Meyer, Ingo Scha¨fer and Klaus Scheller Department of Cell and Developmental Biology, Biocenter of the University, Wu¨rzburg, Germany In late larvae of the blowfly, Calliphora vicina, ar
Trang 1Interaction of the anterior fat body protein with the hexamerin
Immo A Hansen, Susanne R Meyer, Ingo Scha¨fer and Klaus Scheller
Department of Cell and Developmental Biology, Biocenter of the University, Wu¨rzburg, Germany
In late larvae of the blowfly, Calliphora vicina, arylphorin and
LSP-2 proteins, which belong to the class of hexamerins, are
selectively taken up by the fat body from the haemolymph
Hexamerin endocytosis is mediated by a specific
membrane-bound receptor, the arylphorin-binding protein (ABP)
Using the two-hybrid technique, we found that the anterior
fat body protein (AFP) interacts with the hexamerin receptor
AFP, a homologue of the mammalian calcium-binding liver
protein regucalcin (senescence marker protein-30), exhibits a
strong binding affinity for a naturally occurring C-terminal
cleavage fragment of the hexamerin receptor precursor (the
P30 peptide) and other receptor cleavage products that contain P30 Expression of AFP mRNA and protein is restricted to the anterior part of the fat body tissue and to haemocytes in last-instar larvae AFP mRNA occurs in all postembryonic developmental stages Our results suggest that AFP plays a role in the regulation of hexamerin uptake
by fat body cells along the anterior–posterior axis
Keywords: anterior fat body protein; Calliphora vicina; cDNA sequence; hexamerin receptor; yeast two-hybrid system
The construction of adult tissues during the metamorphosis
of holometablous insects requires large amounts of energy
and building blocks Before formation of the puparium, fat
body cells reabsorb proteins and other macromolecules that
have accumulated in the haemolymph during the larval
feeding period The major fraction of incorporated proteins
consists of arylphorins and LSP-2 which belong to the class
of hexamerins, haemocyanin-related proteins, named
according to their composition of six identical or closely
related subunits [1] Although some studies suggest that a
nonspecific, general protein uptake mechanism is
responsi-ble for the incorporation of hexamerins [2], the selectivity of
this process has been demonstrated unambiguously by the
differential clearing of distinct proteins from the
haemol-ymph [3–6] Transport of hexamerins through fat body cell
membranes is controlled by ecdysteroids and mediated by a
specific receptor (for review, see [7]) The hexamerin
receptor of Calliphora vicina was cloned and its
post-translational processing studied in detail Two cleavage
steps, which detach a 45-kDa and a 30-kDa peptide from
the hexamerin-binding N-terminus of the receptor precursor
(Fig 1), have been shown to be connected to activation of
the receptor and initiation of hexamerin endocytosis [8,9]
The principal cell type of the fat body is the trophocyte,
which is morphologically uniform and has long been thought
to have equivalent functions Almost all experiments dealing
with protein expression and sequestration by this tissue have
been performed using the entire fat body [10,11] However, in
both Diptera and Lepidoptera, data are accumulating that show regional differences in fat body function In the corn earworm, Helicoverpa zea, storage proteins are synthesized
by the peripheral fat body fraction, but are taken up and stored only by the perivisceral fat body [12] In the silkworm, Bombyx mori, it has been demonstrated that dorsal and ventral perivisceral fat body contains the most competent cells for sequestering haemolymph proteins compared with peripheral and hind-gut associated fat body tissue [13]
In dipteran insects, differences in both composition and fate of the anterior and posterior fat body have been reported The larval fat body of the fruitfly, Drosophila melanogaster, and the blue blowfly, Calliphora vicina, is organized into a lobed tissue of 2000–3000 polytene cells, which become dissociated from each other during meta-morphosis Roughly half of the cell population survives metamorphosis, indicating a specific degree of differentia-tion during postembryonic life [14,15] An increase in the number of storage protein granules found along the anterior–posterior axis has been described in the fruitfly Drosophila[16], and rapid degradation of the anterior part
of the fat body tissue after pupariation has been reported in the fleshfly Sarcophaga peregrina [17] The authors report the specific expression of anterior fat body protein (AFP) in the trophocytes of the anterior fat body of S peregrina, demonstrating one of the biochemical differences in dipteran fat body tissue
Here we report the tissue-specific expression of AFP and its interaction with the hexamerin receptor This is the first demonstration of a protein–protein interaction of the hexamerin receptor with a nonhexameric partner
E X P E R I M E N T A L P R O C E D U R E S Experimental animals
A strain of C vicina that has been maintained in our laboratory for several decades was used The flies were
Correspondence to I A Hansen, Medizinische Polyklinik der
Universita¨t, Endokrinologie, Josef-Schneider-Str 2, D-97080
Wu¨rzburg, Germany E-mail: i.hansen@medizin.uni-wuerzburg.de
Abbreviations: ABP, arylphorin-binding protein; AFP, anterior fat
body protein; NBT/BCIP, nitroblue tetrazolium
chloride/5-bromo-4-chloro-3-indonyl phosphate
(Received 22 June 2001, revised 22 October 2001, accepted 7 December
2001)
Trang 2reared on bovine meat at 25°C and relative humidity of
65% as previously described [18]
Preparation of fat body tissue and haemocytes
Third-instar larvae were washed in insect saline and
anaes-thetized by cooling on ice for a few minutes The larvae were
dissected by a medial cut, washed with cold insect saline, and
the fat body tissues excised For the isolation of haemocytes,
anaesthetized larvae were dried and transferred to a cold
microscope slide From a small cut in the abdomen,
haemolymph (5–10 lL per larva) was collected by pipette
and transferred to a 1.5-mL Eppendorf tube on ice After
centrigugation at 3000 r.p.m at 4°C, the supernatant was
removed and the pellet containing the haemocytes was
washed twice with ice-cold insect saline and re-centrifuged
Two-hybrid library construction
Total RNA was isolated from dissected fat body tissues of
third-instar larvae (6–7-day-old larvae) using Trizol reagent
(Gibco) following the supplier’s instructions for fatty tissues
One microgram of total RNA was used for cDNA synthesis
with the SMARTTMPCR cDNA Library Construction Kit
(Clontech, Heidelberg, Germany) The cDNA obtained
included two different SfiI restriction sites at the 5¢ and 3¢
ends (SfiI/A, SfiI/B) The two-hybrid library vector pJG4-5
(GenBank accession number U89961) was modified by
introducing the SfiI/A and SfiI/B restriction sites into its
multiple coloning site allowing directed cloning of the
cDNA The ligation reaction was carried out overnight at
16°C The library plasmids were transformed in Escherichia
coliXL1-Blue cells via electroporation and grown on Luria–
Bertani plates containing ampicillin A total of 1.2· 106
independent bacterial clones were obtained and subjected to
plasmid isolation using the QIAfilter Plasmid Mega Kit (Qiagen, Hilden, Germany) One milligram of library plasmids was isolated The cDNA library contains
3.8 · 105individual clones in the correct reading frame The average insert size was 1 kbp
Construction of hexamerin receptor bait proteins for two-hybrid screening
Three hexamerin receptor bait plasmids were constructed according to the natural receptor cleavage products ABP130, ABP96, ABP64 described previously [9] (Fig 1) Using a pBluescript SK+ vector bearing the complete hexamerin receptor cDNA sequence (GenBank accession number X79100) as a template, three cDNA fragments were amplified via PCR using different primer combinations: (1) ABP130: ABP130-5¢(CTCGAGGGTGTTATAATGG ATCGAGGTGGACGAGT)/ABP130-3¢ (CTCGAG ATTCAATTATTTAGTACAAATGGCTAAGAGG CATTT);
(2) ABP96: ABP130-5¢/ABP96-3¢ (CTCGAGAGGCAAC AACAGACGATGAGGCAACTTA);
(3) ABP64: ABP130-5¢/ABP64-3¢(CTCGAGACCAGA GATCTCATCATTATCATTGTAATT)
XhoI restriction sites were attached at the 5¢ ends of the primers PCR was carried out using PfuTurboÒ DNA Polymerase (Stratagene, La Jolla, CA, USA) following the manufacturer’s protocol The PCR products were sub-cloned in pCR-Script Amp vector (PCR-ScriptTM Amp Cloning Kit; Stratagene), excised with XhoI, and finally ligated in the two-hybrid bait vector pEG202 (Origene, Rockville, MD, USA) The orientation and correct insertion were checked by sequencing using the pEG202-seq primer Two-hybrid screening
This was carried out following a standard protocol for LexA-based two-hybrid systems [19] Thirty-one library plasmids that interacted with the baits were isolated from the yeast and transferred into E coli XL1-blue cells and sequenced from the 3¢ and 5¢ end on a Perkin–Elmer 310 sequencer using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin–Elmer)
5¢ RACE 5¢ RACE was performed using the SMARTTM RACE cDNA Amplification Kit (Clontech, Alameda, CA, USA) following the manufacturer’s instructions Two specific primers were used (P1: 5¢-GCCATCGGGCAACAAAT GATCCTTGGGGCTGGTCTTG-3¢; P2: 5¢-GATCGG TTGTACCTTCGACGGGCACAGCAAAACCA-3¢, see Fig 3)
Northern-blot hybridization Total RNA was extracted from freshly prepared tissues using the TriFast Kit (Peqlab, Erlangen, Germany) and subjected to electrophoresis in a 0.8% agarose gel North-ern-blot analysis was performed according to standard procedures [20] As a hybridization probe, we used a digoxygenin-labeled antisense RNA, synthesized from
Fig 1 Scheme of the post-translational cleavage pattern of the
Calli-phora hexamerin receptor [7,9] The primary translation product
con-tains a 17-amino-acid N-terminal signal peptide which is removed
immediately after translation Before reaching the cell membrane, the
receptor precursor is cleaved a second time: a 429-amino-acid
C-ter-minal fragment is removed, giving rise to P45 and ABP96 (807 amino
acids) which comprises the active receptor The onset of arylphorin
reabsorption by the fat body coincides with a third receptor cleavage
which generates ABP64 (554 amino acids) and P30 (253 amino acids).
Only ABP 130, ABP96 and P30 are able to bind hexamerins.
Trang 3linearized AFP cDNA as template using the
DIG-RNA-Labeling Kit (T7; Roche Molecular Biochemicals,
Mann-heim, Germany) Immunodetection was carried out using
antibodies to DIG coupled with peroxidase The blots were
developed by the nitroblue tetrazolium
chloride/5-bromo-4-chloro-3-indonyl phosphate (NBT/BCIP) method
In situ hybridization on cryosections
Seven-day-old anaesthetized larvae received injections of
5 lL 4% paraformaldehyde in NaCl/Pi(7 mMNa2HPO4,
3 mMNaH2HPO4, 130 mMNaCl) and were fixed overnight
in paraformaldehyde/NaCl/Piat 4°C The fixed larvae were
incubated at 4°C for 24 h in Ringer solution (130 mM
NaCl, 4.7 mM KCl, 0.74 mM KH2HPO4, 0.35 mM
Na2HPO4, 1.8 mM MgCl2, pH 7.0) containing 25%
sucrose Longitudinal cryosections (10 lm) were incubated
for 5 min in 0.1M glycine/Tris/HCl buffer (pH 7.0) and
successively for 15 min at room temperature in NaCl/Pi
containing 0.3% Triton X-100 After three wash steps with
NaCl/Pi, the sections were fixed for 2 min in 2%
paraform-aldehyde and then for 10 min in 10 mM Tris/HCl/1 mM
EDTA (pH 7.4) After a 1-h prehybridization, the
heat-denatured DIG-labeled AFP-antisense RNA probe was
added for hybridization overnight at 42°C The slides were
washed according to the following scheme: 3· 10 min with
4· NaCl/Cit; 2 · 10 min with 2 · NaCl/Cit; 10 min
with 0.1· NaCl/Cit; 10 min with 0.05 · NaCl/Cit; 5 min
with NaCl/Tris After incubation for 30 min in nonfat dried
milk-saturated NaCl/Pi, the slides were incubated for 2 h at
37°C with antibodies to DIG After three washes with
NaCl/Tris, the reactive structures were visualized by the
NBT/BCIP method The specimens were mounted in
Mowiol and analyzed under the microscope
Whole-mountin situ hybridization
Last-instar larvae were dissected in ice-cold insect saline by a
cut at the posterior end and upending the complete larvae
The gut was removed and the preparations promptly fixed
in MEMFA (0.1M Mops, 2 mM EGTA, 1 mM MgSO4,
3.7% formaldehyde) for 2 h at room temperature The
tissues were dehydrated with methanol and stored at)20 °C
until used for whole-mount in situ hybridization [21]
Immuno-coprecipitiation with AFP and ABP antibodies
The anti-ABP IgG recognizes the hexamerin (arylphorin)
receptor of C vicina [5] The anti-AFP IgG was provided by
Dr Nakajima and recognizes a 34-kDa AFP in S peregrina
[17] Protein A–Sepharose CL-4B (Pharma Biotech,
Frei-burg, Germany) was suspended in NaCl/Pi The resulting
gel was centrifuged at 1000 g and resuspended in 1 vol
NaCl/Pi (SL) Anterior fat body tissue from 8-day-old
larvae was homogenized in NaCl/Pi containing 0.05%
phenylthiourea and centrifuged for 5 min at 8000 g at 4°C
The supernatant was used for immunoprecipitation SL
(50 lL) was incubated in an Eppendorf cap with 5 lg
anti-ABP IgG at 4°C for 4 h Then, 500 lL fat body
supernatant or 500 lL haemocytes was added and
incuba-ted at 4°C overnight As controls, anti-(rabbit LexA) IgG
was added as an antibody or NaCl/Piwas used instead of
homogenate The incubation mixtures were centrifuged and
the pellet washed eight times with NaCl/Pi The last pellet was suspended in 30 lL sample buffer, heated at 95°C for
2 min, and centrifuged The supernatant (15 lL) was subjected to SDS/PAGE
Western-blot analysis For immunoblots, the heat-denatured proteins were trans-ferred to poly(vinylidene difluoride) membranes (Millipore Corp., Bedford, MA, USA) The membranes were blocked with 10% nonfat dried milk/0.3% Tween 20 in NaCl/Tris and incubated with anti-AFP IgG (0.5 lgÆmL)1in NaCl/ Tris containing 1% BSA) for 2 h at room temperature After three washes in NaCl/Tris, the secondary antibody (goat anti-rabbit IgG conjugated with alkaline phosphatase, diluted 1 : 7500; Promega, Heidelberg, Germany) was added and the blots were incubated for 1 h After three washes, the blots were developed with NBT/BCIP system as described under Northern-blot hybridization
Immunofluorescence analysis Longitudinal cryosections (10 lm) from the same larvae as used for in situ hybridization were blocked at room temperature for 2 h with 3% normal goat serum in 0.5· PAT (1 · NaCl/Pi, 1% albumin, 0.5% Triton X-100) and then incubated overnight at 4°C with anti-AFP IgG (10 lgÆlL)1 in 0.5· PAT) After three washes with NaCl/Pi, the sections were incubated for 2 h at room temperature with a Cy2 (cyanine 2-OSu bisfunctional)-conjugated affinity-purified goat anti-rabbit IgG (1 : 50; Rockland, Gilbertsville, PA, USA) in 0.5· PAT After being thoroughly washed, the sections were analyzed under
a Leica fluorescent microscope and photographed with a Pixera CCD camera The specificity of the AFP immuno-reaction was verified by omitting the primary antibody
R E S U L T S Screening for interaction with hexamerin receptor Using the yeast two-hybrid system and ABP 130, as well as ABP 96 (Fig 1) as a bait, we isolated 27 Ôinteraction positiveÕ yeast clones The library (prey) plasmids of these clones was isolated Sequence analysis of the cDNAs revealed that 17 were hexamerin cDNAs (14 arylphorin and three LSP-2), confirming the ability of the experimental system to identify proteins that interact with the hexamerin receptor Thirteen
of the library plasmids contained cDNAs that encoded nonhexamerin interactors In our database search using
BLAST Xanalysis, nine showed no homology to any known protein We identified three cDNAs that encoded a protein with 93% identity in the deduced amino-acid sequence with the AFP of S peregrina (GenBank accession number BAA99282) Because of the high sequence identity with the Sarcophaga AFP, we named our clone Calliphora AFP (GenBank accession number AY028616) The AFP clones were identified by screening the prey library with ABP130 (once) or ABP96 (twice) as baits
We also examined the ability of AFP to interact with ABP130, ABP96, and ABP64 in the two-hybrid assay We found a strong interaction between AFP and ABP130 and ABP96, but no interaction with ABP64 (Table 1)
Trang 4Immuno-coprecipitation of AFP and hexamerin receptor
To confirm the results demonstrating the interaction of
AFP with different cleavage products of the hexamerin
receptor, we used immuno-coprecipitation as an
indepen-dent method AFP could be precipitated with anti-ABP IgG
and the receptor (ABP) with anti-AFP IgG As can be seen
from Fig 2, anti-AFP IgG precipitated the receptor
cleavage fragment P30, whereas anti-ABP IgG precipitated
AFP
Isolation and sequence analysis of full-length AFP cDNA
The deduced peptide sequences of the isolated AFP cDNAs
did not contain a start methionine and were lacking 40
amino acids at the N-terminus compared with the AFP of
S peregrina (GenBank accession number BAA99282)
5¢-RACE PCR led to an overlapping fragment of 288 bp
The complete 1150-bp AFP cDNA obtained (GenBank
accession number AY028616) had an ORF of 921 bp
starting with an ATG codon at postion 42 and ending with
a TAA codon at position 962 (Fig 3) The predicted protein
is composed of 306 amino acids, with a calculated molecular
mass of 34.3 kDa and a pI of 5.72 Similar searches with the
deduced amino-acid sequence of full-length Calliphora
AFP, tested against the SwissProt database, showed a
93% pairwise amino-acid identity and 97% positivity with
the AFP of S peregrina, and, furthermore, a 75% identity and 85% positivity with the AFP of D melanogaster (GenBank accession number JC7250)
The presence of a stop codon at position 33 in the AFP cDNA ()9 from the start codon) explains why we were not able to isolate a full-length cDNA by two-hybrid screening
A stop codon at this position interrupts the synthesis of a two-hybrid fusion protein, if the full-lenth cDNA is ligated
in the library plasmid
Stage-specific and tissue-specific appearance of AFP
We tested the Sarcophaga antibody to AFP for its ability to recognize a similar protein in Calliphora by immunoblot analysis As shown in Fig 4, a 34-kDa protein was recognized in the anterior as well as the central, but not the posterior, part of the fat body (Fig 4B) A clear signal was also detected in the haemocytes The apparent molec-ular mass of the detected AFP band (34 kDa) corresponds well to that calculated from the amino-acid sequence (34.4 kDa)
Northern-blot analysis confirms the presence of strongly enriched AFP mRNA (1.2 kbp) in the anterior part of the fat body of last-instar larvae (Fig 5B) The mRNA was also present in pupae and adults (Fig 5A), as well as in haemocytes of last-instar larvae (Fig 5B)
The results obtained by immunoblot and Northern-blot analysis were confirmed by immunofluorescence (Fig 6A–C), in situ hybridization of cryosections (Fig 6D), and whole-mount in situ hybridization (Fig 6E,F) A sharp border could be detected between fluorescent cells of the anterior fat body lobe and nonfluorescent cells of the central lobe in the immunofluorescence experiment (Fig 6A) The whole-mount in situ hybridization revealed that the AFP mRNA transcription in the fat body is almost exclusively restricted to the anterior lobes in last-instar larvae (Fig 6E,F) Haemocytes were shown to express the AFP mRNA (Fig 6D) and to synthesize the AFP protein (Fig 6C)
Western blots, using an antibody that recognizes the receptor fragments ABP96, ABP64, P45 and P30, showed that the hexamerin receptor is present in all fat body fractions but not in the haemocytes (Fig 4A)
Fig 2 Immuno-coprecipitation of AFP and the Calliphora hexamerin receptor by antibodies to ABP and AFP demonstrated by Western blotting (A) Proteins were separated by SDS/PAGE (10% gel), transferred to membrane filters, and probed with a polyclonal anti-AFP IgG Fat body extract from 7-day-old larva (H) Fat body proteins after immunoprecipitation with hexamerin receptor antibody (anti-ABP IgG); proteins derived from posterior fat body (pF), or anterior fat body (aF) Controls: K1 ¼ aF, omitting anti-ABP IgG precipitation; K2 ¼ aF using anti-(LexA) IgG instead of anti-AFP IgG; K3 ¼ buffer instead of fat body homogenate The stained 34-kDa band represents AFP AB ¼ ABP or anti-LexA (K2), respectively (B) The separated proteins were probed with a polyclonal anti-ABP IgG aF ¼ proteins from anterior fat body after immunoprecipitation with anti-AFP IgG The stained 30-kDa band represents P30 AB ¼ anti-AFP Visualization of the bands was with a secondary anti-rabbit antibody coupled with alkaline phosphatase followed by NBT/BCIP colour reaction.
Table 1 Interaction of AFP with different fragments of the hexamerin
receptor (see Fig 1) in a two-hybrid experiment AFP binds to ABP130
and ABP96 but not ABP64 The hexamerin, arylphorin, used as a
positive control, binds to all receptor fragments.
Bait
Library plasmid
(Prey)
Reporter gene Leu2 lacZ
ABP130 Arylphorin + +
Trang 5D I S C U S S I O N
AFP, a binding partner of the hexamerin receptor
The fat body is the biochemically most active organ in
insects, with multiple functions such as metabolism of
proteins, carbohydrates and lipids, particularly blood
sugar and haemolymph proteins, such as vitellogenins
and hexamerins The fat body corresponds functionally, at
least in part, to the liver of vertebrates Therefore, this
insect organ is a highly suitable tissue for studies of the
stage-specific and tissue-specific expression of genes,
post-transcriptional regulation of RNA, and post-translational
control of protein biosynthesis One of the most detailed investigations of fat body proteins has been the metab-olism of the storage protein arylphorin, which belongs to the class of hexamerins [5–9] These proteins are synthe-sized in a stage-specific manner and reabsorbed by the fat body Hexamerin uptake has been shown to be due to receptor-mediated endocytosis As in all other dipteran insects investigated so far, the hexamerin receptor of
C vicinais subjected to threefold post-translational cleav-age, which succesively results in the active receptor involved in endocytosis The last cleavage step is initiated
by ecdysteroids, the hormone acting at the post-transla-tional level [7,9]
Fig 4 Tissue-specific appearance of AFP and the hexamerin receptor (A) Extracts of anterior (aF), central (cF), posterior (pF) fat body, hae-mocytes (H) and cell-free haemolyph (s) were analyzed by SDS/PAGE (8% gel) and probed with a polyclonal anti-ABP antibody using the BCIP/ NBT colour reaction The cleavage fragments (ABP96, ABP64, P45, P30) of the hexamerin receptor can be observed exclusively in the fat body but not in the haemocytes and haemolymph (B) Same protein samples as in (A) probed with an anti-AFP IgG Large amounts of the 34-kDa protein (AFP) can be detected in the anterior part of the fat body (aF); substantial less protein is found in the central (cF) fat body and no AFP in the posterior fat body (pF) and within the cell-free haemolymph (s) AFP can also be observed in the haemocytes (H).
Fig 3 Nucleic acid and deduced amino-acid sequences of the cDNA encoding Calliphora AFP The specific primers used for RACE PCR are underlined, and the additional N-terminal sequence obtained by 5¢ RACE is enclosed in shaded boxes Start and termina-tion codons are in bold letters, and the putative polyadenylation signal is double-underlined.
Trang 6Looking for binding partners of the hexamerin receptor,
we constructed and screened a cDNA library of C vicina
RNA from fat body by two-hybrid assays In addition to the
conversant interactors arylphorin and LSP-2, which belong
to the hexamerin family, we identified AFP as a strong
interactor The two-hybrid analysis and the results of the
immuno-coprecipitation revealed that AFP interacts with
P30 and with all cleavage products of the hexamerin
receptor that contain this peptide (ABP130, ABP96), whereas shorter N-terminal fragments of the receptor that
do not include P30 show no interaction with AFP (Table 1) Thus, three cleavage products are possible interactors in vivo: ABP130, ABP96 and P30 However, significant interaction
of AFP and peptides derived from the hexamerin receptor precursor can only take place in the anterior lobe of the fat body because of the large amounts of AFP in this tissue Expression of AFP inC vicina
As in almost all experiments dealing with protein expression and sequestration, these studies were performed using the entire fat body Here we show that AFP is exclusively expressed in the anterior pair of fat body lobes of last-instar larvae, and the median and posterior lobes appear to be free from AFP This region-specific expression pattern also resembles that reported for S peregrina [17] As the anterior part of the fat body is in contact with the ring gland, the ecdysteroid-producing organ, its function may be more under endocrine control than the central and posterior parts, which are provided with hormones circulating in the haemolymph
In addition to its expression in trophocytes of the anterior fat body, AFP was found to be present in another cell type Larval haemocytes contain substantial amounts of AFP mRNA (Fig 5B) and AFP protein (Fig 4B) As these cells never express the hexamerin receptor (Fig 4A), AFP must have different functions in the two cell types Cell-free haemolymph preparations were negative, indicating that AFP is not secreted into the haemolymph AFP does not contain a transmembrane transport signal peptide
Fig 5 Northern blot demonstrating the stage-specific and tissue-specific
appearance of AFP mRNA (A) Stage specificity of AFP mRNA
expression Total fat body RNA (20 lg) isolated from different
developmental stages was applied to each slot A digoxygenin-labeled
antisense AFP RNA probe was used with an alkaline
phosphatase-linked anti-digoxygenin IgG Hybridization resulted in a distinct band
at 1.2 kb RNA was derived from last-instar larvae (4–7: 4–7-day-old
larvae), prepupae (V), and pupae (P) Adult flies (Ad) do not show a
distinct band, indicating weak expression of AFP mRNA (B) Tissue
specificity of AFP mRNA expression Same probe as in (A) Total
RNA was prepared from anterior (aF), central (cF) and posterior (pF)
fat body, and haemocytes (H) of 7-day-old pupae The 1.2-kb signal
was detected in the anterior fat body and the haemocytes A light signal
only appears in the central fat body; no signal was obtained in the
posterior fat body.
Fig 6 Immunostaining and in situ hybridization of Calliphora fat body Longitudinal cryosections (10 lm) of 7-day-old larvae (A–C) were stained with a Cy2-conjugated goat anti-rabbit IgG after incubation with rabbit anti-AFP IgG (A) In the border region of anterior (aF) and central fat body (cF), AFP-immunostaining appears only in the anterior fat body (B) In a single fat body cell of the anterior fat body, AFP immunostaining is restricted to the cytoplasm (C) AFP immunostaining can also be found in the cytoplasm of haemocytes (D) In situ hybridization of longitudinal cryosections with a digoxygenin-labeled antisense AFP RNA probe shows no expression of AFP in muscle (m) and posterior fat body cells (pF) Haemocytes (hc) show high expression of AFP mRNA (E,F) Whole-mount in situ hybridization of upended 7-day-old larvae The anterior fat body exhibits strong expression of AFP mRNA, whereas only weak expression is seen in the central fat body (cF) and no expression in the posterior fat body (pF) or the brain The white bars indicate 50 lm, and the black bars indicate 250 lm.
Trang 7The possible function of AFP
Nothing is known about the function of AFP to date Its
amino-acid sequence contains no conversant domains that
suggest a function In contrast with the mammalian liver
protein, regucalcin, which is assumed to be derived from a
common ancestral gene, AFP has been shown to have no
calcium-binding activity in S peregrina [17] and is
upregu-lated in adult D melanogaster reared at low temperatures
[22]
The interaction of AFP and the hexamerin receptor,
shown in this paper, gives a first clue to a possible function of
this protein From our data, we conclude that it may be
involved in endocytosis of hexamerin by interacting with the
receptor As mentioned above, this molecular interaction can
only occur in the anterior fat body, a tissue known to contain
fewer protein storage granules, particularly fewer hexamerin
storage particles (R Marx, personal communication), than
the central and posterior parts [16] and which rapidly
disintegrates shortly after pupariation [17] We speculate
that, because of the interaction with AFP, most of the
hexamerin receptor is inactivated in the anterior fat body
preventing uptake of storage protein in this part of the tissue
This study opens the way to further experiments in two
distinct areas On the one hand, the binding domains of
AFP and the hexamerin receptor could be mapped in detail
by functional dissection using truncated prey proteins in
two-hybrid experiments On the other hand, the use of
antibodies against AFP in in vitro and in vivo experiments
investigating hexamerin uptake by the anterior fat body
may give insights into the nature of the interactions
described above Such approaches could lead to a better
understanding of the regulation of endocytotic uptake in the
insect fat body and beyond It is possible that hexamerin
endocytosis in insects does not follow the standard scheme
of eukaryotic endocytosis
A C K N O W L E D G E M E N T S
This work was supported by a grant from the Deutsche
Forschungs-gemeinschaft (Sche 195/13) We are indebted to Dr Nakajima for the
gift of antibodies against AFP We thank Anneliese Striewe-Conz and
Dieter Dudaczek for competent technical assistance.
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