©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of an Insect Aminopeptidase N That Serves as a Receptor for Bacillus thuringiensis CryIA(c) Toxin (*)

(Received for publication, April 26, 1995)

Peter J. K. Knight (1) Barbara H. Knowles (2)(§) David J. Ellar (1)(¶)

From the  (1)Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom and the (2)Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Bacillus thuringiensis CryIA(c) insecticidal -endotoxin binds to a 120-kDa glycoprotein receptor in the larval midgut epithelia of the susceptible insect Manduca sexta. This glycoprotein has recently been purified and identified as aminopeptidase N. We now report the cloning of aminopeptidase N from a M. sexta midgut cDNA library. Two overlapping clones were isolated, and their combined 3095-nucleotide sequence contains an open reading frame encoding a 990-residue prepro-protein. The N-terminal amino acid sequence derived from the glycoprotein is present in the open reading frame, immediately following a predicted cleavable signal peptide and a pro-peptide. There are four potential N-linked glycosylation sites. The C-terminal sequence contains a possible glycosylphosphatidylinositol (GPI) anchor signal peptide, which suggests that, unlike most other characterized aminopeptidases, the lepidopteran enzyme is anchored in the membrane by a GPI anchor. This was confirmed by partial release of aminopeptidase N activity from M. sexta midgut brush border membranes by phosphatidylinositol-specific phospholipase C. The deduced amino acid sequence shows significant similarity to the zinc-dependent aminopeptidase gene family, particularly in the region surrounding the consensus zinc-binding motif characteristic of these enzymes.


INTRODUCTION

The target of insecticidal Bacillus thuringiensis crystal -endotoxins is the apical (brush border) membrane of larval midgut cells(1) . In vitro binding assays have demonstrated that the CryIA(c) toxin binds specifically and with high affinity to a single receptor species in brush border membranes prepared from larvae of the susceptible lepidopteran, Manduca sexta(2) . Ligand blotting experiments have identified a single 120-kDa toxin-binding glycoprotein in M. sexta larval midgut membranes as the most likely candidate for the cellular CryIA(c) receptor(3, 4) .

We recently reported the purification of this 120-kDa putative receptor from M. sexta midgut membranes by a combination of protoxin affinity chromatography and anion-exchange chromatography(5) . N-terminal and internal partial amino acid sequences were similar to sequences of the ectoenzyme aminopeptidase N, and the purified 120-kDa glycoprotein displayed aminopeptidase N but not alkaline phosphatase activity. CryIA(c) toxin itself had no apparent effect on aminopeptidase activity over a range of concentrations. In ligand blotting experiments, the purified glycoprotein had the characteristics predicted of the receptor; it bound CryIA(c) toxin in the presence of GlcNAc but not GalNAc, it bound the lectin SBA, but it did not bind CryIB toxin ( (5) and references therein).

The same glycoprotein was partially purified by Sangadala et al.(6) who used isoelectric focusing and immunoaffinity chromatography to obtain a mixture of 120- and 65-kDa midgut brush border proteins from M. sexta. Both (glyco)proteins bound CryIA(c) toxin in ligand blots, although the 120-kDa band was the major toxin-binding component(4) . Enzyme assays revealed both aminopeptidase and alkaline phosphatase activity in the partially purified preparation, and the 120-kDa protein was identified as aminopeptidase N from the partial amino acid sequence. When reconstituted into phospholipid vesicles, the protein mixture increased toxin binding by 35% and enhanced toxin-induced Rb release up to 1000-fold. This important result is the first (and so far only) demonstration that a partially purified receptor can potentiate the action of a toxin in vitro.

Aminopeptidase N (CD13; microsomal aminopeptidase; -aminoacyl-peptide hydrolase (microsomal); EC 3.4.11.2) is a well documented zinc-dependent peptidase that catalyzes removal of N-terminal, preferentially neutral residues from peptides (reviewed in (7) ). This ectoenzyme is commonly found in the brush border membranes of the alimentary tract in a variety of different organisms. Recent reports have shown that a number of coronaviruses and a herpesvirus use aminopeptidase N as a receptor in their target tissue (8, 9, 10) .

Following receptor binding at the midgut epithelium, toxins probably act by opening nonspecific channels or pores in the membrane, which leads to colloid osmotic lysis of midgut cells and ultimately the death of the insect(11) . With the aim of understanding both the biochemical basis for toxin specificity and the mechanism(s) by which membrane insertion and cytolysis occur, we have cloned and sequenced the cDNA of M. sexta aminopeptidase N, a putative CryIA(c) receptor.


EXPERIMENTAL PROCEDURES

Polymerase Chain Reaction Amplification

PCRs()were performed by standard techniques(12) . If the PCR product was to be sequenced, Pfu DNA polymerase (Stratagene) was used in the amplification because of its high fidelity. Otherwise, Taq DNA Polymerase (Promega) was used in all PCRs. Single-stranded cDNA from M. sexta midgut brush border membranes was prepared as described previously(5) .

Oligonucleotide Synthesis and Labeling

Oligonucleotides used as PCR primers and hybridization probes were synthesized on a Millipore Expedite 8909 nucleic acid synthesizer. Oligonucleotide probes were 3` end-labeled with digoxigenin using the digoxigenin oligonucleotide tailing kit from Boehringer Mannheim and were used according to the manufacturer's recommendations.

cDNA Cloning and Sequencing

The M. sexta midgut brush border membrane cDNA library in gt10 was a gift from Dr. J. Van Rie, Plant Genetic Systems, Belgium. The library was screened by the PCR-based microtiter plate technique described by Israel(13) . Briefly, 8000 pfu arranged at 125 pfu/well in an 8 8 well array were screened by PCR (primers 3F and 5R). Two wells tested positive, and the phage from these were titered and rescreened at 4 pfu/well. One PCR-positive well from the secondary screen was selected, individual phage clones were plaque-purified, and phage DNA was prepared by the plate lysis method(12) . Their identity as aminopeptidase N clones was confirmed by Southern blotting.

Subcloning and DNA Sequence Analysis

The APN cDNA insert was excised from the phage vector with EcoRI and subcloned into EcoRI-cut pBluescript II SK(-) (Stratagene). The 5`APN blunt-ended PCR product was subcloned into EcoRV-cut pBluescript II SK(-), and the phage vector sequence was excised on a BamHI fragment. All subcloning operations were performed by standard techniques(12) . DNA was sequenced on an Applied Biosystems Inc. 373 automated DNA sequencer, using an Applied Biosystems Inc. Dye-Deoxy Terminator Cycle sequencing kit. A double-stranded nested deletion kit (Pharmacia Biotech Inc.) was used to generate a set of progressively smaller subclones of APN for sequencing. All clones were completely sequenced on both strands. DNA and protein sequences were assembled and analyzed using the Genetics Computer Group program package (14) and the Lasergene package (DNASTAR).

PI-PLC Digestions and Aminopeptidase N Assay

M. sexta brush border membrane vesicles, prepared as described(15) , were suspended at 2 mg/ml in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, pH 7.4). PI-PLC from Bacillus cereus (Sigma) was added at a final concentration of 2 units/ml and incubated for 90 min at 30 °C. The vesicles were pelleted by centrifugation at 13,000 g for 10 min, the pellet resuspended in the same volume of phosphate-buffered saline, and the supernatant and pellet assayed for aminopeptidase and alkaline phosphatase activity as described(5) . Control release was measured under the same conditions in the absence of PI-PLC. Release by detergents was carried out by the same method, using final concentrations of 0.1% (v/v) Triton X-100 or 0.5% (w/v) CHAPS.


RESULTS

Partial Amino Acid Sequence and PCR

Following purification of aminopeptidase N from M. sexta midgut epithelium, both N-terminal and internal partial amino acid sequences were obtained from the glycoprotein. A possible overlap between the N-terminal sequence and internal amino acid sequence 77 (5) was tested by nested PCR using fully degenerate primers. When subcloned and sequenced, the cDNA sequence confirmed the overlap between the two partial amino acid sequences and also yielded 45 bp of unambiguous aminopeptidase N gene sequence (nt 139-183 in Fig. 2), which was used to design a unique forward PCR primer 3F and an oligonucleotide probe 4F (Fig. 1).


Figure 2: Sequence of M. sexta aminopeptidase N cDNA and deduced amino acid sequence. The putative N-terminal cleavable signal peptide is underlined, and consensus N-linked glycosylation sites are double-underlined. Partial amino acid sequences from the purified protein are broken underlined, and the N-terminal residue of the mature protein determined by Edman degradation is designated by a . The zinc binding/catalytic site (gluzincin motif) is boxed. The GPI signal peptide is dot-underlined, and indicates the probable cleavage/attachment site of the anchor moiety. Repeats in the 3`-untranslated tail are underlined, and the two polyadenylation signals are in capitals.




Figure 1: Aminopeptidase clones and PCR primers. A, relationship between 5`APN (top) and APN (bottom) and location of oligonucleotides used as PCR primers (arrows) and hybridization probes (lines). Primer 3R (brokenline) is a fully degenerate oligonucleotide predicted from partial amino acid sequence, while all other oligonucleotides are designed from a unique cDNA sequence. The scale refers to the position in the combined cDNA sequence (Fig. 2). B, sequence of unique oligonucleotides used in PCR amplifications.



A fully degenerate antisense reverse PCR primer 3R was designed from internal amino acid sequence 68.5, QIVDDVF(5) . This primer was used in conjunction with forward primer 3F to amplify fragments of aminopeptidase N cDNA from M. sexta midgut single-stranded cDNA preparations (Fig. 1). A single 1700-bp PCR product was identified by hybridization with 4F and was gel-purified and directly sequenced. This unambiguous gene sequence was used to design a unique reverse PCR primer 5R (see Fig. 1), situated 345 bp downstream of the unique forward PCR primer 3F.

Isolation of Two Overlapping Clones for Aminopeptidase N

The unique primer pair 3F/5R was used to screen a M. sexta midgut cDNA library in gt10 using the high stringency PCR-based technique of Israel(13) . 8000 phage clones were screened, and one positive recombinant phage, APN, was obtained. Although the 2994-bp cDNA insert (nt 102-3095 in Fig. 2) was found to contain an open reading frame that encoded the N terminus and all eight tryptic peptides derived from the purified protein(5) , no initiating ATG codon was found, indicating that clone APN does not contain the total mRNA sequence. Attempts to obtain the missing 5` end of the mRNA by 5`-rapid amplification of cDNA ends (16) were unsuccessful, and therefore the cDNA library was screened again by nested PCR, using a forward primer (gtLF) sited in gt10 and two nested reverse primers (8R and 9R) at the 5` end of clone APN (see Fig. 1). A single 350-bp PCR product, 5`APN, was obtained containing 148 bp of aminopeptidase N cDNA (nt 1-148 in Fig. 2), including a 47-bp overlap with the 5` end of APN. The new 5` cDNA still did not contain an initiating ATG codon, but it did encode a putative N-terminal cleavable signal peptide (see below).

Nucleotide and Deduced Amino Acid Sequence

Both APN and 5`APN cDNAs were subcloned and sequenced on both DNA strands as described under ``Experimental Procedures.'' The combined 3095-bp nucleotide sequence (Fig. 2) has an in-frame ATG codon at the 5` end of the cDNA (nt 94-96), but this is probably not a start codon since it does not meet the criteria for a Kozak consensus translational initiation site(17) . Therefore, the combined cDNA sequence is presumed to be missing a 5` upstream sequence, including the initiating ATG codon. There is a long open reading frame starting at nucleotide 2 and extending 2970 bp to a TAA stop codon at nucleotide 2971. The short 124-bp 3` noncoding region includes two additional in-frame stop codons and two consensus AATAAA polyadenylation signals contained within a 17-bp repeat (nt 2989-3006 and nt 3064-3081, Fig. 2), which may imply the occurrence of polymorphism in the 3` noncoding region of the mRNA.

The 2970-bp open reading frame encodes a protein of 990 residues (Fig. 2). The N-terminal sequence of the mature (purified) protein determined by Edman degradation extends from Asp to Pro (Fig. 2). The presence of residues upstream of Asp suggests that in M. sexta, aminopeptidase N is synthesized as a larger precursor protein and is trimmed to a mature product by limited proteolysis. There are four consensus Asn-X-Ser/Thr sequences, indicating possible N-glycosylation sites in the protein.

Homology to Other Aminopeptidases

Searches of the SwissProt (EMBL) protein sequence data base with the primary structure of M. sexta aminopeptidase N showed significant similarity to human (31% identity)(18) , rabbit (31% identity)(19) , and rat aminopeptidase N (31% identity)(20) , to aminopeptidase yscII from Saccharomyces cerevisiae (29% identity)(21) , alanine aminopeptidase (pepN) from lactobacterium (27% identity) (22) , and also to mouse (29% identity) (23) and human (28% identity) (24) aminopeptidase A. A multiple sequence alignment between these known aminopeptidase sequences and the M. sexta sequence showed that the most striking similarity was around the characteristic and functionally crucial zinc-binding motif between residues Ile and Phe (Fig. 3A). This sequence classifies the M. sexta protein as a member of the aminopeptidase family of gluzincins, with His, His, and Glu being zinc ligands and Glu being involved in catalysis(25) . An obvious difference in the alignment was the C-terminal 40-60-residue extension of the M. sexta sequence, which includes the GPI anchor signal peptide (Fig. 3B; see below). This feature probably reflects the fact that other membrane-bound aminopeptidases are generally anchored by an N-terminal signal anchor sequence.


Figure 3: Aminopeptidase sequence alignment. Alignment of the deduced amino acid sequences of aminopeptidase N from M. sexta with aminopeptidase N from human(18) , rabbit(19) , and rat(20) , aminopeptidase yscII from S. cerevisiae(21) , alanine aminopeptidase (pepN) from L. delbruckii(22) , and the aminopeptidase A from human (24) and mouse(23) . Letters in the consensus sequence represent residues common to all sequences. Numbers to the left refer to the first residue in each line relative to the start codon of each respective primary sequence. A, highly conserved block including the zinc binding/catalytic site typical of the aminopeptidase family of gluzincins. Conserved sequences are boxed. The gluzincin motif is shown above the consensus sequence, with catalytic residues in boldface and zinc binding ligands in boldface italics. B,M. sexta aminopeptidase N C-terminal extension containing the GPI signal peptide not found in other aminopeptidases.



Membrane Anchoring

In the epithelial cells of mammalian kidney and intestine aminopeptidase N is a type II membrane protein, anchored by an uncleaved N-terminal signal anchor sequence and with a C-terminal extracellular domain(26, 27) . However, treatment of M. sexta brush border membrane vesicles (BBMV) with proteinase K leads to the release into the supernatant of a 100-kDa soluble form of aminopeptidase N, with the same N-terminal amino acid sequence as the membrane-bound form of the protein (data not shown). This suggests that the M. sexta aminopeptidase N is a type I membrane protein, anchored in the membrane by a C-terminal ``stop-transfer'' sequence and with an N-terminal extracellular domain. Such a topology would require an N-terminal cleavable signal peptide to initiate translocation across the endoplasmic reticulum membrane(27) .

A hydropathy plot of the predicted primary structure (Fig. 4A) reveals one region at the N terminus and one at the C terminus with hydropathy averages greater than 1.6 and thus capable of spanning the membrane in a helical conformation(28) . Although there is a third and comparatively shorter hydrophobic region centered around Ala, biochemical analysis of the protein (preceding paragraph) indicates that it is unlikely to be a transmembrane helix. Analysis of the N-terminal hydrophobic region by the weight-matrix method of von Heijne (29) for predicting signal sequence cleavage sites yields a ``score'' of 7.8 for cleavage after residue 15, while all other residues give scores of 1.9 or less. Known cleavage sites in other proteins typically have scores of 6-12. The algorithm gives a correct prediction in 75-80% of cases(29) , and on this evidence it seems probable that the N terminus of the deduced polypeptide is a cleavable signal sequence, with scission by the signal peptidase occurring on the C-terminal side of Thr. According to this predicted topology, the sequence between Thr and Asp (the N terminus of the mature protein as determined by Edman degradation) constitutes a propeptide, the proteolytic release of which might serve to activate pro-aminopeptidase N. Although activation propeptides are a common feature of many proteases, hormones, and growth factors (reviewed in (30) ), there is only one other example of a putative propeptide region in an aminopeptidase, predicted from the cDNA-derived primary structure of aminopeptidase Y in S. cerevisiae(31) .


Figure 4: A, hydropathy plot of the M. sexta aminopeptidase N protein sequence. The method of Kyte and Doolittle (28) was used with averaging over a window of 11 residues. Hydrophobicity resulted in positive and hydrophilicity in negative values. B, schematic diagram of M. sexta aminopeptidase N protein sequence. The predicted N-terminal cleavable signal peptide (openbox) is followed by a predicted pro-peptide (filledbox). The horizontallyhatchedbox represents the gluzincin motif, while at the C terminus there is a predicted O-glycosylated stalk (dottedbox) and the GPI signal peptide (diagonallyhatchedbox). The four potential N-linked glycosylation sites are indicated as knobs.



A closer examination of the C-terminal hydrophobic sequence suggests that it is not the stop-transfer sequence of a type I membrane protein (27) since it lacks charged residues flanking the hydrophobic region, particularly positive charge at the C terminus typically found in such sequences(32, 33, 34) . However, it does show the characteristics of a signal peptide for the addition of a glycosylphosphatidylinositol (GPI) anchor: a C-terminal run of 19 hydrophobic residues (Ile-Ala), preceded by a cluster of three small residues (Gly-Gly, see Fig. 2), which functions as a cleavage/attachment site(35, 36) .

A common diagnostic test for a GPI-anchored protein (37) is to demonstrate its release from the membrane by bacterial PI-PLC. Following incubation of M. sexta BBMV with PI-PLC, 16.0 ± 0.4% of total aminopeptidase N activity was released into the supernatant (n = 4) compared with a release of 4.8 ± 0.9% in the absence of PI-PLC (n = 7). In comparison, PI-PLC released 83.5 ± 2.6% of alkaline phosphatase activity into the supernatant (n = 4), compared with 9.9 ± 3.2% release in the control (n = 4). Differential solubilization by detergents can also be used to predict the presence of a GPI membrane anchor(38, 39) , since only detergents with a high critical micellar concentration (CMC) are able to release significant amounts of GPI-anchored ectoenzymes into the supernatant. Treatment of M. sexta BBMV with 0.5% CHAPS (high CMC) released 78% of the total aminopeptidase N activity into the supernatant (n = 2), while 0.1% Triton X-100 (low CMC) released only 7% of total activity (n = 1). Although PI-PLC releases only a fraction of the total aminopeptidase N activity into the supernatant, this result demonstrates that at least a proportion of the M. sexta enzyme is linked to the brush border membrane by a GPI anchor. A similar study (40) showed that aminopeptidase N in the brush border membrane of the closely related lepidopteran Bombyx mori is also GPI-anchored. PI-PLC caused a maximal 40% release of B. mori aminopeptidase N activity compared with a 90% release of alkaline phosphatase activity.


DISCUSSION

In this study, partial amino acid sequence from aminopeptidase N purified from M. sexta midgut epithelium as a putative B. thuringiensis CryIA(c) toxin receptor was used to isolate M. sexta midgut aminopeptidase N cDNA clones by a PCR-based approach. Analysis of the 990-residue deduced amino acid sequence indicates that it is a large prepro-protein (Fig. 4B). The two pre-regions are the C-terminal GPI signal sequence (residues 968-990) and the (predicted) N-terminal cleavable signal sequence (residues 1-15), while the sequence between the predicted signal peptidase cleavage site and the N terminus of the mature protein (residues 16-35) is presumably a pro-region. Following proteolytic release of these pre- and pro-sequences, the mature polypeptide would then be 934 residues long, with a calculated molecular mass of 105 kDa. A 33-amino-acid long region (residues 935-967) immediately preceding the GPI signal peptide is rich in serine and threonine residues, which are potential O-glycosylation sites, and also in the helix-breaking amino acid proline, commonly found in -turns. By analogy to decay accelerating factor, sucrase/isomaltase, low density lipoprotein receptor, and the mucin protein family (reviewed in (41) ), this region may represent a rigid, O-glycosylated stalk that serves to elevate the active site of the enzyme well above the cell surface. The mature protein sequence also has four consensus N-glycosylation sites, and lectin binding studies have indicated that at least one of these sites is occupied.()The presence of covalently attached carbohydrate may explain the observed difference between the molecular mass of the purified enzyme (120 kDa) and that of the polypeptide predicted from cDNA sequence (105 kDa).

A number of ectoenzymes are now known to possess GPI membrane anchors including acetylcholinesterase, alkaline phosphatase, microsomal dipeptidase, 5`-nucleotidase, trehalase, and aminopeptidase P in mammals (reviewed in (42) and (43) ) and alkaline phosphatase (44) and aminopeptidase N (40) in the midgut of the lepidopteran larva B. mori. It is common to find that treatment of ectoenzymes with PI-PLC releases only a fraction of the total activity. This observation implies that the uncleaved enzyme population is either anchored by a modified GPI structure that is insensitive to PI-PLC (reviewed in (36) ) or by a conventional C-terminal hydrophobic amino acid sequence that arises by alternative splicing of a single mRNA transcript, as is known to be the case with neural cell adhesion molecules(46) . Although M. sexta aminopeptidase N activity is relatively resistant to PI-PLC release, this latter explanation seems unlikely since Northern blot analysis indicates that there is only one aminopeptidase N transcript in M. sexta midgut mRNA preparations.()Therefore the relative resistance of M. sexta aminopeptidase N to PI-PLC cleavage is probably due to modification to the GPI anchor structure itself.

In addition to its role as a receptor for B. thuringiensis CryIA(c) toxin, aminopeptidase N is known to be commandeered as a receptor by human (9) and porcine (8) coronaviruses, and by a human herpesvirus(10) . In the latter two cases, studies demonstrated that the catalytic site and the viral binding site were on different domains and that aminopeptidase enzyme activity was not necessary for viral infection(10, 47) . In our hands CryIA(c) toxin has no effect upon aminopeptidase N activity, which suggests that, like the viruses, the toxin binds at a site distinct from the catalytic site. As an exopeptidase, aminopeptidase N cannot itself be involved in the proteolytic activation (48, 49) of the 133-kDa CryIA(c) protoxin to the 66-kDa active toxin. Nor could it be responsible for cleavage of loop regions within the active toxin(50) , although in theory the enzyme could contribute to any N-terminal trimming reactions following endoprotease cleavage. Thus, it seems that the feature of aminopeptidase N being exploited by both the viruses and CryIA(c) is simply its abundance at the apical membrane of epithelial cells, irrespective of its function as a protease. This does not preclude the possibility that following binding to the aminopeptidase receptor, CryIA(c) toxin may subsequently interact with other membrane components to which aminopeptidase N is functionally linked.

Vadlamudi et al.(51) identified (and subsequently purified) a 210-kDa putative CryIA(b) receptor in the brush border membrane of M. sexta. The same authors (45) recently reported the cloning from M. sexta of this putative CryIA(b) receptor. The cDNA clone encodes a novel cadherin-like glycoprotein which, when expressed in either COS-7 or human embryonic 293 cells, was able to bind CryIA(b) toxin in ligand blotting experiments and in the latter case also in homologous binding assays.

The demonstration (6) that partially purified aminopeptidase, when incorporated in liposomes, requires dramatically less CryIA(c) toxin to induce a given amount of Rb leakage compared with vesicles containing no brush border membrane proteins strongly suggests that the 120-kDa aminopeptidase N glycoprotein functions as a CryIA(c) receptor in vivo. Having cloned M. sexta aminopeptidase N, we are in a position to directly investigate its interaction with CryIA(c) toxin.


FOOTNOTES

*
This study was supported by grants from the Agriculture and Food Research Council (P. J. K. K. and D. J. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X89081[GenBank Link].

§
Royal Society University Research Fellow.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Cambridge, Tennis Court Rd., Cambridge, CB2 1QW, U.K. Tel.: 01223-333651; Fax: 01223-333345.

The abbreviations used are: PCR, polymerase chain reaction; BBMV, brush border membrane vesicles; bp, base pair(s); nt, nucleotide(s); pfu, plaque-forming units; PI-PLC, phosphatidylinositol-specific phospholipase C; GPI, glycosylphosphatidylinositol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CMC, critical micellar concentration.

P. J. K. Knight, unpublished data.

J. C. Martinez, unpublished data.


ACKNOWLEDGEMENTS

We acknowledge the assistance of Drs. L. Packman and C. Hill for oligonucleotide synthesis, supported by the Wellcome Trust, and P. Barker at the Biotechnology and Biological Sciences Research Council, Babraham Institute for automated DNA sequence analysis. We gratefully acknowledge Dr. J. Van Rie for the gift of the gt10 cDNA library, which was used to complete the PCR-derived cDNA sequence. We thank Dr. N. Crickmore and T. Sawyer for technical assistance, and Dr. C. E. Cummings for reviewing the manuscript.


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