Application WO 91/06320. Levels of bivalent antibody expression have been reported to be as high as 1% of the soluble protein in tobacco. It is recognized that as well as antibody molecules, antibody Fragments such as Fab and Fv fragments, can be utilized.
The use of these antibody fragrnents provides the option of reducing the insect specific binding domain derived from a MAb to a very small size.
The genes can be optimized for enhanced expression in plants. See, for example EPA 0359472; EPA 0385962; 'WO 91/16432; Perlak et al. (1991) Proc. Natl. Aced.
Sci.
USA 88:3324-3328;and Murra.y (1989) Nucleic Acids Research 17:477-d98. In this manner, the genes can be synthesized utilizing plant preferred colons. That is, the preferred colon for a particular host is the single colon which most frequently encodes that amino acid in that host. Synthetic genes could also be made based on the distribution of colons a particular host uses for a particular amino acid.
Following this SITBSTIZ'UTE SHEET (Rule 26) approach, the nucleotide sequences can be optimized for expression in any plant and all or any part of the gene sequence may be optimized or synthetic.
Methods for the transformation of plant cells and regeneration of transformed plants are well known in the art. Generally, for the introduction of foreign DNA into plants Ti plasmid vectors have been utilized as well as direct DNA uptake, liposomes, electroporation, micro-injection, and the use of microprojectiles. Such methods have been published. See, for exannple, Guerche et al., (1987) Plant Science 52:111-116;
Neuhause et al., (1987) Theor. Appl. Genet. ?5:30-36; Klein et al., (1987) Nature 327:70-73; Howell et al., (1980) Science 208:1265; Horsch et al., (1985) Science 227:
1229-1231; DeBlock et al., (1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988);
and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc.
(1989). See also, EPA 0193:!59 and EPA 0451878A1. It is understood that the method of transformation will depend upon the plant cell to be transformed.
The components of a~i expression cassette containing the sequence of interest may be modified to increase expression in the plant or plant cell For example, truncated sequences, nucleotide substitutions or other modifications may be employed.
See, for example Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; Murray et al.
(1989) Nucleic Acids Research 17:477-498; and WO 91/16432. The construct may also include any other necessary regulators such as terminators, (Guerineau et al., (1991), Mol. Gen. Genet., 22;6:141-144; Proudfoot, (1991), Cell, 64:671-674;
Sanfacon et al., (1991), Genes Dev., 5:141-149; Mogen et al., {1990), Plant Cell 2:1261-1272; Munroe et al., (1990), Gene, 91:151-158; Ballas et al., (1989), Nucleic Acids Res., x7:7891-7903; Joshi et al., (1987), Nucleic Acid Res., 15:9627-9639);
plant translational consensus sequences (Joshi, C. P., (1987), Nucleic Acids Research, 15:6643-b653), introns (Luehrsen and Walbot, (1991), Mol. Gen. Genet., 225:81-93) and the like, operably linked to the nucleotide sequence. For tissue specific expression, the nucleotide sequences of t:he invention can be operably linked to tissue specific promoters.
SUBSTITUTE SHEET (Rule 26) Accordingly, it is to b~e understood that the embodiments of the invention herein described are merely illustratiive of the application of the principles of the invention.
Reference herein to details of the illustrated embodiments are not intended to limit the scope of the claims, which themselves recite those features regarded as essential to the invention.
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(i) APPLICANT: Granados,, Robert R

Wang, Ping IO (ii) TITLE OF INVENTION: A Novel Invertebrate Intestinal Mucin cDNA and Related Products and Methods.

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{iii) HYPOTHETICAL: NO

C)O (iv) ANTI-SENSE: NO

SI1BSTTTUTE SHEET (Rule 26) 2, (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE:
S (A) ORGANISM: Trichoplusia ni (F) TISSUE TYPE: Peritrophic Membrane (vii) IMMEDIATE SOURCE:
(B) CLONE: IIM1~9 (xi) SEQUENCE DESCRIP'.fION: SEQ ID NO:1:

GTAACGTTAA GTGAAAAGAA TAAIf:CAGCGA ACAAGTTATG ATAAAGACCC60 GACGGCCCTC GGGCTCGTCG CCG(:GCGTCC TC~AAGTCAGC GACGCGGAGA120 AGAACCCCGC

TGCCTCACGA

CACCGAGAGA

CTGTGCTCCT GGTACCGAAT TCAFvGTTCTC CGCTCAGACT TGTGTTCACG300 CCGCTTTAGC

CGGATGCACC CTGCCAGGAC CTCC:AGCTGA GACAACCCAG GCCCCAGCAA360 CCCAACAACC ACCCAGGCCC CAAC:CAC:AAC TACTCAGGCC CCTACTACAA420 CCACCCAGGC

CCCAACCACA ACCACCCAGG CCCC'AACCAC CACCCAGGCC CCAACCACCA480 CCCAGGCCCC

CTCAGGCCCC

AGGCCCCAAC

CAACTACCCC

TGTTGCCCAA

ACTGCAACCT

CTTCTACCAG TGCTCCAACG GTTACACCTT CGAACJ~GAGG TGCCCTGAGG900 GACTCTACTT

CCCCGCACCC CCAGTCACAG AAGC~rAACGA AGACGAAGAC ATTGACATCG1020 GAGACCTCCT

CGACAATGGA TGCCCAGCTA ACTTC:GAAAT CGACTGGCTC TTGCCCCACG1080 GAAACCGTTG

GAGCCGGCAC

GCACCCTCCC

CGGCGGCGAG AGCGAAGAAG TTGA(:GTCGA CGAGGATGCC TGCACCGGCT1260 SS GGTACTGCCC

CACGGAACCC ATTGAATGGG AGCC(:CTCCC CAACGGCTGC CCTGCCGACT1320 TCAGCATCGA

CCACCTCCTC CCCCACGAGA GCGAC:TGCGG CCAGTATCTA CAGTGTGTCC1380 ATGGACAGAC

GO TATCGCAAGA CCTTGCCCTG GAAAC:CTGCA CTTCAGTCCT GCCACACAGT1440 CCTGTGAGTC

TCCTGTGACC GCTGGTTGCC AAGT7.'TTCGA GTGCGATTCT GACAACCAGT1500 GCACATCGAC

TGCTGCCCCG A(',AGCTGCTC CAACGGCTGC CCCAACGGCT GCCCCAACGG1560 CTGCCCCAAC

SUBSTITUTE SHEET (Rule 26) TGCCGCACCC TCCACCGTGG TCC'CACCTGC AACGCCACCC GCAACTGCAG CCCCAGTCCC 1620 S CTCCTACTAC

CCACCGCAGC

CCGCTGCCCC

CAG,AAATCCC

CCCCCAACAC

CACAGTCACT GTACCACCCA CTGf:TGCCCC TACTACCGCA GCACCTGCCC1980 AGTCACTGTA CCACCCACTG CTG(:CCCCAC TGCAGCTCCC CCTACCGTCG2090 CACATGCACC

CAACACCACA GCTGCCCCGG TAA(:TACAAC CAGCGCACCA GCTACCACAC2100 CTGAAGATGA

2O TGACATCGAC CCCCCTCTCC CCAF1CGACCC CATCAACCCT TGCGTTGi4AG2160 AATGCAACGT

TTTGCCCTGG (',CTCACGCTG ACTGCGACAA ATACTGGGTC TGTGACGGCpI2220 ACAACCAAGT

TTGCApaCGTC GGTTGCGTGA GGAGCAACAT TCAGATGTCT GAAAGCTACG2340 AAGGRGTCCA

GGTCTTCATC CCATGGAACA AACT'AGATGA AGACATCAGA CAGGCGCTGA2400 ACTTTGAGTT

AAAAA

(2) INFORMATION FOR SEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:
3S (A) LENGTH: 2821 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: not relevant 4O (ii) MOLECULE TYPE: cDINA

(iii) HYPOTHETICAL: NO

4S (iv) ANTI-SENSE: NO

(v) FRAC~IENT TYPE: N-terminal (vi) ORIGINAL SOURCE:

SO (A) ORGANISM: Trichoplusia ni (D) DEVELOPMENTAI: STAGE: larva (F) TISSUE TYPE: peritrophic membrane SS (xf) SEQUENCE DESCRIPTION: SEQ ID N0:2:

GAAAA~hrAATA ACCAGCGAAC AAGTT'ATGAT AAAGACCCTC CTATTCCTGA60 CGGCCCTCGG

ACGACTGCAC

GTGCTCCTGG

S1~TBST1TZJTE SHEET (R~le 26) GoATGCACCCT

CAACAACCAC

CAACCACAAC

CACCCAGGCC CCAACCACCA CCCiAGGCCCC AAICCACCACC CAGGCCCCAA480 CTACCACTCA

GGCCCCTACT ACTACCACTC AGG(:CCCAAC CACAACCACT CAGGCCCCTA540 CCACAACCAC

CCAGGCCCCA ACCACCACCC AGG(:CCCAAC TACCACCCAG GCCCCAACTA600 CCACTCAGGC

CCCAACTACA ATCACCCAGG CTGC:AACTAIC CCCGGCCGCA ACTACCCCGG660 CCGCAACTAC

IS CCCGGCCGCA ACTACCCCTG CCGC:GACAAC CCCCGCTGCA ACTACCCCAG720 GTGTTCCTGC

ACCCACTTCA GCCCCAGTCT GGCC;CCCGAT CTGTGAACTG TTGCCCAATG780 GTTGCCCAGC

TGACTTCGAC ATCCACTTGT TGAT'TCCCCA CGACAAGTAC TGCAACCTCT890 TCTACCAGTG

CTCCAACGGT TACACCTTCG AACA.GAGGTG CCCTGAGG(',A CTCTACTTCA900 ACCCCTACGT

CCAGCGCTGC GACTCTCCTG CTAA.CGTTGA ATGCGACGGC GAAATCAICCC960 CCGCACCCCC

AGAATGGATG

ACAAGTATTA

CCAGTGCGTC CACGGTAACT TGGT.AGAGAG GCGTTGTGGA GCCGGCACCC1140 ACTTCAGTTT

TGAACTTCAG CAATGTGACC ACATGf.~AGCT CGTTGGCTGC ACCCTCCCCG1200 GCGGCGAGAG

CGAAGAAGTT GACGTCGACG AGGA'TGCCTG CACCGGCTGG TACTGCCCCA1260 CGGAP,CCCAT

ACCTCCTCCC

CCACGAGAGC GACTGCGGCC AGTA'.CCTACA GTGTGTCCAT G,GACAGACTA1380 TCGCAAGACC

TTGCCCTGGA AACCTGCACT TCAG"PCCTGC CACACAGTCC TGTGAGTCTC1440 CTGTGACCGC

TGGTTGCCAA GTTTTCGAGT GCGA7.'TCTGA CRACCAGTGC ACATCGACTG1500 CTGCCCCGAC

CCGCACCCTC

CTACAACCGC

AATTCCTACT CCGGCCCCCA CCGCT'GCCCC CACCGCAGCT CCTACTAICTG1680 CTGCCCCTGA

ATCCCCAACC AC:TGTCACAG TAGCR,CCTAC TGCTGCTCCC ACCGCAGCCC1740 SO CTACTACTGC

CCGCTGCCCC

CTACTGTCAC

CAGTCACTGT

TGACTGCACC

CCACAGCTGC

CCCGGTAACT ACAACCAGCG CACCAhCTAC CACACCTGAA GATGATGACA2160 TCGACCCCCC

S1:TBSTITLITE SHEET (Rule 26) S TGAGGGTCTC CAGTTCAACC CCACTACTAA GACCTGTC~i4C TTCGCTTGCA2390 ACGTCGGTTG

CGTGAGGAGC AACATTCAGA TG'TCTGAAAG CTACGAAGGA GTCCAGGTCT2400 TCATCCCATG

C~AACA74ACTA GATGAAGACA TC;pGACAGGC GCTGAACTTT GAGTTGTAAA2960 CCTACTTAAA

TTAATGAAGG TTTTGTTTTA TT'L'TTGAGTT ATTATTCCAA TGGGCGGGAA2520 AGTCCGCCAT

TATTGGGTCT TGCCAGTTTT GAc;GAAACCT TTTTTTTTAC TACCAACATT2580 CTTGTGAACC

IS CATATTTATT ACGTATTAAA CA'.PCGTGATT TGAAAAACGT TAiCATGATTT2640 TTTCATTAAT

TCGAAACTGG

CAATTTTGGA TTGGAATAAT CA~1CAAATGG TTAAGAAAAA AAACGATTTC2760 TTAAAAATGT

AAAAAAAAAA

A

2S (2) INFORMATION FOR SEQ ID N0:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 788 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein 3S (iii) HYPOTHETICAL: NO

(ivy ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:
(A) ORGANISM: T:richoplusia ni (F) TISSUE TYPE'.: peritrophic membrane 4S (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met Ile Lys Thr Leu Leu Phe Leu Thr Ala Leu Gly Leu Val Ala Ala SO Arg pro Glu Val Ser p,sp A~,a Glu Lys Asn Pro Ala Leu His Glu Pro His Pra Asp Cys Pro P'ro Ala Glu Gln His Trp Leu Leu Pro His Glu SS
Tyr Asp Cys Thr Lys Phe Tyr Tyr Cys Glu Tyr Gly Leu Lys Phe Ile Ala Pra Arg Asp Cys Ala Pro Gly Thr Glu Phe Lys Phe Ser Ala Gln Thr Cys Val His Ala Ala Leu Ala Gly Cys Thr Leu Pro Gly Pro Pro SiTBSTTI'UTE SHEET (Rule 26) Ala Glu Thr Thr Gln Al~a Pro Ala Thr Thr Gln Ala Pro Thr Thr Thr Gln Ala Pro Thr Thr Thr Thr Gln Thr Thr Thr Thr Ala Pro Gln Ala Pro Thr Thr Thr Thr Gln Ala Pro Thr Gln Ala Pro Thr Thr Thr Thr Thr Gln Ala Pro Thr Thr Thr Gln Thr Thr Thr Thr Ala Pro Gln Ala Pro Thr Thr Thr Thr Gln Ala Pro Thr Thr Gln Ala Thr Thr Pro Thr IS

Thr Thr Gln Ala Pro Thr Thr Thr Pro Thr Thr Thr Gln Ala Gln Ala Pro Thr Thr Ile Thr Gln Ala Ala Pro Ala Ala Thr Thr Thr Thr Pro Ala Ala Thr Thr Pro Ala Ala Thr Ala Ala Thr Thr Thr Pro Pro Ala Ala Thr Thr Pro Gly Va7. Pro 5er Ala Pro Val Ala Pro Thr Trp Pro Pro Ile Cys Glu Leu Leu Pro Asn Pro Ala Asp Phe Gly Cys Asp Ile His Leu Leu Ile Pro His Asp Lys Asn Leu Phe Tyr Tyr Cys Gln Cys Ser Asn Gly Tyr Thr Phe~ Glu Pro Glu Gly Leu Gln Arg Cys Tyr Phe Asn Pro Tyr Val Gln Arcf Cys Ala Asn Val Glu Asp Ser Pro Cys Asp Gly Glu Ile Ser Pro Ala Pro Pro Glu Gly Asn Glu Val Thr Asp Glu Asp Ile Asp Ile Gly Asp Leu Leu Gly Cys Pro Ala Asp Asn Asn Phe Glu Ile Asp Trp Leu Leu Pro His Arg Cys Asp Lys Gly Asn Tyr Tyr Gln Cys Val His Gly Asn Leu Val Arg Cys Gly Ala Glu Arg Gly Thr His Phe Ser Phe Glu Leu Gln Gln His Ile Glu Leu Cys Asp Val Gly Cys Thr Leu Pro Gly Gly Glu Ser Val Asp Val Asp Glu Glu Glu Asp Ala Cys Thr Gly Trp Tyr Cys Pro Pro Ile Glu Trp Thr Glu Glu Pro Leu Pro Asn Gly Cys Pro Ala Asp Ile Asp His Leu Phe Ser Leu Pro His Glu Ser Asp Cys Gly Gln Tyr Cys Val His Gly Leu Gln Gln Thr S1:TBSTIZ'LTTE SHEET (Rule 26) Ile Ala Arg pro Cys Pro Gly Asn Leu His Phe Ser Pro Ala Thr Gln Ser Cys Glu Ser Pro Val Thr Ala Gly Cys Gln Val Phe Glu Cys Asp Ser Asp Aan Gln Cys Thr Ser Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Ser Thr Val Va1 Pro Pro Ala Thr Pro Pro Ala Thr Ala Ala Pro Val Pro Pro Thr Thr Ala Ile Pro Thr Pro Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Thr.Ala Ala Pro Glu Ser Pro Thr Thr Val Thr Val Pro Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Thr Ala Val Pro Glu Ile 56s Pro Ile Thr Val Thr Ser Ala pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala pro Thr Ala Ala Pro Thr Thr Ala Val Pro Glu Ile Pro Thr Thr Val Thr Ser P.ro Pro Thr Ala Ala Pro Thr Thr Ala Ala Pro Ala Pro Asn Thr Thr 'Val Thr Val Pro Pro Thr Ala Ala Pro Thr Thr Ala Ala Pro Ala Pro ~Asn Thr Thr Val Thr Val Pro Pro Thr Ala Ala Pro Thr Ala Ala Pra 1?ro Thr Val Ala His Ala Pro Asn Thr Thr Ala Ala Pro Val Thr Thr 7'hr Ser Ala Pro Ala Thr Thr Pro Glu Asp Asp Asp Ile Asp Pro Pro Leu Pro Asn Asp Pro Ile Asn Pro Cys Val Glu Glu Cya Asn Val Leu Faro Trp Ala His Ala Asp Cys Asp Lys Tyr Trp Val Cys Asp Gly Asn 74.sn Gln Val Leu Val Val Cys Ser Glu Gly Leu Gln Phe Asn Pro Thr Thr Lys Thr Cys Asp Phe Ala Cys Asn Val Gly Cys Val Arg Ser Asn Ile Gln Met Ser Glu Ser Tyr Glu Gly Val Gln Val Phe Ile Pro Trp Asn Lys Leu Asp Glu Asp Ile Arg Gln Ala Leu S1UBST1TUTE SHEET (Rule 26) Asn Phe Glu Leu S (2) INFORMATION
FOR
SEQ
I:D
N0:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 80T amino acids (B) TYPE; amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein IS (iii) HYPOTHETICAL; NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: T:richoplusia ni (F) TISSUE TYPE: peritrophic membrane PCTlUS99/14220 2S (xi) SEQUENCE DESCRIP7CION: SEQ ID N0:4:
Met Ile Lys Thr Leu I:eu Phe Leu Thr Ala Leu Gly Leu Val Ala Ala Arg Pro Glu Val Ser Asp A1a Glu Lys Asn Pro Ala Leu His Glu Pro His Pro Asp Xaa Pro Fro Ala Glu Gln Xaa Xaa Leu Leu Pro Xaa Glu 3S Tyr Asp Cys Thr Lys P~he Tyr Tyr Cys Glu Tyr Gly Leu Lys Phe Ile Ala Pro Arg Asp Cys Ala Pro Gly Thr Glu Phe Lys Phe Ser Ala Gln Thr Cys Val His Ala Ala Leu Ala Gly Cys Thr Leu Pro Gly Pro Pro Ala Glu Thr Thr Gln Ala Pro Ala Thr Thr Gln Ala Pro Thr Thr Thr 4S loo l05 110 Gln Ala Pro Thr Thr Tlzr Thr Gln Ala Pro Thr Thr Thr Thr Gln Ala SO Pro Thr Thr Thr Thr Gln Ala Pro Thr Thr Thr Gln Ala Pro Thr Thr Thr Gln Ala Pro Thr Thr Thr ~ln Ala Pro Thr Thr Thr Thr Gln Ala SS 195 1~'0 155 160 Pro Thr Thr Thr Thr Gl.n Ala Pro Thr Thr Thr Thr Gln Ala Pro Thr Thr Thr Gln Ala Pro Thr Thr Thr Gln Ala Pro Thr Thr Thr Gln Ala Pro Thr Thr Ile Thr Gl.n Ala Ala Thr Thr Pro Ala Ala Thr Thr Pro SUBSTITUTE SHEET (Rule 26) Pro Ile Cys Glu Leu Ala Ala Thr Thr Pro :Ala Ala Thr Thr Pro Ala Ala Thr Thr Pro Ala Ala Thr Thr Pro Gly Val Pro Ala Pro Thr Ser Ala Pro Val Trp Pro Pro Ile Cys Glu Leu Leu Pro Asn Gly Cys Pro Ala Asp phe Asp Ile His Leu Leu Ile Pro His Asp Lys Tyr Cys Asn Leu Phe Tyr Gln Cys Ser Asn Gly Tyr Thr F~he Glu Gln Arg Cys Pro Glu Gly Leu Tyr Phe Asn Pro Tyr Val Gln J9,rg Cys Asp Ser Pro Ala Asn Val Glu Cys Asp 29« 295 Gly Glu Ile Ser Pro A.la Pro Pro Val Thr Glu Gly Asn Glu Asp Glu Asp Ile Asp Ile Gly Asp Leu Leu Asp Asn Gly Cys Pro Ala Asn Phe Glu Ile Asp Trp Leu Leu Pro His Gly Asn Arg Cys Asp Lys Tyr Tyr Gln Cys Val His Gly Asn Leu Val Glu Arg Arg Cys Gly Ala Gly Thr His Phe Ser Phe Glu Leu Gln Gln Cys Asp His Ile Glu Leu Val Gly Cys Thr Leu Pro Gly Gly Glu Ser Glu Glu Val Asp Val Asp Glu Asp 385 3<.~0 395 900 Ala Cys Thr Gly Trp Tyr Cys Pro Thr Glu Pro Ile Glu Trp Glu Pro Leu Pro Asn Gly Cys Pra Ala Asp Phe Ser Ile Asp His Leu Leu Pro His Glu Ser Asp Cys Gl.y Gln Tyr Leu Gln Cys Val His Gly Gln Thr Ile Ala Arg Pro Cys Pro Gly Asn Leu His Phe Ser Pro Ala Thr Gln Ser Cys Glu Ser Pro Val Thr Ala Gly Cys Gln Val Phe Glu Cys Asp Ser Asp Asn Gln Cys Thr Ser Tht Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Ser Thr Val Val Pro Pro Ala Thr Pro Pro Ala Thr Ala Ala Pro Val Pro Pro Thr Thr Ala Ile Pra Thr Pro Ala Pro Thr Ala Ala pro Thr Ala Ala Pro Thr Thr Ala Ala Pro Glu Ser Pro Thr Thr Val Thr Val Pro SITBSTITUTE SHEET (Rule 26) 595 5fi0 555 560 Pro Thr Ala Ala Pro Thr Ala Ala pro Thr Thr Ala Val Pro Glu Ile s Pro Ile Thr Val Thr Se:r Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Thr Ala Val Pro Glu Ile Pro Thr Thr Val Thr Ser Pro Pro Thr Ala Ala Pro Thr Thr Ala Ala Pro Ala Pro Asn Thr Thr Va.l Thr Val pro Pro Thr Ala Ala Pro Thr Thr Ala Ala Pro Ala Pro Asn Thr Thr Val Thr Ala Pro Pro Thr Ala Ala Pro Thr Thr Ala Ala Pro Ala Pro Asn Thr Thr Val Thr Val Pro Pro Thr Ala Ala Pro Thr Ala Ala Pro Pro Thr Val Ala His Ala Pro Asn Thr Thr Ala Ala Pro Val Thr Thr Thr Ser Ala Pro Ala Thr Thr Pro Glu Asp Asp Asp Ile Asp Pro pro Leu Pro Asn Asp Pro Ile Asn Pro Cys Val Glu Glu Cys Asn Val Leu Pro Trp Ala His Ala Asp Cys Asp Lys Tyr Trp Val Cys As;p Gly Asn Asn Gln Val Leu Val Val Cys Ser Glu Gly Leu Gln Phe Assn Pro Thr Thr Lys Thr Cys Asp Phe Ala Cys Asn Val Gly Cys Val Ar~g Ser Asn Ile Gln Met Ser Glu Ser Tyr Glu Gly Val Gln Val Phe Il~e Pro Trp Asn Lys Leu Asp Glu Asp Ile Arg Gln Ala Leu Asn Phe Glu Leu S~UBSTIT'UTE SHEET (Rule 26)

Claims (19)

What is claimed is:

1. A transformed virus, microorganism, cell, plant, or animal capable of expressing an inverterbrate intestinal mucin protein, comprising an expression vector wherein said expression vector comprises a gene encoding said invertebrate intestinal mucin protein operably linked to an expression control sequence.

2. The transformed virus, microorganism, cell, plant or animal of claim 1 wherein said microorganism is Escherichia coli.

3. A recombinant DNA sequence comprising a DNA sequence that codes for an invertebrate intestinal mucin protein.

4. The recombinant DNA sequence of claim 3, wherein the nucleic acid sequence of said recombinant DNA sequence is selected from the group consisting of:
a) a cDNA sequence as shown in SEQ. ID. No. 1; and b) a cDNA sequence as shown in SEQ. ID. No. 2.

5. The recombinant DNA sequence of claim 3, wherein said invertebrate intestinal mucin protein has an amino acid sequence selected from the group consisting of:
a) an amino acid sequence as shown in SEQ. ID. No. 3; and b) an amino acid sequence as shown in SEQ. ID. No. 4.

6. A method of producing an invertebrate intestinal mucin protein or peptide, comprising:
a) transforming a host cell with an expression vector comprising a promoter operatively linked to a nucleotide sequence which codes for a predetermined protein or peptide of an invertebrate intestinal mucin protein;
b) culturing said host cell under favorable conditions for expression of said invertebrate intestinal mucin protein;

c) lysing said host cell; and d) recovering said invertebrate intestinal mucin protein.

7. The method of claim 6 wherein said expression vector further comprises a gene encoding for glutathione-S-transferase.

8. A gene expression vector containing a recombinant DNA sequence encoding a Trichoplusia ni invertebrate intestinal mucin protein sequence.

9. The method of claim 7, wherein said expression vector is a recombinant plasmid adapted for insertion into and transformation of bacteria.

10. The method of claim 7, wherein said expression vector is a recombinant plasmid adapted for insertion into and transformation of a plant.

11. A fusion protein that includes a first protein linked to a second protein that comprises at least a functional domain of a Trichoplusia ni invertebrate intestinal mucin protein.

12. A pure preparation of an invertebrate intestinal mucin protein.

13. A transformed microorganism, cell, plant, or animal capable of expressing an antibody directed against an invertebrate intestinal mucin protein comprising an expression vector, wherein said expression vector comprises a gene encoding said antibody that binds to an invertebrate intestinal mucin protein sequence.

14. The microorganism, cell, plant, or animal of claim 13, wherein said antibody specifically binds to a chitin binding region of said invertebrate intestinal mucin protein.

15. An antibody produced by the microorganism, cell, plant, or animal of claim 13.

16. A purified and isolated antibody that specifically binds to an invertebrate intestinal mucin protein.

17. The antibody of claim 16, wherein said antibody specifically binds to a chitin binding region of said invertebrate intestinal mucin protein.

18. A recombinant DNA molecule comprising a nucleotide sequence which codes for a fusion protein that includes a first protein linked to a second protein that comprises at least a functional chitin binding domain of a Trichoplusia ni invertebrate intestinal mucin protein.

19. A method of protecting a plant against an insect pest that includes an invertebrate intestinal mucin protein in said insect pest's midgut, comprising:
a) transforming a microorganism, cell, plant, or animal capable of expressing an antibody directed against said invertebrate intestinal mucin protein with an expression vector, wherein said expression vector comprises a gene encoding said antibody that binds to an invertebrate intestinal mucin protein; and b) placing said transformed microorganism, cell, plant, or animal in an environment that includes a plant to be protected and said insect pest;
wherein said antibody blocks the activity of the invertebrate intestinal mucin protein.