The gut of most insects is lined with a membrane called the
peritrophic membrane (PM) (
)or peritrophic matrix, which is
composed of chitin, proteoglycans, and protein(1) . There are
two types of insect PMs. Type 1 PM is synthesized from the digestive
epithelial cells that line the gut of the insect, whereas type 2 PM is
synthesized as a continuous tube from a specialized organ, the cardia
(or proventriculus) situated in the anterior midgut region of the
insect. The functions of this semi-permeable membrane are not entirely
clear but are likely to be crucial for the protection of underlying
digestive epithelial cells from bacterial damage and parasite invasion,
as well as facilitating the digestive process due to the
membrane's ability to partition digestive enzymes and ingested
food between the endo- and ecto-PM spaces(1) . The PM is the
first barrier in insects encountered by a number of ingested viral and
protozoal
organisms(1, 2, 3, 4, 5, 6, 7, 8) ,
some of which use blood- or tissue-feeding insects as vectors for
transmission to vertebrate hosts, where these organisms cause
considerable morbidity and mortality. There is evidence that the PM of
insect vectors can affect the survival and virulence of these
pathogenic organisms(8, 9, 10, 11) .
Proteins bound to PM have been reported to account for a
considerable proportion (35-55%) of the total mass of the PM in
many insects(1) . A subset of these proteins is the strongly
bound or integral PM proteins, which can only be released from the PM
by strong denaturants (12) . These latter proteins, which we
have named ``peritrophins,'' are probably of central
importance for the maintenance of the structural and protective
biological functions of the PM and may be involved in determining the
ultrafilter character of the membrane(13) . Disruption of the
functions of these integral PM proteins by chemical or immunological
means could lead to novel mechanisms of insect control. Indeed, it is
possible to vaccinate sheep against the tissue- and blood-feeding
larvae of the fly Lucilia cuprina using isolated PM
proteins(12, 14) . It was demonstrated that antibodies
to a PM protein, peritrophin-44, inhibited the free movement of small 6
nm gold particles from the gut lumen across the PM to the underlying
digestive epithelial cells. The primary mechanism of action of the
vaccine on the larvae was therefore postulated to involve the
antibody-mediated blockage of the pores in the PM and the subsequent
starvation of the larvae(14) . The larvae of the related flies Chrysomya bezziana (Old World Screwworm) and Cochliomyia
hominivorax (New World Screwworm) cause a similar cutaneous
myiasis in a wider spectrum of vertebrate hosts including
man(15) . We have taken advantage of the ability of L.
cuprina larvae to shed their type 2 PM continuously from the hind
gut and our ability to culture these larvae in vitro to allow
the isolation of sufficient quantities of PM for detailed study of one
of the most abundant integral PM proteins. This is the first
characterization of a PM protein from any insect.
EXPERIMENTAL PROCEDURES
Laboratory chemicals were analytical grade and were generally
purchased from Sigma or Ajax Chemical Company (Auburn, Australia).
Newborn calf serum and gentamicin were purchased from Commonwealth
Serum Laboratories (Parkville, Australia). Endoproteinase Lys-C,
endoproteinase Glu-C, endoglycosidase H, and N-glycosidase F
were purchased from Boehringer Mannheim, and Zwittergent 3-14 was
from Calbiochem. Biotinylated lectins were obtained from Vector
Laboratories Inc. (Burlingham, CA) and Pierce.
Preparation of Peritrophin-44
The laboratory culture of L. cuprina larvae and harvesting of PM from these larvae have
been described elsewhere(12, 16) . The PM (dry weight,
1 g) was obtained from 320,000 larvae cultured in vitro in 20
batches and was stored at -70 °C in PBS (20 mM Na
PO
/140 mM NaCl, pH 7.2)
containing 2.5 mM benzamidine and 5 mM EDTA. The PM
was homogenized and progressively washed by centrifugation (50,000
g, 30 min, 4 °C) with 40 ml of each of the
following solutions: water; 100 mM Tris-HCl, pH 7.5/150 mM NaCl/5 mM EDTA/5 mM benzamidine/0.1 mM phenylmethylsulfonyl fluoride (PMSF); and 20 mM Tris-HCl,
pH 7.4/140 mM NaCl (TBS)/0.1 mM PMSF containing 2%
Zwittergent 3-14. The detergent-washed PM was then extracted with
40 ml of TBS containing 6 M urea and 0.1 mM PMSF. The
extracted proteins were concentrated and subjected to gel permeation
chromatography (0.5 ml/min) on a column of Superose 12 (1.6
50
cm; Pharmacia Biotech Inc.) equilibrated with 50 mM Tris-HCl,
pH 7.5/6 M urea/1 mM EDTA/1 mM benzamidine.
A major peak containing proteins of M
44,000-48,000 was pooled, dialyzed against 20 mM Tris-HCl, pH 8.5/4 M urea/1 mM benzamidine/1
mM EDTA, and subjected to anion exchange chromatography on a
MonoQ column (Pharmacia) equilibrated with the dialysis buffer. A
0-250 mM nonlinear NaCl gradient in the same buffer
separated peritrophin-44 from one other protein, peritrophin-48.
Approximately 600 µg of purified peritrophin-44 was obtained from 1
g of dry weight of PM. Once solubilized from PM with urea,
peritrophin-44 remained soluble in TBS in the absence of urea. Purified
and denatured peritrophin-44 (2 µg) was deglycosylated by
incubation with N-glycosidase F (0.1 unit) or endoglycosidase
H (0.1 unit) essentially according to the instructions issued by the
manufacturer of these enzymes. Protein concentration determinations
were made using the Pierce BCA kit with bovine serum albumin as a
standard. Samples containing urea or Zwittergent 3-14 were
diluted before measurements to reduce interference of these agents in
the assay. The protein standards were measured in the same buffer.
SDS-Polyacrylamide Gel Electrophoresis and Lectin
Blots
Protein samples were analyzed by SDS-polyacrylamide gel
electrophoresis (PAGE) and lectin blots according to previously
described procedures(12, 17) . The M
of PM proteins separated by SDS-PAGE were measured by
interpolation of a plot of log molecular weight of the standard
proteins against their relative mobility. Densitometry was carried out
with a Hoeffer Scientific Instruments scanning densitometer. The
biotinylated lectins and their specificities (in brackets; (18) ) which were used included: lentil lectin (Man, Glc),
concanavalin A (Man, Glc), and wheat germ lectin (GlcNAc, NeuAc).
Localization of Peritrophin-44 on Peritrophic
Membrane
Ovine anti-serum to a purified recombinant glutathione S-transferase-peritrophin-44 fusion protein
(GST-peritrophin-44) was prepared essentially according to a published
procedure(12, 20) . The GST-peritrophin-44 contained
amino acids 35-321 of peritrophin-44(19) . Anti-serum
raised to GST-peritrophin-44 did not react with other PM proteins.
Animals were maintained under veterinary supervision in accordance with
the Australian code of practice for the care and use of animals for
experimental purposes (21) . Immunogold localizations on
freshly dissected PM from second instar larvae were performed
essentially according to instructions issued by the manufacturer of the
donkey anti-sheep antibody conjugated to 10 nm colloidal gold particles
(Biocell Research Laboratories, Cardiff, UK). The processed samples
were transferred to 10% heat-inactivated normal horse serum (0.5 h, 56
°C), followed by the introduction of a 1:500 dilution of either
sheep anti-GST-peritrophin-44 serum or prevaccination serum for 1.5 h.
After washing, the sections were transferred to a 1:100 dilution of the
donkey anti-sheep antibody conjugated to 10 nm colloidal gold particles
for 1.5 h. This was followed by extensive washing, and then the
sections were stained lightly (
2 min) in 2% aqueous uranyl
acetate/0.08 M lead citrate and examined and photographed in a
Phillips EM 300 transmission electron microscope. Immunofluorescence
localization of peritrophin-44 on PM was performed with a 1:1000
dilution of anti-GST-peritrophin-44 serum according to a previously
described method(12) .
Peptide Amino Acid Sequences from
Peritrophin-44
Purified peritrophin-44 (100 µg) was reduced,
alkylated, and digested with either endoproteinase Glu-C or
endoproteinase Lys-C, and the released peptides were purified and
sequenced(22, 23) . A total of four independent
preparations of peritrophin-44 were used for the generation of
peptides. The amino-terminal sequences of purified, reduced, and
alkylated peritrophin-44 and peritrophin-48 (as well as untreated
peritrophin-44 and peritrophin-48) were directly determined. Amino acid
sequences were obtained from an Applied Biosystems model 471A protein
sequencer.
Oligonucleotide Synthesis, Preparation of Genomic DNA,
and Construction of a cDNA Library from First Instar L. cuprina
Larvae
The peptide amino acid sequences obtained from
peritrophin-44 were used to design degenerate oligonucleotide primers
(Pharmacia Gene Assembler Plus oligonucleotide synthesizer), which were
used in conjunction with the polymerase chain reaction (PCR) (24) to amplify DNA coding for a fragment of peritrophin-44
from genomic DNA. Primers were designed from the amino acid sequences
of two peptides from peritrophin-44 i.e. PM30022 and PM2704.
The former peptide amino acid sequence represented a region near the
amino terminus of peritrophin-44 (PDGFIADP) and was used to design the
sense primer (1536-fold redundancy),
5`-CC(G/A/T/C)GA(T/C)GG(G/A/T/C)TT(T/C)AT(T/
C/A)GC(G/A/T/C)GA(C/T)CC-3`. The antisense primer (512-fold redundancy)
devised from the internal peptide sequence (GMAYNYGG) was
5`-CC(G/A/TC)CC(G/A)TA(G/A)TT(G/A)TA(G/A/T/C)GCCAT(G/A/T/C)CC-3`.
Bracketed nucleotides show alternatives at a specific position. Genomic
DNA was prepared from L. cuprina first instar
larvae(25) . The production of cDNA and the construction of a
gt-11 cDNA library from first instar larvae of L. cuprina have been described elsewhere(23) .
Production of a Genomic DNA Fragment Specific for
Peritrophin-44
PCR was performed on 1 µg of genomic DNA in
the presence of 100 µl of 10 mM Tris-HCl, pH 8.3/50 mM KCl/2.5 mM MgCl
/500 µM of each
dNTP/1 µM of each oligonucleotide primer/1 µl of
perfect match enhancer (Stratagene; 1 unit)/0.5 unit of AmpliTaq
(Promega). Amplification took place over 40 cycles, each of which
consisted of denaturation for 150 s at 94 °C, annealing for 150 s
at 59 °C, and extension for 150 s at 72 °C. Appropriate
controls were also included(24) . Samples of the PCR reaction
were analyzed on 1% agarose gels. A unique band of 860 bp was produced
when either genomic DNA or cDNA was used in the PCR reaction,
suggesting the absence of introns in the genomic DNA. The L.
cuprina PCR product derived from genomic DNA (500 ng) was isolated
and sequenced on both strands using standard
procedures(23, 26) .
Isolation of a cDNA Clone Coding for
Peritrophin-44
The cDNA library in
gt11 was screened with
the 860-bp DNA fragment amplified by PCR (described above) from genomic
DNA and labeled with
digoxigenin-11-2`-deoxyuridine-5`-triphosphate (Boehringer
Mannheim). The cDNA library was transferred in duplicate to Hybond
N+ positively charged nylon membranes (Amersham Corp.), denatured,
neutralized, and alkali-fixed in a modification of the
manufacturer's instructions. Hybridization was performed at 42
°C for 16 h in 50% formamide/5
SSPE(27) /5
Denhardt's solution/0.1% SDS/2% blocking reagent (Boehringer
Mannheim) after a prehybridization step of 4 h. Filters were washed
twice (each 5 min) in 2
SSC (26) containing 0.1% SDS at
room temperature and once in 1
SSC containing 0.1% SDS for 15
min at 65 °C. Eight positive plaques were detected by
anti-digoxigenin Fab fragment conjugated to alkaline phosphatase
(Boehringer Mannheim) and Fast Violet stain(27) . The insert
from one positive clone contained two EcoRI fragments of 439
and 1031 bp that were subcloned separately into pBluescript KS(+)
(Stratagene) and sequenced on both strands using either a Promega
TaqTrack kit or a U. S. Biochemical Corp. Sequenase 2.0 kit. Internal
sequences were determined with the aid of internal sequencing primers.
Data base searches and alignments were performed on the ANGIS computer
system (The University of Sydney, Sydney, Australia).
Reverse Transcriptase-PCR
The guts from third
instar L. cuprina larvae were excised and carefully dissected
into the cardia, midgut, and hindgut. Each tissue was derived from a
separate individual. First strand cDNA was prepared from total RNA
derived from each of these tissues (28) and various life stages
and was then subjected to PCR specific for the amplification of DNA
coding for peritrophin-44. The sense oligonucleotide primer was
5`-GCAATGAAAGAACTACAAATAACAAC-3` and the antisense primer was
5`-TAAGATGTTTGGTTTATGTCGCAGG-3`. The conditions used for PCR were
identical to those described above. This combination of oligonucleotide
primers specifically amplified a 1100-bp DNA product from tissues
expressing mRNA coding for peritrophin-44. The total RNA prepared from
each tissue was initially treated with DNase 1 to ensure the absence of
any genomic DNA. This was verified using a PCR approach with primers
specific for a L. cuprina gene (peritrophin-95) that contains
an intron. The quantity of first strand cDNA used in PCR to examine the
tissue-specific expression of peritrophin-44 directly reflected the
total content of first strand cDNA derived from each tissue. For the
examination of the developmental expression of peritrophin-44, the same
quantity of first strand cDNA (0.9 ng) was used for all of the samples
except eggs (3.2 ng). Direct comparison of the relative quantity of
total cDNA in each of these developmental stage samples was also made
using a PCR approach specific for cDNA coding for
-actin. In this
instance the sense primer was 5`-CAGATCATGTTTGAGACCTTCAAC-3` and the
antisense primer was 5`-G(G/C)CCATCTC(C/T)TGCTCGAA(G/A)TC-3`. A 350-bp
-actin DNA fragment was amplified from each cDNA sample using the
following conditions: one cycle of 2 min at 95 °C, 1 min at 60
°C, and 1 min at 72 °C; 33 cycles of 1 min at 95 °C, 1 min
at 60 °C, and 1 min at 72 °C; and one cycle of 2 min at 95
°C, 1 min at 60 °C, and 1 min at 72 °C. The
-actin
expression level in eggs was markedly less than other tissues. The
reasons for this are not clear. DNA fragments amplified from cardia and
midgut using primers specific for peritrophin-44 were also cloned and
sequenced to confirm that they coded for peritrophin-44.
Reacetylated Chitosan Affinity Chromatography
Crab
shell chitosan was reacetylated (29) and homogenized in a
Waring blender for 30 s, and 3 ml were poured into a column and
equilibrated with TBS. Purified peritrophin-44 (10 µg) was dialyzed
against the same buffer and applied to the reacetylated chitosan
affinity column, which was washed (0.5 ml/min) with 10 ml of TBS and
sequentially eluted with 10 ml of TBS/20 mM Glc, 10 ml TBS/20
mM methyl
-D-mannopyranoside, 10 ml TBS/20
mM GalNAc, 10 ml TBS/20 mM GlcNAc, and finally 10 ml
of 50 mM sodium acetate, pH 3.0. The peritrophin-44 eluted in
each wash was concentrated in a Centricon 10K cell (Amicon) to 200
µl, and 40-µl samples were subjected to SDS-PAGE. A Hoefer
Scientific Instruments GS 300 scanning densitometer was used to measure
the relative quantities of peritrophin-44 in each lane of the
silver-stained gel. There was a linear relationship between the
quantity of peritrophin-44 (0-2 µg) loaded onto the SDS-PAGE
and the scanned area of the silver-stained band measured by
densitometry. Negative control experiments were performed under similar
conditions using bovine serum albumin (100 µg) and soybean trypsin
inhibitor (100 µg). Neither of these proteins bound to the
reacetylated chitosan affinity column. A positive control experiment
was performed using wheat germ lectin (100 µg) under the same
conditions. Wheat germ lectin bound very strongly to the reacetylated
chitosan affinity column and was only eluted using the pH 3.0 acetate
buffer.
Intrinsic Fluorescence
The intrinsic fluorescence
of peritrophin-44 (dialyzed against PBS) was measured in a Perkin-Elmer
LS50B luminescence spectrometer at 25 °C. All solutions were
filtered (0.22-µm filter) before use. Excitation was at 280 nm, and
both slit widths were 5 nm. Spectra were corrected for the small
background signal from the appropriate solvent but remain uncorrected
for the variation in detector efficiency with wavelength. The final
concentration of peritrophin-44 was 10 µg/ml (0.23
µM). The effect of a range of tri-N-acetyl
chitotriose concentrations (0-1 mM) on the intrinsic
fluorescence intensity (emission at 347 nm; excitation at 280 nm) of
peritrophin-44 (10 µg/ml) was measured and analyzed by the method
of Scatchard(30) . Briefly, the fractional change in intrinsic
fluorescence (r) at a specific concentration of
tri-N-acetyl chitotriose was defined as: r = (F
- F
)/F
, where F
is the intensity in the absence of
tri-N-acetyl-chitotriose, F
is the
intensity at specific tri-N-acetyl chitotriose concentration
(s), and F
is the total change in intensity at a
saturating concentration of tri-N-acetyl chitotriose. The
Scatchard analysis plotted r/s versus r. The slope of
this line is a measure of the association binding constant for the
interaction (i.e. K
). The maximum correction for
dilution effects in this experiment was 6%. Sugars such as Glc, methyl
-D-mannopyranoside, and GalNAc each at a concentration of
1 mM had no significant effect on the intrinsic fluorescence
spectrum of peritrophin-44.
RESULTS AND DISCUSSION
Integral Peritrophic Membrane Proteins
The PM
was progressively extracted with a series of buffers with increasing
strengths of solubilization. The detergent (2% Zwittergent 3-14)
and then 6 M urea extractions of the PM solubilized 9 and 22
mg protein/g dry weight of PM, respectively. A subsequent 6 M guanidine HCl extract of the PM solubilized a further 4 mg
protein/g dry weight of PM. The total protein extracted was 35 mg
protein/g dry weight of PM, which was substantially less than the value
of 350-550 mg/g dry weight for the total protein content of PM
reported for some other higher dipteran insects(1) . The reason
for this difference is not clear but may reflect species-specific
differences or indicate the presence of a substantial quantity of
protein covalently attached to the PM that was not extracted by any of
the buffers used in the present study. Another significant difference
from previous studies was the use of PM obtained by larval culture
rather than by direct dissection from larval or adult guts. The latter
PM probably contains substantial quantities of contaminating proteins
from the ingested proteins present in the gut of these insects. PM
obtained by larval culture should be relatively free of these
contaminating proteins because of the progress of this PM through the
digestive environment of the gut before its extrusion from the larvae
and subsequent collection.The SDS-PAGE profile of the detergent
extract showed the presence of two predominant proteins (55,000 and
40,000 Da) but in a background of a large number of lower abundant
proteins (Fig. 1a). The sample also shows a strong
general background staining that may indicate the presence of
nonproteinacious material. The proteins in the detergent extract are
probably representative of the proteins loosely bound to the PM or
entrapped within the PM and are unlikely to be intimately involved with
the maintenance of PM structure. Of primary interest in this study is
the limited number of urea-extracted integral PM proteins
(peritrophins) that are likely to be the major structural proteins of
the PM as well as being intimately involved in the functions of the PM. Fig. 1b (lanes 2-4) shows a
silver-stained SDS-PAGE profile of the proteins extracted by the 6 M urea buffer. This extract contained 6 major proteins of M
= 95,000, 65,000, 48,000, 44,000, 35,000,
and 33,000 (measured under reducing conditions). Two of the integral PM
proteins of M
= 44,000 and 48,000 (i.e. peritrophin-44 and peritrophin-48) were the most abundant in this
extract, together representing >70% of the total urea-extractable
protein (measured by densitometry). The size of the largest peritrophin (M
= 95,000, peritrophin-95), unlike the
other PM proteins, was variable ranging between 85,000 and 95,000
depending on the percentage of acrylamide used in the SDS-PAGE (result
not shown). The PMs from larvae of Calliphora erythrocephala, Sarcophaga barbata, and Trichoplusia ni contain a
similar repertoire of major integral PM
proteins(6, 13) , suggesting that these proteins may
occur in many insect PMs. None of the major L. cuprina proteins was extracted by the nonurea-containing buffers initially
used to wash the PM as determined by comparative SDS-PAGE and specific
immunoblots. A number of lower abundance proteins in a regular ladder
of higher molecular weights (i.e. M
> 100,000)
was also present in the 6 M urea extract from L. cuprina PM. This can be seen in the higher loadings of this sample in lanes 3 and 4 of Fig. 1b. The
relative quantities of these minor high molecular weight bands were
variable and depended on the age of the sample and the time taken for
SDS-PAGE. One possibility is that these bands are due to nonspecific
polypeptide associations mediated by reoxidation of cysteine residues
during the actual running time of the electrophoresis, despite the
sample being initially reduced.
Figure 1:
Proteins extracted from L. cuprina larval PM. Silver-stained SDS-PAGE profile of
proteins extracted from PM. a, proteins extracted by TBS
containing 2% Zwittergent 3-14 and 0.1 mM PMSF. Lane
1, molecular mass standards; lane 2, proteins (5 µg)
extracted from PM with TBS containing 2% Zwittergent 3-14 and 0.1
mM PMSF. b, proteins extracted from Zwittergent
3-14 washed PM with TBS containing 6 M urea and 0.1
mM PMSF. Lane 1, molecular mass standards; lanes
2-4, proteins (1-, 5-, and 10-µg loadings, respectively)
extracted from PM with TBS containing 6 M urea and 0.1 mM PMSF. The major integral PM proteins, peritrophin-95,
peritrophin-65, peritrophin-48, peritrophin-44, peritrophin-35, and
peritrophin-33 are represented on the figure as PM95, PM65, PM48, PM44, PM35, and PM33, respectively. The protein samples were all initially
reduced.
Purification and Glycosylation of
Peritrophin-44
The prevalence of peritrophin-44 and
peritrophin-48 in the PM suggested that they play a major role in the
structure and function of the PM. Antibodies to peritrophin-44 ingested
by larvae block the pores in the PM, indicating that this protein is
probably involved in determining the porosity of PM(14) . This
possibility is consistent with the uniform distribution of
peritrophin-44 in PM (see below) and the strong interaction between
peritrophin-44 and the PM. Peritrophin-44, the most abundant integral
PM protein, was purified from the 6 M urea extract of
detergent-washed PM by Superose 12 gel permeation chromatography and
MonoQ anion exchange chromatography (Fig. 2a, lanes
2 and 5). The lack of focus of proteins such as
peritrophin-44 on SDS-PAGE can indicate that the protein is
glycosylated. To test this possibility, purified peritrophin-44 was
incubated with N-glycosidase F or endoglycosidase H, which
specifically removes N-linked oligiosaccharides(31) .
Both enzymes significantly reduced the size of peritrophin-44 (Fig. 2a, lanes 3 and 6,
respectively), thereby confirming the presence of oligosaccharides on
this protein. N-Glycosidase F caused a shift in the size of
peritrophin-44 to an M
= 37,500, whereas
endoglycosidase H caused a marginally smaller change to an M
= 40,000. The former enzyme removes
intact N-linked oligosaccharides by cleavage between the
asparagine and the first core GlcNAc. This enzyme readily removes most N-linked oligosaccharides. Endoglycosidase H cleaves between
residues of the core N,N`-diacetylchitobiose, thereby
leaving one GlcNAc residue still attached to the protein.
Endoglycosidase H is specific for most high mannose oligosaccharides
but is unable to remove complex oligosaccharides.
Figure 2:
Purification and glycosylation of
peritrophin-44. a, silver-stained SDS-PAGE profile showing
purified peritrophin-44 before and after deglycosylation. Lanes 1 and 4, molecular mass standards; lanes 2 and 5, purified peritrophin-44 (2 µg); lane 3,
peritrophin-44 (2 µg) after deglycosylation with N-glycosidase F (0.1 unit); lane 6, peritrophin-44 (2
µg) after deglycosylation with endoglycosidase H (0.1 unit). b, reactivity of biotinylated lectins with peritrophin-44.
Purified peritrophin-44 was subjected to SDS-PAGE, transferred to
nitrocellulose by electroblotting, and probed with a range of
biotinylated lectins. Lanes 1, 3, 5, and 7 contained blue molecular mass standards, and lanes
2, 4, 6, and 8 contained 2 µg of
peritrophin-44. Lane 2, reactivity with biotinylated lentil
lectin; lane 4, biotinylated lentil lectin after preincubation
of the lectin with 0.3 M methyl
-D-mannopyranoside for 30 min at 25 °C; lane
6, biotinylated concanavalin A; lane 8, biotinylated
concanavalin A after preincubation with 0.3 M methyl
-D-mannopyranoside for 30 min at 25 °C. c,
reactivity of biotinylated lectins with deglycosylated peritrophin-44.
Peritrophin-44 (2 µg) was incubated with N-glycosidase F
(0.1 unit) or endoglycosidase H (0.1 unit), subjected to SDS-PAGE,
transferred to nitrocellulose, and probed with biotinylated lentil
lectin. Lanes 1 and 4, blue molecular mass standards; lanes 2 and 5, purified peritrophin-44 (2 µg); lanes 3 and 6, peritrophin-44 (2 µg) pretreated
with N-glycosidase F (0.1 unit) or endoglycosidase H (0.1
unit), respectively.
To further
characterize the glycosylation of peritrophin-44, it was subjected to
SDS-PAGE, transferred to nitrocellulose by electro-blotting, and then
probed with a range of biotinylated lectins (Fig. 2b).
Both biotinylated lentil lectin and biotinylated concanavalin A reacted
with peritrophin-44 (Fig. 2b, lanes 2 and 6, respectively). The binding of lentil lectin was completely
inhibited by preincubation of this lectin with 0.3 M methyl
-D-mannopyranoside (lane 4), whereas the binding
of concanavalin A was partially reduced by the same sugar (lane
8). Both of these lectins have similar, although nonidentical
specificities for terminal oligosaccharides containing Glc and/or Man
residues(18) . Biotinylated wheat germ lectin, Phaseolus
vulgaris E lectin, peanut agglutinin, Sophora japonica lectin, or Pisum sativum lectin did not bind to
peritrophin-44 (result not shown). Incubation of peritrophin-44 with N-glycosidase F completely inhibited the binding of
biotinylated lentil lectin (Fig. 2c). However,
treatment of peritrophin-44 with endoglycosidase H, while clearly
removing some oligosaccharides as evidenced by a mobility shift of the
protein (Fig. 2a), had no effect on the binding of
biotinylated lentil lectin (Fig. 2c). One explanation
for this result is that peritrophin-44 contains two classes of attached
oligosaccharides. The first class consists of a high mannose
oligosaccharide that is reactive with biotinylated lentil lectin and
that is removed by either endoglycosidase H or N-glycosidase
F. The second class is a complex oligosaccharide that also reacts with
biotinylated lentil lectin but can only be removed by N-glycosidase F.
Resistance of Peritrophin-44 to Proteolysis
The PM
is bathed in relatively high concentrations of proteolytic enzymes
particularly trypsins and chymotrypsins, which are present in the
larval gut to digest ingested
food(16, 23, 32, 33, 34) .
Proteins, such as peritrophin-44, which are an integral component of
the PM should be exposed to these proteases and therefore subject to
proteolysis. However, immunoblots of 6 M urea extracts from
freshly dissected PM and PM obtained by larval culture using an
anti-serum to GST-peritrophin-44 identified a single 44-kDa
immunoreactive protein. Therefore, there was no evidence of significant
processing of peritrophin-44 as it slowly passed through the gut while
attached to the growing PM. The reason why peritrophin-44 was not
subject to extensive proteolysis in this harsh proteolytic environment
was not immediately clear. One possibility is that peritrophin-44 is
inherently resistant to digestive gut proteases. Indeed, peritrophin-44
was totally resistant to proteolysis by endoproteinase Lys-C over 2 h
at 37 °C (Fig. 3, lanes 2-6). However, after
initial reduction with 5 mM dithiothreitol, peritrophin-44 was
readily digested by this protease in less than 5 min (Fig. 3, lanes 7-11). This result demonstrates that disulfide
bonds in peritrophin-44 play an important role in protecting this
protein from proteolysis. The oligosaccharides attached to
peritrophin-44 may also help protect this protein from proteases.
Another possible explanation for the lack of digestion of
peritrophin-44 in vivo could involve its lack of ready
accessibility to digestive gut proteases either because of a protective
envelope of chitin within the PM or because peritrophin-44 is present
on the ecto- rather than the endo-PM surface. The former surface may
not be as readily exposed to digestive proteases.
Figure 3:
Resistance of peritrophin-44 to
proteolysis. Time course of digestion of peritrophin-44 with
endoproteinase Lys-C. Peritrophin-44 (5 µg) was incubated with
endoproteinase Lys-C (0.25 µg) in TBS for 0, 5, 20, 60, and 120 min
at 37 °C (lanes 2-6, respectively). Lanes
7-11 were identical to lanes 2-6 except that
the peritrophin-44 was preincubated with 5 mM dithiothreitol
for 20 min before the addition of endoproteinase Lys-C. These samples
were then analyzed by SDS-PAGE. Lane 1 contained molecular
mass standards. All samples were also reduced immediately before
SDS-PAGE.
Immunolocalization of Peritrophin-44 on Peritrophic
Membrane
Fig. 4shows the immunolocalization of
peritrophin-44 on PM obtained from freshly dissected larvae. There is
strong and uniform immunogold labeling of the entire PM (Fig. 4b). The corresponding control using
prevaccination serum showed very little gold labeling of the PM (Fig. 4a). The resistance of peritrophin-44 to
proteolysis in vivo, therefore, was not due to a privileged
location of this protein on the ecto-PM surface where digestive
proteases may not have ready access. The electron-lucent and
electron-dense layers in the PM, which have been reported
previously(1) , can also be discerned in the electron
micrographs. At least for peritrophin-44, one of the major integral PM
proteins, there was no correspondence between its location and these
layers. Fig. 4d shows the immunofluorescence
localization of peritrophin-44 on freshly dissected PM. Again,
peritrophin-44 was uniformly distributed throughout the entire PM.
There was little or no fluorescence associated with the corresponding
prevaccination control serum (Fig. 4c). Specific
antibodies bound to peritrophin-44 on intact, unfixed PM during the
immunofluorescence localization experiments, thereby demonstrating that
this protein was exposed on the PM surface and not protected by a
chitin envelope. Therefore, peritrophin-44 should also be accessible to
digestive proteases. Consequently, the resistance of peritrophin-44 to
proteolysis in vivo is primarily due to the inherent stability
of the protein, which is mediated by intramolecular disulfide bonds.
The uniform distribution of peritrophin-44 in the PM and its relative
abundance are consistent with a major role of this protein in
maintaining the structure of the PM.
Figure 4:
Localization of peritrophin-44 on PM.
Peritrophin-44 was localized to freshly dissected PM by immunogold
labeling (a and b) and immunofluorescence labeling (c and d) using anti-serum to a recombinant
GST-peritrophin-44 fusion protein. Controls using prevaccination serum
are shown in a and c. b and d show
the localization of peritrophin-44 using anti-serum raised to purified
GST-peritrophin-44. B, bacterium; ECPS, ecto-PM
space; ENPS, endo-PM space. The scale bars in a and b represent 500 nm. The PM used for the
immunofluorescence localization of peritrophin-44 was freshly dissected
from L. cuprina larvae.
Amino-terminal Amino Acid Sequences of Peritrophin-44 and
Peritrophin-48
The amino-terminal amino acid sequences of
purified peritrophin-44 and peritrophin-48 were directly determined (Fig. 5). There were 9 identical and a further 6 conserved
positions in the first 29 amino acids of these two sequences (i.e. 52% similarity). The probability that the sequence alignment is a
chance occurrence is 4.5 e
(SEQDP computer program;
ANGIS computer system, Sydney). Of particular note is the conservation
of 3 cysteine residues at positions 8, 23, and 29. Cysteines are often
conserved in related extracellular proteins because of their
involvement in intramolecular disulfide bonds(35) . The
sequence information indicates that there is significant amino acid
sequence similarity between peritrophin-44 and peritrophin-48,
suggesting that they belong to a common family of proteins. Searches of
the National Biomedical Research Foundation and Swiss Protein sequence
data bases did not reveal any significant similarities of these
sequences with other proteins.
Figure 5:
Related amino-terminal amino acid
sequences of two major integral PM proteins from L. cuprina larvae. The amino-terminal amino acid sequences of purified
peritrophin-44 and peritrophin-48 were directly determined. Identical
residues are boxed. A single space was introduced into the
peritrophin-44 sequence to optimize the alignment. PM44,
peritrophin-44; PM48,
peritrophin-48.
Isolation of cDNA Clones Coding for
Peritrophin-44
The cDNA coding for peritrophin-44 was isolated
and sequenced to gain further information about the structure and
function of this protein. Peritrophin-44 was reduced, alkylated, and
digested with endoproteinase Lys-C or endoproteinase Glu-C, and the
released peptides were purified and sequenced. These peptide amino acid
sequences (and the amino-terminal sequence of peritrophin-44) were used
to design suitable, degenerate oligonucleotide primers that were used
in the PCR in conjunction with either cDNA or genomic DNA to amplify in
both cases a 860-bp DNA fragment; a result that indicated that introns
were not present in the genomic DNA fragment. The latter DNA fragment
was sequenced and shown to contain a single open reading frame. The
deduced amino acid sequence contained extensions of the peptide
sequences used to design the oligonucleotide primers used in the PCR
reaction as well as 10 additional peptide sequences that were obtained
directly from purified peritrophin-44. Some of these peptide sequences
were overlapping. The 860-bp genomic DNA fragment was then used to
screen a L. cuprina larval cDNA library constructed in
gt-11. Eight positive clones were isolated.
Complete Nucleotide Sequence of Peritrophin-44
The
nucleotide sequence obtained from one cDNA clone containing the longest
insert (1470 bp) had an open reading frame of 1068 nucleotides that
coded for a protein of 356 amino acids (M
=
38,700; Fig. 6). A poly(A) signal sequence (AATAAA; (36) ) was located 69 nucleotides after the stop codon,
although there was no poly(A) tail. Translation of the nucleotide
sequence revealed 20 of the 23 peritrophin-44 peptide amino acid
sequences (5 of the 20 identified peptide amino acid sequences are not
shown in Fig. 6because they were substantially or fully
redundant with peptides already listed in the figure). Three low
abundance peptide sequences from one of the four independent
peritrophin-44 protein preparations (each of which were used to
generate peptides) were not located within the deduced amino acid
sequence. Recently, these three peptide sequences were located in the
amino acid sequence of peritrophin-48. This result indicated that one
of the four peritrophin-44 preparations also contained small amounts of
peritrophin-48. There were 10 positional differences (out of a total of
177 unique positions) between the peritrophin-44 peptide amino acid
sequences and the corresponding amino acid positions deduced from cDNA.
Several of these differences (6 out of 10) were conservative
substitutions and none involved cysteine residues (see below). These
minor differences in sequence may reflect allelic variations because
the cDNA, genomic DNA and purified peritrophin-44 were obtained from
large numbers of individuals (16,000, 16,000, and 320,000 individuals,
respectively). Alternatively, there may be a family of highly related
peritrophin-44 genes. The existence of extensive intramolecular
disulfide bonds (see Fig. 3) could maintain the tertiary
structure of peritrophin-44 and therefore its principal function, while
accommodating limited amino acid substitutions in flexible loops on the
surface of the protein. Other gene sequences from L. cuprina,
such as the excretory and secretory chymotrypsin, LCTb, also show
sequence variation(23) .
Figure 6:
Nucleotide and deduced amino acid
sequences of peritrophin-44 determined from cDNA. Underlining denotes the identification of peptide amino acid sequences.
Differences between peptide amino acid sequences and the deduced amino
acid sequence are shown below the indicated peptides as lowercase letters. Cysteines are in bold type and circled. A potential polyadenylation signal sequence is underlined twice. and the amino-terminal signal sequence of
the protein is underlined with a broken line. Two
potential N-linked glycosylation sites are boxed. The numbers at the end of each line refer to the nucleotide (upper) and amino acid (lower)
sequences.
The amino acid sequence of
peritrophin-44 deduced from cDNA contained a typical amino-terminal
signal sequence of 23 amino acids (37) . The amino terminus of
the mature protein was verified by direct amino acid sequencing. The
calculated molecular mass of the mature protein is 36,300 Da, which is
significantly less than that measured by SDS-PAGE (M
= 44,000) and consistent with the size of peritrophin-44
after deglycosylation with N-glycosidase F (M
= 37,500; Fig. 2). The calculated pI of the mature
protein is 5.2. Apart from the amino-terminal signal sequence, there
are no other regions in the protein which are strongly hydrophobic. The
sequence contains 2 potential N-linked glycosylation consensus
sites (NX(S/T)X, where X is any amino acid
except proline; (38) ), which is consistent with the knowledge
that the native protein contains two classes of oligosaccharides (Fig. 2). The most striking feature of the deduced amino acid
sequence is the abundance of cysteine residues (32 cysteines in the
mature protein, or approximately 10 mole percent). The abundance of
cysteine residues, their relatively close spacing, and the presence of
appropriate potential
-turns in the secondary structure of the
protein (39) strongly suggest that these cysteines are involved
in extensive intramolecular disulfide bonding(35) . Indeed, the
results shown in Fig. 3directly support this conclusion.
Domain Structure of Peritrophin-44 and Similarities with
Other Proteins
Peritrophin-44 contains 5 nonidentical but
related domains, each of approximately 70 amino acids (Fig. 7).
The characteristic feature of each of these domains is a common
register of 6 cysteine residues (except domain 2, which contains 8
cysteines). This register consists of the following consensus sequence
C-X
-C-X
-C-X
-C-X
-C-X
-C
(where X is any amino acid except cysteine). There is a small
amount of additional interdomain amino acid sequence similarity between
each of these cysteine residues. In particular, there is strong
conservation of an aromatic amino acid between cysteines 1 and 2, 2 and
3, and 4 and 5 (Fig. 7a). The conservation of the
aromatic amino acids in each of these domains suggests that these
residues are important for expression of the function of
peritrophin-44. Fig. 7b shows a diagrammatic
representation of the domain structure of peritrophin-44.
Figure 7:
Related cysteine-rich domains in
peritrophin-44. a, five contiguous domains from peritrophin-44 (PM44-I-PM44-V) each containing six cysteines (except
domain 2, which contained an additional two cysteines) were aligned.
The conserved cysteine residues are boxed, and conserved
aromatic amino acids (in at least four out of five domains) are
indicated by asterisks. Three similar cysteine-rich domains
from B. malayi (Bm), M. sexta (Ms),
and Chelonus sp. (Ch) chitinases as well as one
hypothetical cysteine-rich domain from the baculovirus A.
californica nuclear polyhedrosis virus (Bv) are also
shown. Gaps have been introduced at appropriate positions in the
sequences to optimize the alignment. b, schematic
representation of the domain structure of peritrophin-44. The solid
box represents the signal sequence, whereas the shaded boxes denote the five cysteine-rich domains in peritrophin-44. Potential N-linked glycosylation sites are represented by the y-shaped symbols.
The
National Biomedical Research Foundation amino acid sequence data base
was searched using the FASTA program (40) for proteins that
showed significant amino acid sequence similarity to peritrophin-44. A
number of extracellular cysteine-rich proteins (e.g. epidermal
growth factor precursor, laminin, and fibulin) showed similarity to
peritrophin-44, but this was primarily due to the prevalence of
cysteines in these proteins rather than overall sequence similarity.
The characteristic 6-cysteine register in each of the 5 domains of
peritrophin-44 was not present in any of these proteins. Moreover,
there were no sequence similarities with any proteins derived from
insect cuticle that lines the crop and hindgut of this insect. Cuticle
proteins typically have no cysteine residues and are not glycosylated (41) . The cysteine-rich domains are the major architectural
feature of peritrophin-44 and related proteins would be expected to
contain a similar arrangement of cysteine residues.
The computer
program Scrutineer (42) was used to search the GenPep protein
sequence data base (translated sequences from Genbank) for proteins
containing the 6-cysteine domain consensus sequence described above (or
minor variations thereof). There were only four matches, namely the
cysteine-rich, carboxyl-terminal domains in chitinases from Brugia
malayi (a parasitic nematode; (43) ), Manduca sexta (tobacco hornworm, an insect; (44) ), and Chelonus sp. (an endoparasitic wasp; (45) ) as well as a small
hypothetical polypeptide from Autographa californica nuclear
polyhedrosis virus (i.e. baculovirus; Genbank accession number
L22858) (Fig. 7a). Each of the chitinases contains only
one of these domains, which is located immediately adjacent to the
carboxyl terminus. This region is not part of the catalytic domain of
the chitinases and has no identified function.
Plant chitinases do
not contain this particular 6-cysteine domain, but rather one group,
the class 1 plant chitinases, contain an analogous domain containing 8
cysteines, which is strongly related to each of the cysteine-rich
domains in wheat germ lectin. The single 8-cysteine wheat germ
lectin-like domain is located at the mature amino termini of this class
of plant chitinases and has been shown to mediate binding of class 1
chitinases to chitin(46, 47) . By analogy with the
plant chitinases, it is likely that the single 6-cysteine domain at the
carboxyl-terminal end of each of the three animal chitinases listed
above and also the multiple 6-cysteine domains within peritrophin-44
mediate binding of these proteins to chitin. The major component of
insect PM is chitin, a linear polymer of
-1,4-linked
GlcNAc(1) . Thus, it is likely that the domain structure of
peritrophin-44 reflects its capacity to interact with chitin within the
PM. Moreover, the multiple cysteine-rich domains in peritrophin-44 may
allow multi-site binding to the GlcNAc polymer that makes chitin
thereby producing a noncovalent binding interaction of considerable
strength. This possibility may explain the necessity for strong
denaturants such as 6 M urea to solubilize peritrophin-44 from
PM.
The baculovirus polypeptide sequence was deduced from an open
reading frame and has no known function. The predicted polypeptide
consists of an apparent amino-terminal signal sequence of 23 amino
acids followed by one copy of the 6-cysteine domain (76 amino acids).
Both of these features suggest that the protein is secreted. There is
also additional sequence similarity between this hypothetical
polypeptide and peritrophin-44 based on the conservation of specific
aromatic amino acids. It is interesting to note that baculoviruses
infect specific insect species via the insect gut and cross the PM
during this process. It is possible that this viral polypeptide binds
to chitin within the insect PM and facilitates the movement of the
virus across the PM.
Effect of Tri-N-Acetyl Chitotriose on the Intrinsic
Fluorescence Spectrum of Peritrophin-44
The intrinsic
fluorescence spectrum of purified native peritrophin-44 after
excitation at 280 nm was measured in the presence and the absence of
tri-N-acetyl chitotriose to determine whether this
oligosaccharide bound to peritrophin-44 (Fig. 8a). In
the absence of the oligosaccharide, the wavelength of maximal emission
was 347 nm, which is characteristic of tryptophan emission in a polar
environment (48) and suggests substantial exposure of the
single tryptophan residue on the surface of peritrophin-44.
Tri-N-acetyl chitotriose, at a saturating concentration of 2.8
mM, caused significant quenching (
16% at 347 nm) of the
intrinsic fluorescence spectrum of peritrophin-44 without significantly
affecting the wavelength of maximal emission. The decreased
fluorescence emission indicated that the oligosaccharide had bound to
peritrophin-44 and perturbed the environment(s) of some of the aromatic
amino acids within peritrophin-44 either directly through local contact
or via alteration in the efficiency of energy transfer between tyrosine
residues and the single tryptophan residue in this protein. Titration
of the change in intrinsic fluorescence of peritrophin-44 with a range
of tri-N-acetyl chitotriose concentrations indicated that the
binding was saturable. A Scatchard plot (30; Fig. 8b)
was linear (r = -0.95), indicating a single class
of specific binding sites with an association constant K
= 5.5 ± 2.3 mM
. A
number of sugars including Glc, methyl
-D-mannopyranoside, and GalNAc, each at a concentration
of 1 mM, had no significant effect on the intrinsic
fluorescence spectrum of peritrophin-44. GlcNAc at a concentration of 1
mM caused approximately 6% quenching of the fluorescence
spectrum. The binding constant for the interaction of GlcNAc with
peritrophin-44 could not be accurately determined from this relatively
small change in intrinsic fluorescence.
Figure 8:
Intrinsic fluorescence spectra of
peritrophin-44 in the absence and the presence of
tri-N-acetyl-chitotriose. a, intrinsic fluorescence
spectra of peritrophin-44. The intrinsic fluorescence spectra of
peritrophin-44 (10 µg/ml) in the absence (solid line) or
the presence (dotted line) of 2.8 mM tri-N-acetyl-chitotriose in PBS were measured after
excitation at 280 nm. All spectra were corrected for the relatively
small signal arising from PBS or PBS containing 2.8 mM tri-N-acetyl chitotriose. b, Scatchard plot for
the binding of tri-N-acetyl-chitotriose to peritrophin-44. r is the fractional change in intrinsic fluorescence of
peritrophin-44 at a specific concentration of
tri-N-acetyl-chitotriose (s).
Binding of Peritrophin-44 to Reacetylated
Chitosan
The binding of peritrophin-44 to a reacetylated
chitosan affinity column was directly measured to further test the
proposal that peritrophin-44 binds chitin. Reacetylated chitosan is an
insoluble heterogeneous mixture of GlcNAc linear polymers. The
peritrophin-44 present in various washes from the affinity column was
analyzed by SDS-PAGE (Fig. 9). The majority of the
peritrophin-44 (
80%) bound to the reacetylated chitosan affinity
column and was specifically eluted by either 20 mM GlcNAc (Fig. 9, lane 5) or subsequently by a pH 3.0 acetate
buffer (Fig. 9, lane 6). The sugars Glc, methyl
-D-mannopyranoside, and GalNAc each at 20 mM concentrations in TBS did not elute peritrophin-44 from the
reacetylated chitosan affinity column (lanes 2-4). These
results indicate that peritrophin-44 specifically binds to reacetylated
chitosan and are also consistent with the results shown in Fig. 8. A small quantity of the applied peritrophin-44 did not
bind and was found in the initial TBS wash of the reacetylated chitosan
affinity column. Although this unbound peritrophin-44 is difficult to
see in lane 1 of Fig. 9, higher loadings of this sample
(not shown) indicate that it represents approximately 10% of the
applied protein. This fraction may represent peritrophin-44, which was
irreversibly denatured by the harsh conditions required to initially
extract the protein from PM. The ability of GlcNAc to elute only a
fraction (
70%) of the total peritrophin-44 bound to the
reacetylated chitosan affinity column (the remaining 30% was eluted by
the low pH buffer) may reflect the polydispersity of the latter with
respect to GlcNAc polymer lengths and degrees of reacetylation. Indeed,
the affinity of peritrophin-44 for GlcNAc polymer may depend on the
length of the polymer (as for wheat germ lectin; (48) ). GlcNAc
may efficiently elute peritrophin-44 from one population of small
GlcNAc polymers, whereas a low pH buffer may be required to remove
peritrophin-44, which had bound more strongly to longer polymers. The
specificity of the interaction between peritrophin-44 and reacetylated
chitosan was also demonstrated with control experiments, which
demonstrated that bovine serum albumin and soybean trypsin inhibitor
did not bind to the reacetylated chitin affinity column (result not
shown). Thus, peritrophin-44 binds specifically to reacetylated
chitosan (Fig. 9) and tri-N-acetyl chitotriose (Fig. 8). GlcNAc (0.3 M) did not elute peritrophin-44
directly from freshly isolated PM.
Figure 9:
Binding of peritrophin-44 to reacetylated
chitosan. Purified peritrophin-44 (10 µg) was added to a
reacetylated chitosan affinity column, which was then progressively
washed with TBS, Glc, methyl
-D-mannopyranoside, GalNAc,
and GlcNAc (each in TBS). Finally, the reacetylated chitosan affinity
column was washed with 50 mM sodium acetate, pH 3.0. Each
eluate was concentrated, and samples were subjected to SDS-PAGE and
then stained with silver. Lane 1, TBS wash; lane 2,
20 mM Glc eluate; lane 3, 20 mM methyl
-D-mannopyranoside eluate; lane 4, 20 mM GalNAc eluate; lane 5, 20 mM GlcNAc eluate; lane 6, 50 mM sodium acetate buffer, pH 3.0
eluate.
Wheat germ lectin has been used
extensively as a probe for detection of chitin associated with PMs
because of the ability of this lectin to strongly bind GlcNAc polymers
including reacetylated chitosan(1) . This lectin is composed of
two subunits, each containing four very similar 8-cysteine domains (A-D). One region in domain A and an identical region in
domain B of wheat germ lectin have the sequence CSQYGYC. A similar
sequence is present in the first cysteine-rich domain of
peritrophin-44, i.e. CQSYGYC. Although this similarity, by
itself, is not particularly significant, it is noteworthy that in the
wheat germ lectin sequence, the serine and both tyrosines are important
ligands directly involved in binding GlcNAc (49) . Moreover,
there is absolute conservation of a tyrosine residue in the central
position of this sequence similarity throughout all of the
peritrophin-44 cysteine-rich domains (Fig. 7), suggesting that
this position has an important involvement in the function of this
protein. The intrinsic fluorescence spectra of both wheat germ lectin
and peritrophin-44 are altered by GlcNAc polymers although in opposite
directions ((48) ; Fig. 8a). Thus, there are a
number of functional and structural parallels between wheat germ lectin
and peritrophin-44 including a small region of sequence similarity, the
presence of multiple cysteine-rich domains, the ability to bind chitin
and reacetylated chitosan, a similar relationship with the
chitin-binding domains of chitinases, and the ability to bind to PM.
However, peritrophin-44 does not have wheat germ lectin-like
agglutination abilities (result not shown).
Expression of Peritrophin-44
Microscopic
examination of the gut of many insects has indicated that the cardia is
the primary site for synthesis of type 2 PMs(1) . This small
group of highly specialized cells (
1000) is usually situated in
the anterior midgut region. However, it is not clear whether the
proteins associated with this class of PM are synthesized in the cardia
or produced by midgut cells and then added to the PM in a subsequent
maturation step. To differentiate between these two possibilities,
reverse transcriptase-PCR specific for peritrophin-44 was performed on
first strand cDNA made from RNA derived from various gut tissues
dissected from individual larva. Fig. 10a shows the
amplification of a peritrophin-44-specific DNA fragment (1100 bp) from
first strand cDNA derived from cardia (lane 2) and to a much
smaller extent from midgut (lane 3). There was no expression
of peritrophin-44 mRNA in hindgut (lane 4), crop (lane
5), salivary gland (lane 6), malphigian tubules (lane
7) or trachea (lane 8). The quantity of first strand cDNA
used in PCR for each sample was directly related to the total mRNA
content from that tissue. Thus, the dominant expression of
peritrophin-44 in cardia is further underscored by the knowledge that
this tissue contains relatively few cells (
1000) compared with
each of the other tissues tested, particularly the midgut. It is
therefore likely that the PM is produced predominantly from the larval
cardia in a near mature form containing a full complement of integral
PM proteins.
Figure 10:
Expression of peritrophin-44. Reverse
transcriptase-PCR was used to locate the site of synthesis and
developmental expression pattern of the mRNA coding for peritrophin-44. a, tissue-specific expression of peritrophin-44. Lane
1, standards; lane 2, first strand cDNA from cardia; lane 3, midgut; lane 4, hindgut; lane 5,
crop; lane 6, salivary gland; lane 7, malphigian
tubules; lane 8, trachea; lanes 9 and 10, no
DNA controls; lane 11, cloned peritrophin-44 cDNA (positive
control). Each PCR contained 10% of the total first strand cDNA derived
from each tissue. b, developmental expression of
peritrophin-44. Each sample contained the same quantity of first strand
cDNA (0.9 ng) except the egg sample (3.2 ng). Lane 1,
standards; lane 2, first instar larvae; lane 3,
second instar larvae; lane 4, third instar larvae; lane
5, pupae; lane 6, adults; lane 7, eggs; lane
8, no cDNA control. c, corresponding
-actin controls
for each of the cDNA samples shown in b.
All three larval instars express peritrophin-44 (Fig. 10b, lanes 2-4), whereas virtually
none is expressed in pupae or eggs (lanes 5 and 7).
There is a very small level of expression of peritrophin-44 in adult
flies (lane 6). These results indicate that peritrophin-44
expression is primarily restricted to larvae. The relative level of
expression (per unit mass of total first strand cDNA) in the three
larval instars is constant even though these larvae are enormously
different in size. This suggests that peritrophin-44 is constitutively
expressed and that the rate of growth of the larvae is directly linked
to the rate of growth of the PM. Adult L. cuprina also produce
a type 2 PM from cardia(1) . Specific immunoblots of adult fly
tissues and isolated PM failed to detect peritrophin-44 (result not
shown). This result is consistent with the very low level of expression
of peritrophin-44 in adult flies detected by reverse transcriptase-PCR
and indicates that the structure of the larval and adult type 2 PMs
from L. cuprina are very different. The reasons for this are
not clear. One possibility is that the differential level of expression
of peritrophin-44 reflects the different nutritional emphasises of
larvae and adults. Larvae feed on live ovine tissue and fluid exudate,
whereas the adult fly feeds on plant nectar and to a lesser extent on
proteinacious material from dead and decaying animal sources.
The
relative abundance of peritrophin-44 in PM, its strong interaction with
the PM, and its uniform distribution throughout the PM strongly
indicate that this protein is intimately involved in the major
functions of the PM. Scanning electron micrographs of the PM (1) shows the presence of a meshwork of chitin fibrils that
probably define the porosity of the PM, which is finely controlled with
a size exclusion limit of approximately 9 nm. (
)Antibodies
to peritrophin-44 block the pores in the PM and prevent nutrients from
passing from the gut lumen to the underlying digestive epithelial
cells, thereby starving the larvae(14) . This result suggests
that peritrophin-44 is directly involved in the determination of the
porosity of the PM. Such a function is also consistent with the uniform
distribution of peritrophin-44 throughout the PM. Peritrophin-44 could
be multi-valent for binding to chitin fibrils in the PM, thereby
cross-linking and locking the fibrils together while simultaneously
dictating the permeability characteristics of the PM. This could have
important implications for the nature of the digestive process in the
gut of this insect by the partitioning of ingested proteins and
digestive enzymes between the ecto- and endo-PM spaces.