close

Вход

Забыли?

вход по аккаунту

?

Secretion of the type 2 peritrophic matrix protein peritrophin-15 from the cardia.

код для вставкиСкачать
76
Eisemann et al.
Archives of Insect Biochemistry and Physiology 47:76–85 (2001)
Secretion of the Type 2 Peritrophic Matrix Protein,
Peritrophin-15, From the Cardia
Craig Eisemann, Gene Wijffels, and Ross L. Tellam*
Molecular Animal Genetics Centre, CSIRO Livestock Industries, Gehrmann Laboratories,
The University of Queensland, St Lucia, Queensland, Australia
The midgut of most insects is lined with a peritrophic matrix,
which is thought to facilitate digestion and protect the midgut
digestive epithelial cells from abrasive damage and invasion by
ingested micro-organisms. The type 2 peritrophic matrix is
synthesised by a complex and highly specialised organ called
the cardia typically located at the junction of the cuticle-lined
foregut and midgut. Although the complex anatomy of this small
organ has been described, virtually nothing is known of the molecular processes that lead to the assembly of the type 2
peritrophic matrix in the cardia. As a step towards understanding the synthesis of the peritrophic matrix, the synthesis and
secretion of the intrinsic peritrophic matrix protein, peritrophin15 has been followed in the cardia of Lucilia cuprina larvae
using immuno-gold localisations. The protein is synthesised by
cardia epithelial cells, which have abundant rough endoplasmic reticulum, Golgi, and vesicles indicative of a general secretory function. Peritrophin-15 is packaged into secretory vesicles
probably produced from Golgi and transported to the cytoplasmic face of the apical plasma membrane. The vesicles fuse with
the plasma membrane at the base of the microvilli and release
peritrophin-15 into the inter-microvilli spaces. The protein then
becomes associated with the nascent peritrophic matrix, which
lies along the tips of the epithelial cell microvilli. It is proposed
that peritrophin-15 binds to the ends of chitin fibrils present in
the nascent peritrophic matrix, thereby protecting the fibril from
the action of exochitinases. Arch. Insect Biochem. Physiol. 47:76–
85, 2001. © 2001 Wiley-Liss, Inc.
Key words: peritrophic membrane; peritrophic matrix; Lucilia cuprina,
peritrophin-15; cardia
INTRODUCTION
The peritrophic matrix (PM or peritrophic
membrane) is a semi-permeable extracellular
matrix that often lines the midgut of insects separating the contents of the gut lumen from the digestive epithelial cells lining the midgut. The PM
is thought to have assorted functions including
the facilitation of the digestive process in the in© 2001 Wiley-Liss, Inc.
Contract grant sponsor: L.W. Bett Trust; Contract grant sponsor: International Wool Secretariat; Contract grant sponsor:
Australian Centre for International Agricultural Research.
*Correspondence to: Ross L. Tellam, Molecular Animal Genetics Centre, CSIRO Livestock Industries, Gehrmann Laboratories, Research Rd, The University of Queensland, St Lucia
4067. QLD, Australia. E-mail: Ross.Tellam@tag.csiro.au
Received 18 December 2000; Accepted in revised form 8 February 2001
Secretion of Peritrophin-15
sect gut and protection of midgut epithelia from
ingested abrasive particles and potentially invasive micro-organisms (Peters, 1992; JacobsLorena and Oo, 1996; Tellam, 1996; Terra, 1996;
Lehane, 1997). The importance of the PM in the
growth and development of insects is demonstrated by experiments showing that agents binding to the PM, such as specific antibodies or
lectins, cause marked inhibition of insect growth
(East et al., 1993; Eisemann et al., 1994; Casu et
al., 1997; Fitches and Gatehouse, 1998; Tellam
and Eisemann, 1998; Zhu-Salzman et al., 1998).
It was postulated that these agents decrease the
permeability of the PM, thereby leading to starvation of the insect (Eisemann et al., 1994; Casu
et al., 1997).
There are two distinct forms of PM that have
been defined based on their sites of synthesis (Peters, 1992). Type 1 PM is synthesized by all midgut epithelial cells and forms a bag-like structure
containing the ingested meal. Typically, type 1 PM
is produced in direct response to the ingestion of
a meal (e.g., blood-fed adult mosquitoes and black
flies) but can also be constitutively produced. Type
2 PM is constitutively produced from a small
highly specialized organ, the cardia, typically located in the anterior midgut region at the junction of the foregut and midgut. This PM is an
open-ended sleeve-like structure and often more
highly structured than type 1 PM (Peters, 1992;
Tellam, 1996; Lehane, 1997). The type 2 PM is
characterized by a distinctive lamellar appearance
upon examination by electron microscopy. The
rapid rate of growth of many insect developmental stages is accompanied by the corresponding
rapid production of type 2 PMs. Indeed, type 2
PM growth rates as fast as 7.2 mm/h have been
measured in vitro (Peters, 1992). Although the
cardia is a relatively small organ typically consisting of only a few hundred PM synthesising
cells, it must be able to synthesise relatively large
quantities of the components of the PM to accommodate this rapid growth. Therefore, the cardia
cells actively synthesising PM must be heavily
committed to the synthesis of the PM components
and their assembly into a nascent PM. These components include chitin, intrinsic structural proteins or peritrophins, and proteoglycans (Peters,
1992; Tellam, 1996; Tellam et al., 1999). There is
77
still considerable debate regarding the relative
amounts of these components. Chitin has been
thought to be a significant constituent of type 2
PMs; however, this view has recently been challenged (Tellam and Eisemann, 2000).
The PM varies enormously in its synthesis,
structure, and organization in different species
of insects (Peters, 1992). Further, different life
stages of the same insect often produce different
types of PM, e.g.. larval mosquito (type 2 PM)
and adult mosquito (type 1 PM). This suggests
that the PM has been readily adapted to the specialized diets and developmental programs of each
insect species. In keeping with this view of evolutionary plasticity of the PM, the amino acid sequences of peritrophins are generally poorly
conserved even between relatively closely related
insects such as the higher Dipteran flies Lucilia
cuprina, Chrysomya bezziana, and Drosophila
melanogaster (Tellam et al., 1999). For example,
the percent identity of deduced amino acid sequences of the intrinsic PM protein peritrophin48 from any pair of these species only ranges
between 32–42% (Schorderet et al., 1998; Vuocolo
et al., 2000). In contrast, proteins unrelated to
the PM but from the same species generally have
a much higher percent identity, typically greater
than 70% (e.g., chymotrypsins, actin, NOTCH,
chitin synthase; unpublished results). Despite this
amino acid sequence variability, the major architectural feature of the peritrophin-48 proteins,
which is dictated by a multiple domain structure
characterised by extensive intradomain disulphide bonding, is highly conserved (Tellam et al.,
1999). Indeed, one of the characteristic features
of all peritrophins is the presence of multiple domains with extensive intradomain disulphide
bonding (Tellam et al., 1999).
The PM is thought to consist of a chitin fibril
meshwork embedded with glycoproteins and
proteoglycans (Peters, 1992). However, very little
is known of the constituents of the PM and how
they interact to form the highly ordered structure of this semi-permeable matrix. Virtually
nothing is known of the molecular mechanisms
underlying the synthesis of PM, i.e., where the
components are synthesised, the spatial and temporal order of their secretion, and the assembly
of these components into a highly structured PM.
78
Eisemann et al.
To partially address some of these issues, we have
followed the synthesis and secretion of the type
2 PM protein, peritrophin-15, in the cardia of larvae of the higher Dipteran fly, L. cuprina.
Peritrophin-15 has a molecular mass of 8
kDa and can only be solubilized from PM using
strong denaturants such as 6 M guanidine HCl
or 5% SDS (Wijffels et al., 2001). The protein is a
major component of the core complex of the PM
and is only synthesised by the larval cardia. The
amino acid sequences of orthologs of peritrophin15 from L. cuprina, C. bezziana, and D. melanogaster have been determined (Fig. 1; Wijffels et
al., 2001). In contrast to all other peritrophins
characterised to date, the deduced amino acid sequences of peritrophin-15 orthologs are relatively
conserved ranging between 50 and 80% identity
for any pair of sequences. It is likely that the relatively greater conservation of the sequence of this
peritrophin reflects its fundamental role in the
core assembly of the PMs from these species. Also,
unlike other PM proteins, peritrophin-15 is not
glycosylated (as determined by biochemical and
bioinformatics analyses) and consists of a single
domain structure. The characteristic feature of
the amino acid sequence is the absolute conservation of 6 cysteines whose register defines the
peritrophin-C type domain (Tellam et al., 1999).
The cysteines probably form 3 intramolecular
disulphide bonds. In addition, there is strong conservation of aromatic amino acids located between
cysteines 2 and 3, 4 and 5, and 5 and 6. A recombinant form of the protein has been expressed as
a soluble protein in bacteria, purified and shown
to bind chitin in vitro (Wijffels et al., 2001). The
presence of conserved aromatic amino acids in
peritrophin-15 is consistent with this function
(Elgavish and Shaanan, 1997). In the current
study, specific serum raised to the recombinant
Fig. 1. Alignment of the amino acid sequences of peritrophin-15 from three higher Dipterans. Only the mature
polypeptide sequences are shown (Wijffels et al., 2001). The
absolutely conserved cysteine residues are boxed. DmPM15a,
D. melanogaster peritrophin-15a; LcPM15, L. cuprina
peritrophin-15; CbPM15, C. bezziana peritrophin-15. D.
protein has been used for immuno-gold localizations to follow the synthesis and secretion of
peritrophin-15 in the cardia epithelial cells from
L. cuprina larvae.
MATERIALS AND METHODS
Culture of L. cuprina Larvae
Laboratory populations of L. cuprina, which
had originated from fly-struck sheep, were maintained on an artificial medium for up to 10 generations. Eggs were collected by placing small
trays of minced liver covered with fine nylon
gauze, inside cages of adult L. cuprina for 4–5 h.
The eggs were then incubated overnight at 16°C
and 100% relative humidity before being transferred to 34°C until hatching. Larvae were reared
to third instar on a diet containing 10% w/v skim
milk powder and 2% w/v brewer’s yeast in 1% w/
v agar gel.
Production and Purification of Recombinant
Hexahis-Peritrophin-15
The recombinant protein, hexahis-peritrophin-15 derived from the C. bezziana peritrophin15 sequence (Wijffels et al., 2001), was expressed
using the pQE9 vector (Qiagen) essentially according to the manufacturer’s instructions. Only the
DNA encoding the mature form of the protein was
used. The soluble protein was purified by Ni-NTA
affinity chromatography (Qiagen).
Production of Serum to Hexahis-Peritrophin-15
Recombinant hexahis-peritrophin-15 (70 µg)
was homogenized in Montanide ISA70 (Seppic,
Paris, France) and subcutaneously injected into a
rabbit. The rabbit received two further injections
4 and 6 weeks after the primary immunization.
Serum was collected 2 weeks after the final im-
melanogaster contains two peritrophin-15-like sequences. For
simplicity, only one of these is shown. The asterisks show
positions where an amino acid is absolutely conserved in
the three orthologs. Dashes have been introduced in the sequences to optimise the alignment.
Secretion of Peritrophin-15
munization. The “Australian code of practice for
the care and use of animals for scientific purposes”
was followed for all procedures involving the rabbit. Immuno-blots and ELISAs confirmed the specificity of the antibody. Although the serum was
raised to C. bezziana hexahis-peritrophin-15, it reacted strongly with L. cuprina peritrophin-15 in
immuno-blots and ELISAs as would be expected
for two proteins with 82% amino acid sequence
identity (results not shown).
Immuno-Gold Localization
Cardiae were dissected from third instar larvae in PBS and fixed in 4% paraformaldehyde
and 0.3% glutaraldehyde in PBS for 1 h at room
temperature. The dissected cardiae were washed
in PBS, dehydrated through an ethanol series (30,
50, 70, 90%) and embedded in medium grade LR
White embedding resin (London Resin Co., Reading, Berkshire, UK), which was then polymerised
in air-tight gelatin capsules at 50°C overnight. Ultra-thin sections were cut longitudinally through
individual cardia on an LKB Ultrotome Nova ultramicrotome and the sections taken up on Butvarcoated copper grids. Sections on grids were
processed by transferring to drops of buffer A (PBS
with 0.5% ovalbumin [Sigma] and 0.1% Tween 20)
containing: (1) 10% normal goat serum (1.5 h); (2)
pre- or post-vaccination rabbit serum to recombinant hexahis-peritrophin-15 (diluted 1/500) for 1
h; (3) goat anti-rabbit Ig antibody conjugated to
10-nm-diameter colloidal gold particles diluted 1/
100 (British Biocell International) for 1.5 h all at
room temperature. The sections were washed (3×
15 min) in drops of buffer A after steps (2) and
(3) and finally in distilled water. Each section was
then stained (5 min each) in 2% aqueous uranyl
acetate and 0.1 M lead citrate and examined in a
JEOL 1010 transmission electron microscope.
79
tains the oesophagus surrounded by highly modified foregut including a cuticular lining and this,
in turn, is surrounded by specialized midgut cells
that synthesise the PM. Only a single PM is produced by the larval cardia. In contrast, the adult
cardia simultaneously produces 3 PMs (Binnington, 1988). The region of the cardia marked F in
Figure 2 represents the region where sections were
examined in detail for the presence of peritrophin15 using immuno-gold localizations. In total, the
sections encompassed the cuticular lining contributed by the foregut, the adjacent PM, and the underlying epithelial cells characterised by extensive
microvilli. The order of the following figures has
been arranged to progressively show the spatial
aspects of peritrophin-15 production, secretion, and
addition to the nascent PM.
The epithelial cells underlying the nascent
PM in the cardia are characterised by extensive
rough endoplasmic reticulum, Golgi, and secretory vesicles, collectively indicative of a secretory
cell (Fig. 3). The vesicle labeled in Figure 3a is
surrounded by membranous layers, probably
RESULTS
The cardia from the larvae of higher Dipterans is a small highly specialized organ located at
the junction of the cuticle-lined foregut (oesophagus) and midgut, and formed by intussusception
of both tissues. For orientation, Figure 2 shows a
diagrammatic representation of a sagittal section
through the cardia of a higher Dipteran larva
(Binnington, 1988; Peters, 1992). The cardia con-
Fig. 2. Schematic representation of a sagittal section through
the larval cardia of a higher Dipteran. B, bacteria; Cu, cuticle;
F, formation zone of the PM; FG, foregut; IC, imaginal cells; O,
oesophagus; PM, peritrophic matrix. The region marked F represents the sections in the PM formation zone examined for the
synthesis of peritrophin-15. Reproduced from Peters (1992) with
permission of the publisher, Springer-Verlag, Berlin.
80
Eisemann et al.
Fig. 3. Immuno-gold localization of peritrophin-15 in the
cytoplasm of cardia epithelial cells from third instar L.
cuprina larvae. The sections were from the cardia PM formation zone. a: Pre-vaccination control. b–d: Post-vaccination serum. Arrows denote examples of gold particles
associated with the rough endoplasmic reticulum and
vesicles. G, Golgi; M, mitochondria; N, nucleus; RER, rough
endoplasmic reticulum; V, intracellular vesicle. The bars represent (a) 267 nm; (b) 267 nm; (c) 133 nm; (d) 133 nm.
Golgi. This suggests that the vesicle is in intimate contact with Golgi and possibly derived from
it. Close examination of the immuno-gold-labeled
cells reveals the presence of gold particles associated with the rough endoplasmic reticulum, Golgi,
and vesicles (Fig. 3b–d). The labeling of the rough
endoplasmic reticulum is consistent with the production of a protein destined for secretion. Not
all vesicles were labeled to the same density and
a minority contained no gold particles (e.g., Fig.
3c,d). The vesicles, therefore, may have contained
multiple cargoes and in some cases no peritroph-
in-15. Figure 3a is a control using pre-vaccination serum. This section shows virtually no gold
labeling, thereby demonstrating the specificity of
the immuno-localization.
Figure 4 shows concentrations of heavily labeled vesicles immediately underlying the base
of microvilli at the apical plasma membrane of
the cardia epithelial cells. The vesicles are generally larger than those present further within
the cell and often contain internal “nodes.” These
are particularly discernible in the pre-vaccination
control shown in Figure 4a but are also apparent
Secretion of Peritrophin-15
81
Fig. 4. Immuno-gold localization of peritrophin-15 in intracellular vesicles adjacent to the apical plasma membrane
of cardia epithelial cells. a: Pre-vaccination control. b–d:
Post-vaccination serum. Arrows denote examples of intrac-
ellular vesicles labeled with gold particles. M, mitochondria;
MF, microfilaments; MV, microvilli; RER, rough endoplasmic reticulum; V, intracellular vesicle. The bars represent
(a) 267 nm; (b) 267 nm; (c) 133 nm; (d) 133 nm.
in the labeled vesicles shown in Figure 4b–d.
Again, this suggests multiple cargoes in the
vesicles. Often the vesicles are associated with a
microfilament extending from the base of a microvillus back into the cell (Fig. 4c). The microfilament may be a means of transporting the vesicles
from their site of synthesis to the cell periphery
or may be involved in organization of vesicles at
the cell membrane as a prelude to fusion of the
vesicle with the plasma membrane and subse-
quent exocytosis. The vesicles appear to fuse with
the cell membrane lying between two microvilli
and, therefore, presumably disgorge their vesicle
contents between the microvilli (Fig. 4c,d). There
is no evidence that any of the vesicles travel
within the microvillus to its tip before membrane
fusion. What appear to be the remnants of the
vesicles, which now are much larger and contain
little or no gold labeling, are present extracellularly between the microvilli (Fig. 4a,d). It is not
82
Eisemann et al.
clear whether these putative vesicle remnants are
membrane bound. Further, there is no indication
that the putative vesicle remnants contain peritrophin-15 or any other cargoes. The putative
vesicle remnants progressively move through the
inter-microvillar spaces to the tips of the microvilli immediately adjacent to the nascent PM.
Here, they are considerably smaller in size (Fig.
5a). Close examination of the inter-microvilli
spaces reveals the presence of gold particles but
at relatively low density (Fig. 5c). This would be
expected if the contents of the intracellular
vesicles were diluted into the larger extracellular spaces associated with the microvilli.
Fig. 5. Immuno-gold localization of peritrophin-15 on nascent PM. a: Pre-vaccination control. b,c: Post-vaccination
serum. Solid black arrows denote examples of gold particles
associated with the PM, intracellular vesicles, and microvilli.
Broken white arrows denote the positions of the cuticle (C),
microvilli (MV), vesicles (V), and peritrophic matrix (PM) in
(a) and (b). The bars represent (a) 350 nm; (b) 350 nm; (c)
133 nm.
Secretion of Peritrophin-15
Figure 5a and b show sections of the PM formation zone encompassing the cuticle, nascent
PM, and microvilli of the cardia epithelial cells.
The microvilli that characterize these cells are
relatively long (~ 2–3 µm). The sections show labeled vesicles strongly concentrated at the apical
face of the plasma membrane, presumably awaiting membrane fusion and secretion of their contents. Also clearly discernible are the putative
vesicle remnants between the microvilli adjacent
the cell proper and smaller remnants at the distal ends of the microvilli. The microvilli extend
directly to the nascent PM, which is strongly and
uniformly labeled with gold (Fig. 5c). In contrast,
the highly defined cuticle, which is located immediately adjacent to the nascent PM but opposite the microvilli, shows no gold labeling. This
is consistent with the view that peritrophin-15 is
restricted in its final location to only the larval
PM. Indeed, tissue-specific immuno-blots have
demonstrated the presence of peritrophin-15 in
larval cardia and PM but not in any other larval
tissues (Wijffels et al., 2001). The PM at the position in the cardia where the sections were taken
does not show the marked lamellar appearance
that characterises the fully mature PM isolated
from larval midgut. Presumably, the PM visualized in these sections is immature.
DISCUSSION
The current study has demonstrated that
peritrophin-15 is synthesised by epithelial cells
in the PM formation zone of the cardia. These
cells package peritrophin-15 into secretory vesicles, which originate from deep within the cell,
probably derived from Golgi. The vesicles are then
transported to the apical face of the cell where
they concentrate at the base of microvilli. The process of vesicular movement and organization at
the plasma membrane may be facilitated by microfilaments originating from the base of the microvilli. The vesicles presumably fuse with the
plasma membrane and release their cargoes into
the inter-microvillar space via a process of exocytosis. Peritrophin-15 is strongly associated with
the nascent PM, which is situated at the tips of
the microvilli of the cardia epithelial cells. What
is not clear is how the protein moves from the
base of the microvilli to the PM. This may be a
83
passive diffusional process that relies on the binding of peritrophin-15 to the nascent PM and the
subsequent movement of the PM away from this
site thereby creating a concentration gradient of
peritrophin-15 in the intermicrovillar spaces. Alternatively, pulsatile contractions of the cardia
may facilitate such movement (Peters, 1992).
The process of intracellular vesicular transport of peritrophin-15 followed by its exocytosis
at the base of microvilli also describes the production and secretion of a peritrophin (IIM) from
midgut cells of a lepidopteran insect producing
type 1 PM (Harper and Granados, 1999). Thus,
the same mechanisms of peritrophin synthesis
and secretion may underlie the formation of both
type 1 and type 2 PMs. The cardia may be an
evolutionary adaptation in some insects, allowing synthesis of relatively large quantities of
peritrophins in a highly localized gut region that
acts as an organising centre for the nascent PM.
One of the significant differences between type 2
and type 1 PMs is the greater level of structural
organization in the former PMs (Peters, 1992).
One putative function of peritrophin-15 is to
cap the ends of chitin polymers or fibrils in the
PM, thereby possibly regulating the lengths of the
polymer and also protecting its ends from attack
by exochitinases (Wijffels et al., 2001). This model
was based on the evidence that peritrophin-15
bound strongly to chitin but at low stoichiometries
(~1 molecule of peritrophin-15 to 10,000 GlcNAc
molecules) in vitro and that the protein was probably a monomer consisting of a single protein domain. This model is consistent with the presence
of an immature PM at the position in the cardia
where peritrophin-15 is secreted and presumably
added, as well as the uniform distribution of
peritrophin-15 throughout the nascent PM.
It is interesting to note the complete absence
of peritrophin-15 in the cuticle layer lying adjacent to the nascent PM but opposite the microvilli
of the cardia epithelia cells. The cuticle, like the
PM, is also thought to contain chitin (Hackman,
1974). Substantial analyses of cuticular proteins
from various sources have never revealed the
presence of peritrophin-like proteins (Anderson
et al., 1995). The cuticular proteins in their mature state nearly always contain no cysteine residues, a feature that easily distinguishes the two
groups. Presumably, the chitin present within the
84
Eisemann et al.
cuticle layer in the cardia is not available for binding peritrophin-15. The cuticle layer at this position in the PM could be relatively mature and
bound cuticle proteins may block any binding sites
for peritrophin-15. Alternatively, chitin present in
the PM and cuticle may be in very different conformations, which could dictate the specific binding interactions with the different families of
proteins associated with these structures.
It will be important to follow up the current
studies with multiple labeling for several PM proteins to determine whether these proteins are made
simultaneously by the same cells and secreted from
common or different vesicles or whether there is a
spatially distinct distribution of synthesis along the
cardia epithelial cells that allows the progressive
and ordered addition of specific PM proteins to the
nascent PM. Of particular importance will be the
determination of those cells producing chitin destined for inclusion in the PM. Presumably, chitin is
the principal component of the nascent PM onto
which peritrophin-15 is added. Cardia epithelial
cells located further toward the origins of the PM
formation zone of the cardia could be producing
chitin that assembles into the nascent PM, which
then progressively moves as it is synthesised toward the posterior of the cardia. Another possibility is that chitin is synthesised by the same cells
producing peritrophin-15. Here, the tips of the microvilli may be sites for chitin synthesis and secretion. However, these regions contain no specialized
structures normally associated with chitin production at least in those cells synthesising cuticular
chitin (Locke, 1996). Alternatively, the epithelia cells
of the PM formation zone could package chitin in
the same secretory vesicles that carry peritrophin15 and perhaps other peritrophins as well. The preassembled contents of these vesicles could be
released into the inter-microvillar spaces and then
finally assemble into the nascent PM at the tips of
microvilli. To distinguish between these models it
will be necessary to localise chitin- and peritrophinsynthesising cells. The detection of chitin in tissue
sections is not easy (Tellam and Eisemann, 2000).
The results obtained from the traditional method
of localisation of chitin using gold-labeled wheat
germ lectin are difficult to interpret as many
peritrophins are glycoproteins with strong affinity
for this lectin (East et al., 1993; Casu et al., 1997;
Tellam et al., 1999). Perhaps the best way of
characterising the cells synthesising chitin is to
identify those that express chitin synthase. The recent molecular characterization of insect chitin synthase should facilitate the localization of this
enzyme in the insect cardia (Tellam et al., 2000).
The current study has followed the exocytosis and assembly of the integral PM protein,
peritrophin-15, onto newly formed PM in the cardia of L. cuprina larvae. This is a small step in a
complex assembly process involved in the production of PM from a small but highly specialized
group of cells, the cardia. Many questions still
remain unanswered. For example: what is the full
repertoire of specific components, particularly the
peritrophins, present in the PM; where is chitin
produced; what is the conformation of chitin in
the PM; what controls the order, if any, of the
addition of specific components to the PM; what
specific protein-protein, protein-chitin, and protein-oligosaccharide interactions are required for
assembly of the PM; and what are the precise
molecular and biological functions of the PM? The
recent determination of the Drosophila genomic
sequence and the molecular characterization of
several insect PM proteins (Tellam et al., 1999;
and our unpublished results) now enable the identification of the majority of the principal protein
components of the PM from higher Dipterans.
These proteins are major constituents of the PM
and studies of their synthesis and assembly into
the PM will provide valuable insights into the
structure and functions of the PM.
ACKNOWLEDGMENTS
We thank the Australian wool growers
through the International Wool Secretariat for
financial support.
LITERATURE CITED
Andersen SO, Hojrup P, Roepstorff P. 1995. Insect cuticular
proteins. Insect Biochem Mol Biol 25:153–176.
Binnington KC. 1988. Ultrastructure of peritrophic membrane-secreting cells in the cardia of the blowfly,
Lucilia cuprina. Tissue Cell 20:269–281.
Casu R, Eisemann C, Pearson R, Riding G, East I, Donaldson
A, Cadogan L, Tellam RL. 1997. Antibody-mediated
inhibition of the growth of larvae from an insect causing cutaneous myiasis in a mammalian host. Proc Natl
Acad Sci 94:8939–8944.
Secretion of Peritrophin-15
85
East IJ, Fitzgerald CJ, Pearson RD, Donaldson RA, Vuocolo
T, Cadogan LC, Eisemann CH, Tellam RL. 1993.
Lucilia cuprina: Inhibition of larval growth induced
by immunization of host sheep with extracts of larval
peritrophic membrane. Int J Parasitol 23:221–229.
Schorderet S, Pearson RD, Vuocolo T, Eisemann C, Riding
GA, Tellam RL. 1998. cDNA and deduced amino acid
sequences of a peritrophic membrane glycoprotein,
‘peritrophin-48’, from the larvae of Lucilia cuprina.
Insect Biochem Mol Biol 28:99–111.
Eisemann CH, Donaldson RA, Pearson RD, Cadogan LC,
Vuocolo T, Tellam RL. 1994. Larvicidal activity of
lectins on Lucilia cuprina: mechanism of action.
Entomol Exp Appl 72:1–10.
Tellam RL. 1996. The peritrophic matrix. In: Billingsley PF,
Lehane MJ, editors. The biology of the insect midgut.
London: Chapman and Hall. p 86–114.
Elgavish S, and Shaanan B. 1997. Lectin-carbohydrate interactions: different folds, common recognition principles. Trends Biochem Sci 22:462–467.
Fitches E, and Gatehouse JA. 1998. A comparison of the
short and long term effects of insecticidal lectins on
the activities of soluble and brush border enzymes of
tomato moth larvae (Lacanobia oleracea). J Insect
Physiol 44: 1213–1224.
Hackman RH. 1974. Chemistry of the insect cuticle. In:
Rockstein M, editor. The physiology of insecta. New
York: Academic Press. p 215–270.
Harper MS, Granados RR. 1999. Peritrophic membrane
structure and formation of larval Trichoplusia ni with
an investigation on the secretion patterns of a PM mucin. Tissue and Cell 31: 202–211.
Tellam RL, Eisemann CH. 1998. Inhibition of growth of
Lucilia cuprina larvae using serum from sheep vaccinated with first-instar larval antigens. Int J Parasitol
28:439–450.
Tellam RL, Eisemann CH. 2000. Chitin is only a minor component of the peritrophic matrix from larvae of Lucilia
cuprina. J Insect Biochem Mol Biol 30:1189–1201.
Tellam RL, Wijffels G, Willadsen P. 1999. Peritrophic matrix proteins. Insect Biochem Mol Biol 29:87–101.
Tellam RL, Vuocolo T, Johnson S, Jarmey J, Pearson R. 2000.
Insect chtin synthase: cDNA sequence, gene organization and expression. Eur J Biochem 267:6025–6043.
Terra WR. 1996. Evolution and function of insect peritrophic
membrane. Ciencia Cultura L Braz Assoc Advan Sci
48:317–324.
Jacobs-Lorena M, Oo MM. 1996. The peritrophic membrane
of insects. In: Beaty B, Marqquardt W, editors. Biology of disease vectors: a molecular, physiological, and
populational approach. Denver: University Press of
Colorado. p 318–332.
Vuocolo T, Eisemann C, Pearson R, Willadsen P, Tellam RL.
2001. Identification and molecular characterisation of a
peritrophin gene, ‘C.b. peritrophin-48’ from the myiasis
fly Chrysomya bezziana. Insect Mol Biol 30:1189–1201.
Lehane MJ. 1997. Peritrophic matrix structure and function. Annu Rev Entomol 42: 525–550.
Wijffels G, Eisemann C, Riding G, Pearson R, Jones A, Tellam
RL. 2001. A novel family of chitin binding proteins from
insect type 2 peritrophic matrix: cDNA sequences, chitin
binding activity and cellular localization. J Biol Chem
(in press, published online February 2001).
Locke M. 1996. The role of plasma membrane plaques and
Golgi complex vesicles in cuticle deposition during the
moult/intermoult cycle. In: Hepburn HR, editor. The
insect integument. London: Elsevier Scientific Publishing Company. p 237–257.
Peters W. 1992. Peritrophic membranes. Zoophysiology, Vol
130. Berlin: Springer-Verlag.
Zhu-Salzman K, Shade RE, Koiwa H, Salzman RA, Narasimhan M, Bressan RA, Hasegawa PM, Murdock LL.
1998. Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia
lectin II. Proc Natl Acad Sci USA 95:15123–15128.
Документ
Категория
Без категории
Просмотров
0
Размер файла
519 Кб
Теги
cardio, matrix, peritrophic, peritrophin, protein, secretion, typed
1/--страниц
Пожаловаться на содержимое документа