In the shrimp, Metapenaeus ensis, FAMeT mRNA is expressed throughout ovarian maturation in the nerve and eyestalksuggesting a possible role of FAMeT in the regulation of reproduction [18
Trang 1Function and cellular localization of farnesoic acid
Y I N Silva Gunawardene1, S S Tobe2, W G Bendena3, B K C Chow1, K J Yagi2and S.-M Chan1 1
Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong, China;2Department of Zoology,
University of Toronto, Ontario, Canada;3Department of Biology, Queen’s University, Kingston, Ontario, Canada
The isoprenoid methyl farnesoate (MF) has been
impli-cated in the regulation of crustacean development and
reproduction in conjunction with eyestalkmolt inhibiting
hormones and ecdysteroids Farnesoic acid
O-methyl-transferase (FAMeT) catalyzes the methylation of
farne-soic acid (FA) to produce MF in the terminal step of MF
synthesis We have previously cloned and characterized
the shrimp FAMeT In the present study, recombinant
FAMeT (rFAMeT) was produced for bioassay and
anti-serum generation FAMeT is widely distributed in shrimp
tissues with the highest concentration observed in the
ventral nerve cord Interestingly, an additional larger
protein in the eyestalkalso showed immunoreactivity to
anti-FAMeT serum FAMeT was localized in the
neuro-secretory cells of the X-organ-sinus gland complex of the
eyestalk As shown by RT-PCR, FAMeT mRNA is
constitutively expressed throughout the molt cycle in the
eyestalkand the ventral nerve cord To show that our cloned gene product had FAMeT activity, we demon-strated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition
to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalkneuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland
Keywords: shrimp; farnesoic acid O-methyltransferase; methyl farnesoate; neuropeptides; juvenile hormone
Methyl farnesoate (MF), the sesquiterpenoid precursor of
the insect juvenile hormone III (JH III), is produced and
released by the mandibular organs of decapod crustaceans
[1] The physiological function for MF is not well
understood in crustaceans However, by analogy to the
established functions of JH III in insects, MF has been
suggested to play an important role in the regulation of
growth and reproduction in crustaceans [2–4] In some
crustaceans, circulating titer and biosynthesis of MF
appear to be positively correlated with the maturation of
the ovary [5,6] MF has also been suggested to play a role
in delaying the onset of molting in larval crustaceans [7,8]
This evidence implicates MF in both crustacean growth
and reproduction
Farnesoic acid O-methyltransferase (FAMeT; also
known as S-adenosyl-methionine:farnesoic acid
O-methyl-transferase) is the enzyme that catalyses the final step in
the MF biosynthetic pathway in crustaceans [9,10] FAMeT, also present in the insect corpora allata, carries out the methylation of farnesoic acid (FA) to yield MF using the cofactor S-adenosyl-L-methionine [9,11] Varia-tions in activity of the O-methyltransferase during devel-opment appear to be an important component in the regulation of JH biosynthesis in insects [10,12] The rate-limiting step controlling JH III biosynthesis in the cock-roach larva is catalyzed by FAMeT [13] Neuropeptides released from the eyestalkX-organ sinus gland complex appear to modulate the MF synthesis in the crustaceans similar to the allatostatins repressing JH biosynthesis by the corpora allata in insects [14–16] This negative regulation of MF biosynthesis occurs in part through the inhibition of FAMeT activity [9,17] Furthermore, studies in insects have also demonstrated that allatostatins are able to suppress the production of JH by modulating O-methyltransferase and/or epoxidase activity [10] Thus,
in both insects and crustaceans, O-methyltransferase plays
a role in the regulation of MF synthesis and thereby mediates an effect on vitellogenesis and metamorphosis of these animals through MF In the shrimp, Metapenaeus ensis, FAMeT mRNA is expressed throughout ovarian maturation in the nerve and eyestalksuggesting a possible role of FAMeT in the regulation of reproduction [18] Coincidentally, the high levels of FAMeT mRNA tran-scripts and protein in the eyestalkand the ventral nerve cord in shrimp parallels the expression of some neuro-peptides of the crustacean hyperglycemic hormone (CHH)/gonad inhibiting hormone (GIH)/ molt inhibiting hormone (MIH) family [18,19]
Correspondence to S M Chan, Department of
Zoology, The University of Hong Kong, Pokfulam Road,
Hong Kong, China Fax: + 852 2299 0864,
E-mail: chansm@hkucc.hku.hk
Abbreviations: MF, methyl farnesoate; FAMeT, Farnesoic acid
O-methyltransferase; FA, farnesoic acid; JH III, insect juvenile
hormone III; IPTG, isopropyl thio-b- D -galactoside; CHH,
crustacean hyperglycemic hormone; MIH, molt inhibiting
hormone; GIH, gonad inhibiting hormone.
(Received 7 March 2002, revised 4 June 2002,
accepted 17 June 2002)
Trang 2To understand the role of MF in the growth and
reproduction of crustaceans, considerable effort has been
invested in studying the regulation of the biosynthesis of
MF [1,4,6] Although FAMeT has been implicated as a
potential point in the regulation of MF biosynthesis, the
biochemical and biological properties of the gene encoding
the FAMeT have not been characterized Thus, the study of
the enzymes of the MF biosynthetic pathway will enable us
to better understand the role of MF in shrimp as well as its
probable structural and functional relationship with the
crustacean neuropeptides We have previously reported on
the expression, cloning and characterization of the cDNA
encoding the FAMeT in the shrimp Metapenaeus ensis [18]
In the lobster, putative FAMeT clones have also been
isolated (GenBankno U25846 and GenBankno
AF249871) but the function of these genes has not yet been
demonstrated Database searches also suggest that a
homologue for this gene exists in the Drosophila genome
[18] Although there is a report on the cytosolic nature of
O-methyltransferase in the locust corpora allata, the cellular
localization/distribution of the enzymes of the MF
biosyn-thetic pathway remain unidentified [11] In, this study, we
expressed recombinant (r) FAMeT in bacteria and
deter-mined its biological function by a radiochemical assay
Antiserum raised against rFAMeT was used to localize
native FAMeT at the cellular level
E X P E R I M E N T A L P R O C E D U R E S
Construction of the shrimp FAMeT expression plasmid
The DNA fragment encoding the FAMeT coding sequence
(nucleotides 75–917; GenBankaccession no AF 333042)
was amplified using the primers FAOMX-F4(forward: 5¢-C
CGGGATCCATGGCTGACAACTGGCCTGCC-3¢) and
FAOMX-R6(reverse: 5¢-CGGGGTACCTTAGAATTCG
AACTTCCACTT-3¢) by PCR Following amplification the
cDNA fragment was digested with BamHI and KpnI and
ligated into the BamHI, KpnI-digested vector pREST-A
(Invitrogen, Groningen, the Netherlands) The ligated
product (pREST-A/rFAMeT) was transformed into E coli
XL1-blue for DNA sequence determination The pREST-A/
rFAMeT construct was transformed into BL21(DE3) cells
for protein expression Overnight culture of a single colony
was diluted (1 : 200) with Luria–Bertani medium and
incubated at 37C with vigorous shaking to an D600 of
0.3–0.5 IPTG was added at a final concentration of 1 mM
to induce the protein expression for a period of 4 h The
protein expression and cell growth was monitored by taking
1 mL of the bacterial culture at 1-h intervals The protein
extract was analyzed by 12% SDS/PAGE and Western blot
analysis using Ni-nitrilotriacetic acid–alkaline phosphatase
conjugate or mouse anti-histidine IgG as the first antibody
and a goat anti-mouse IgG–alkaline phosphatase conjugate
as the secondary antibody
Purification of rFAMeT
Bacteria were pelleted by centrifugation and resuspended
in 15 mL binding buffer (20 mM Tris/HCl pH 7.9,
0.5 mM NaCl and 5 mM imidazole) The cells were
homogenized with a Polytron and centrifuged at 5700 g
for 15 min The pellet was suspended in 15 mL denatured
binding buffer (8M urea in binding buffer) and agitated for 2 h at room temperature Following centrifugation as above, the supernatant was collected and loaded onto a Ni-nitrilotriacetic acid–agarose (Qiagen, Hilden, Germany) affinity column (pre-equilibrated with denatured binding buffer) Denatured binding buffer was used to wash the column until zero absorbency (280 nm) was observed in the eluate The fusion protein was eluted with an elution buffer (6 mMTris/HCl, pH 7.9, 0.15MNaCl and 300 mM imidazole, 8M urea) and dialyzed in a buffer (0.01% SDS, 0.1% Tween 20, 0.1· NaCl/Tris (0.8 g NaCl, 0.02 g KCl and 0.3 g Tris base in 1 L of water, pH 7.4) at
4C overnight A second and a third dialysis were performed for 10 h in 0.1· NaCl/Tris and 0.1 · NaCl/Pi (0.8 g NaCl, 0.02 g KCl, 0.144 g Na2HPO4 and 0.024 g
KH2O4 in 1 L of water, pH 7.4), respectively A protein assay kit (Bio-Rad) was used to determine the protein concentration
Production of anti-rFAMeT serum Purified rFAMeT (50 mgÆmL)1 in NaCl/Pi) was mixed with equal volume of complete Freund’s adjuvant (Gibco)
by homogenization with a Polytron The emulsion was injected into a New Zealand white rabbit subcutaneously
at three different sites During the injections and subse-quent handling of the animal, principles of laboratory animal care and specific national laws were followed The rFAMeT used for the second and third booster injections were mixed with an equal volume of incomplete Freund’s adjuvant The second and third injections (100 lg) were given at 10-day intervals A weekafter the third injection, the antibody titer was monitored by SDS/PAGE and Western blot analysis The anti-rFAMeT serum was prepared from the rabbit whole blood by centrifugation
at 800 g for 15 min and the supernatant was stored
at)80 C in aliquots
Detection of FAMeT in shrimp tissue protein extracts Shrimp (8–10 g) were purchased from a local sea food market and immediately transferred to sea water aquaria held at ambient photoperiod and temperature The molt staging of the animals was performed by pleopod setogenesis [20] and the female reproductive stage was determined by the gonadal-somatic index Different tissues from the shrimps were dissected under NaCl/Pi and homogenized in 1 mL per 50 mg (wet tissue weight)
of homogenization buffer (0.1MNaCl, 0.05MTris, 0.1% Tween-20, 1 mM phenylmethanesulfonyl fluoride and
1 lgÆmL)1 aprotinin; pH 7.8) The homogenate was centrifuged at 3000 g for 5 min The supernatant was collected and centrifuged at 17 000 g for 40 min This successive supernatant containing the total protein extract was analyzed by 10% SDS/PAGE and Western blot detection with anti-rFAMeT serum as the primary antibody (1 : 20 000) and the goat anti-rabbit IgG– alkaline phosphatase conjugate as the second antibody (1 : 5000) The negative control was detected with preimmune rabbit serum (1 : 1000) as the primary antibody To test for specificity of the anti-rFAMeT serum duplicate Western blots of shrimp protein extracts were detected with anti-rCHH and anti-rMIH serum [23]
Trang 3Signals were visualized by adding
5-bromo-4-chloro-3-indolyl phosphate/Nitro Blue tetrazolium/alkaline
phos-phatase detection buffer and the colour reaction was
terminated with running tap water Protein concentration
in the shrimp extracts was determined using a Bio-Rad
protein assay kit
Immunocytochemical detection of FAMeT
in shrimp eyestalk
Different tissues from the shrimp were dissected under
cold NaCl/Pi The rigid cuticle containing most of the
retina and the lamina layer of the eyestalkwas removed
and the tissues were immediately fixed in Bouin’s fixative
at 4C for 24 h, dehydrated and embedded in paraffin
Consecutive 7-lm sections were mounted onto slides and
dried at 37C overnight Tissue sections were
deparaf-finized in xylene (twice for 5 min) and rehydrated
through an ethanol series The endogenous peroxidase
was removed by incubating the tissue sections in 0.3%
H2O2 in methanol for 30 min at room temperature and
rinsed briefly with NaCl/Pi The immunocytochemical
staining of the tissue sections were performed with a
Vectastain Elite ABC kit (Vector Laboratories, CA, USA)
according to the manufacturers instructions Tissue
sec-tions were blocked in normal blocking serum (provided
with the kit) for 30 min at room temperature After
blotting the excess blocking serum, the sections were
incubated in the anti-rFAMeT serum (1 : 3000 dilution in
NaCl/Pi buffer) overnight at 4C The control sections
were incubated in preimmunized rabbit serum (1 : 400
dilution) The slides were washed with a large volume of
NaCl/Piunder gentle agitation twice for 5 min each and
incubated in a biotinylated secondary antibody solution
(diluted according to the manufacturers instructions) for
30 min at room temperature After the washing of the
slides for 5 min in a large volume of NaCl/Pi, the
sections were incubated for 30 min with Vectastain Elite
ABC reagent according to the manufacturers instructions
The slides were washed with NaCl/Pi for 5 min and
incubated in the peroxidase substrate solution containing
0.01% H2O2 and 1.39 mM 3,3¢-diaminobenzidine (Sigma,
St Louis, MO, USA) The colour reaction was terminated
by rinsing the slides in running tap water The sections
were dehydrated through increasing concentrations of
ethanol, cleared with xylene and mounted with coverslips
in DPX mountant (Sigma)
RT-PCR and Southern blot detection of FAMeT
Nervous tissue and eyestalks were extracted for total
RNA [21] RNA quality was monitored by agarose gel
electrophoresis The first strand cDNA was synthesized by
reverse transcription in a buffer containing 1–5 lg total
RNA, 2 pmol of gene specific primer, 2 mM dNTP mix,
2.5 mM MgCl2 and 1 U of Superscript II reverse
tran-scriptase (Life Technologies, USA) at 42C for 3 h A
pair of primers (forward: FAOM F45¢-CCGGGATCCA
TGGCTGACAACTGGCCTGCC-3¢; reverse: FAOM R6
5¢-CGGGGTACCTTAGAATTCGAACTTCCACTT-3¢)
PCR mix consisted of 2 lL of the RT reaction mix, 2 mM
MgCl2, 2 mM of dNTP, 1 U of Taq DNA polymerase
and 10 pmol of each primer in 1· PCR buffer PCR
included denaturation at 95C for 1 min, annealing at
55C for 1 min and extension at 72 C for 1 min for 35 cycles and an extension at 72C for 10 min for the completion of the DNA synthesis PCR products were analyzed on a 1% agarose gel and blotted onto a nylon membrane (Amersham) and cross-linked with UV irradi-ation The membrane was hybridized overnight at 42C
to a DIG-labeled nonradioactive cDNA probe of 843 bp
in a buffer (5· NaCl/Cit, 0.5% SDS, 5 · Denhardt’s solution and denatured salmon sperm DNA (100 lgÆmL)1) with 50% formamide) The membrane was then washed twice in 2· NaCl/Cit with 0.1% SDS for 5 min at room temperature and finally at 68C in 0.5· NaCl/Cit containing 0.1% SDS twice for 15 min Positive signals were detected with DIG–alkaline phosphatase alkaline conjugate by adding Nitro Blue tetrazolium (5%) and 5-bromo-4-chloro-3-indolyl phos-phate (5%) to the detection buffer (0.1M Tris/HCl,
pH 9.5, 0.1M NaCl)
Enzyme preparation Transformed bacteria BL21 (DE3) containing the expres-sion plasmid construct was cultured as stated above Following induction with isopropyl thio-b-D-galactoside (IPTG) the bacterial culture was grown at 37C with shaking for 2.5 h The bacterial cells were pelleted by centrifugation at 800 g for 20 min The bacteria pellet was then resuspended in NaCl/Pi buffer containing 1 mM phenylmethanesulfonyl fluoride and 1 lgÆmL)1 aprotinin and the cells lysed by a Polytron homogenizer Following centrifugation at 10 000 g for 30 min at 4C, the superna-tant was used to test for O-methyltransferase activity The BL21 (DE3) with expression vector only was used as a negative control to checkfor any bacterial O-methyltrans-ferase activity
Methyltransferase assay The O-methyltransferase activity of rFAMeT was assayed according to the procedure of Reibstein & Law and Feyereisen et al [11,22] with some modifications The enzyme preparations were incubated in a final volume of
100 lL of 100 mM phosphate buffer (pH 7.2, containing
1 mM EDTA, 1 mM 2-mercaptoethanol and 1% BSA) with 0.4 lM [3H]S-adenosyl-L-methionine (SAM), 30 lM farnesoic acid and 1.0 mM unlabeled SAM for 45 min at
37C The reaction was stopped by the addition of
200 lL methanol and 100 lL 1% Na-EDTA Cold carriers (20 lg JH III and 20 lg MF) were added to the reaction mixture prior to extraction with chloroform (2· 750 lL) After passing the chloroform phase through anhydrous Na2SO4, the extracts were dried under N2, redissolved in diethyl ether and applied to TLC plates (Mercksilica gel 60 F254 plastic sheet) The samples were then focused twice with methanol After development in a solvent system of toluene/ethyl acetate/acetic acid (85 : 15 : 1, v/v/v), the bands corresponding to MF and
JH III were cut out and assayed for radioactivity by liquid scintillation spectrometry The absolute enzyme activity of rFAMeT was obtained by subtracting background and bacterial activity from the above results of the rFAMeT assay
Trang 4R E S U L T S
Bacterial expression of the recombinant FAMeT
The rFAMeT protein was detected by using either the
Ni-nitrilotriacetic acid–alkaline phosphatase conjugate or
the mouse anti-(His-tag) IgG [23] At different time points
(0–4 h) after induction with IPTG, the recombinant protein
of the expected size was detected on Western blots of the
protein samples obtained from the cell pellets (Fig 1A, II)
The maximal expression of the rFAMeT in E coli was
observed 2.5 h after the IPTG induction (Fig 1A, I,II)
Furthermore, the expression of rFAMeT in the positive
expression constructs is specific because no positive bands
were detected in the bacterial controls (Fig 1A, II) The
molecular mass of the detected rFAMeT band was
35 kDa, which was the expected size of the fusion protein
Purification of rFAMeT and production
and specificity of anti-rFAMeT serum
Due to the presence of expressed rFAMeT in inclusion
bodies, the protein remained insoluble following lysis of the
bacterial pellet At high concentrations of denaturing
reagents, such as urea and guanidine thiocyanate, most of
the inclusion bodies were soluble in solution Western blot
analysis at different steps of the purification process
indicated that this method enables the complete purification
of rFAMeT with no contaminants (data not shown) To
renature the protein following elution, the protein was first
dialyzed in NaCl/Tris to remove urea and imidazole and the
protein was further dialyzed against NaCl/Tris and NaCl/Pi
to remove the detergent The yield for rFAMeT in the
E coli pRSET expression system was approximately
3 mgÆL)1 Antiserum against Ni-nitrilotriacetic acid affinity-purified rFAMeT was raised by injecting rabbits with the renatured protein The titer and specificity of the antiserum were determined by Western blot analysis (Figs 1B, I,II) The results indicated that the antiserum was highly specific as it detected only the rFAMeT in the cell lysate (Fig 1B, II) with a dilution of 1 : 160 000
Tissue distribution and immunolocalization
of native FAMeT in shrimp The results of Western blot analysis indicated the presence
of FAMeT in the ventral nerve cord, epidermis, eyestalk, heart, tail muscle, mandibular organ, ovary and testis (Fig 2A,B) The strongest signal was observed for the ventral nerve cord whereas no positive signals were detected for the hepatopancreas and the mid gut (Fig 2A,B) The molecular mass (32 kDa) of the protein was in accordance with that predicted from the amino-acid sequence deduced from the cDNA [18] In addition, a larger protein of approximately 44 kDa unique to the eyestalk was detected
in both the male and the female shrimps (Fig 2A,B) To determine whether the distribution of the FAMeT varied between adult and juvenile shrimp, total protein of the eyestalkand ventral nerve cord of the juvenile shrimps were analyzed The results indicate that FAMeT is present in both developmental stages in the eyestalkand the ventral nerve cord (Fig 2C) Furthermore, the larger protein (i.e
44 kDa) was also observed in the juvenile eyestalk (Fig 2C)
Fig 1 SDS/PAGE and Western blot analysis of the expression of rFAMeT protein in pREST-A/BL21 (DE3) (A) and titering of rabbit antiserum against rFAMeT protein by Western Blot (B) (A) (I) Cell lysates were prepared at different induction time points and proteins analyzed on a 12% SDS/PAGE that was stained with Commassie Blue (II) A 1 : 1000 dilution of the Ni-nitrilotriacetic acid–alkaline phosphatase conjugate was used
in the Western blot analysis to detect the His-tag rFAMeT fusion protein The numbers on top indicate the induction time All lanes show the soluble fraction except 4P, which indicate the insoluble fraction of the lysed cell pellet The lysate of only bacteria BL21 (DE3) or pREST-A vector transformed BL21 (DE3) were used as controls (B) (I) 200 ng of rFAMeT proteins (lanes 1 and 4–10) and whole bacterial lysate (lane 2) were analyzed by 12% SDS/PAGE and transferred to Hybond-C membrane (II) Individual lanes were immunostained with different dilutions of rabbit antiserum against rFAMeT PM, prestained standard protein marker The last lane was the Western blot with preimmune rabbit antiserum.
Trang 5Immunocytochemistry of serial sections of the eyestalk
revealed the presence of the FAMeT in the sinus gland, the
X-organ neurosecretory cell clusters together with the
lamina ganglionaries and the nerve fibers of the three
ganglia of the eye: the medulla externa, medulla interna and
the medulla terminalis (Fig 3A) The sinus gland, located
between the medulla externa and interna of shrimp eyestalk
show strong immunoreactivity to anti-rFAMeT serum
(Fig 3A) The neurosecretory cells and the laminar
struc-ture of the onion bodies of the X-organ [24] show the
highest immunoreactivity to the anti-rFAMeT serum in the
M ensiseyestalk(Fig 3B)
Expression of the FAMeT at different molting stages The expression of the FAMeT mRNA during the molt cycle was analyzed to ascertain whether the expression of FAMeT gene is related to the molting cycle in the shrimp Because of the limited quantity of RNA obtained from ventral nerve cord and eyestalkof a single shrimp, RT-PCR was employed
to study the expression of FAMeT The results revealed that FAMeT mRNA is expressed throughout the intermolt (stage C), premolt (stage D) and postmolt (stages A, B) stages in both adult and the juvenile shrimp in the eyestalktissue (Figs 4A,B) Similarly, FAMeT is also expressed in the ventral nerve cord of both juvenile and adult animals during all stages of the molt cycle (Fig 4C,D) However, there is no significant variation in the level of expression in the ventral nerve cord and the eyestalkduring the molt cycle Further analysis indicated that the FAMeT mRNA is expressed in adults of both sexes throughout the molting cycle (Fig 4B) However, in the eyestalkof juvenile premolt animals the FAMeT mRNA is expressed only in some shrimps (Fig 4A)
To further confirm the RT-PCR results, a Southern hybrid-ization was carried out on the RT-PCR products with a cDNA-specific probe (Fig 4A–D, III) and b-actin RT-PCR amplification was used as an internal control to show the integrity of the RNA samples (Figs 4A–D, II)
Fig 2 Western blot analysis of proteins from different tissues of the
M ensis (A) Detection of FAMeT in M ensis Lanes are protein
samples from different tissues of the Female shrimp (B) Detection of
M ensis FAMeT native protein in different tissues of the male shrimp.
The last lane indicates protein sample from testis (C) Detection of
M ensis FAMeT native protein in ventral nerve cord and eyestalk
tissues of the juvenile shrimp.
Fig 3 Cellular localization of FAMeT in shrimp (A) Immunolocal-ization of FAMeT in eyestalksections (B) Higher magnifications (see rectangle in A) XO, X-organ; SG, sinus gland; ME, medulla externa;
MI, medulla interna; MT, medulla terminalis; LAM, lamina gangli-onaris; NGF, nerve and glial fibers; AT, axonal tract; ON, optic nerve;
OB, onion bodies of the X-organ; NSC, neurosecretory cells of the X-organ.
Trang 6Radiochemical assay for the functional analysis
of rFAMeT
To obtain a large quantity of FAMeT for the functional
assay, we expressed the rFAMeT in a pRSET bacterial
expression system The O-methyltransferase assay was
carried out with the rFAMeT to checkfor presence of
enzyme activity As shown in Fig 5, MF production
increased in relation to the increasing amounts of rFAMeT
However, when the protein quantity was increased beyond
17.5 lg, the enzyme activity was relatively constant (Fig 5)
The data shown in Fig 5 indicated the absolute enzyme
activity for each of the samples following the subtraction of
bacterial or background activity
D I S C U S S I O N
The insect JH-related compound MF is produced and
released by the mandibular organ of crustaceans [5,25,26]
MF is thought to be involved in both reproduction and molting in the crustaceans [5,7] FAMeT catalyzes the methylation of FA to MF as the terminal step in the MF biosynthetic pathway Changes in O-methyltransferase activity during development in insect corpora allata have been suggested to be important components in the regula-tion of JH biosynthesis [9] Hence, the present study was carried out to determine the biochemical function of the shrimp FAMeT gene and cellular distribution of native FAMeT enzyme, in an attempt to investigate the biochemi-cal and biologibiochemi-cal properties and the regulation of MF production in M ensis Using the bacterial expression system, a recombinant protein of35 kDa was produced (Fig 1A, II) This protein is 3 kDa larger than the predicted molecular mass of the FAMeT protein (32 k Da) as a result
of the addition of several amino acids at the N-terminal end
of the fusion protein The rFAMeT was purified through an affinity column with the aide of the series of His6-tag residues in the fusion protein, which function as a metal binding domain The His6-tag residues further assist the subsequent analysis of the purification by Western detection
of the fusion protein with Ni-nitrilotriacetic acid conjugate The results from the Western blot indicate the anti-rFAMeT serum specifically detected rFAMeT in the cell lysate demonstrating the high specificity of the antiserum (Fig 1B, II) and the wide distribution in nature of the FAMeT in shrimp (Fig 2) The anti-rFAMeT serum detects the highest level of the FAMeT in ventral nerve cord tissue, whereas FAMeT level is lower in the epidermis, eyestalk, heart, tail muscle, mandibular organ, ovary and testis (Figs 2A,B) This observation agrees with the results of our earlier study on the expression of the FAMeT mRNA [18] However, in contrast to results from RT-PCR [18], FAMeT
is not present in the hepatopancreas by Western blot analysis (Figs 2A,B) Northern blot analysis [18] suggests that FAMeT mRNA level is much lower in hepatopancreas than ventral nerve cord, tail muscle or testes and thus, FAMeT expression in this tissue may be below the sensitivity of this assay Alternatively, FAMeT may be
Fig 5 Methyl farnesoate production by different quantities of
rFA-MeT Each point represents the mean ± STDEV of five
determina-tions.
Fig 4 RT-PCR detection of the FAMeT transcripts at different molting stages in
M ensis eyestalk (ES) and ventral nerve cord (VNC) (A) Expression of the FAMeT of
M ensis at different molting stages in the juvenile eyestalk (B) Expression of the FAMeT of M ensis in the eyestalkat different molting stages in the adult males and females (C) Expression of the FAMeT of M ensis at different molting stages in the adult ventral nerve cord (D) Expression of the FAMeT of
M ensis at different molting stages in the juvenile ventral nerve cord (A–D, I), RT-PCR detection of M ensis FAMeT transcripts at different molting stages of the shrimp; (A–D, II), RT-PCR detection of the shrimp b-actin gene using gene-specific primers; and (A–D, III) Southern blot analysis of FAMeT RT-PCR products with cDNA coding region probe.
Trang 7secreted from the hepatopanceas However, extensive work
will be required to determine the definite role of FAMeT in
the shrimp hepatopancreatic tissue Surprisingly, the
man-dibular organ elicits a weaksignal for FAMeT in
compar-ison to ventral nerve cord and other tissues (Fig 2A,B)
This suggests that the last step of MF biosynthesis catalysed
by FAMeT enzyme occurs in many tissues FA has been
implicated as a major product of release from the
mandib-ular organ in the crustaceans [26] Similar reports have also
been documented in the insects where the JH-acids released
from the corpora allata are converted to the corresponding
JHs in the male accessory reproductive glands [27] The wide
distribution of the FAMeT in different shrimp tissues
further confirms our earlier speculation that the major part
of the FA is secreted from the mandibular organ and
transported to target tissues for final conversion to MF [18]
However, it is also possible that FAMeT methylates
substrates other than FA in tissues apart from mandibular
organ In M ensis, FAMeT is also found in the juvenile
ventral nerve cord and eyestalktissues (Fig 2C) In insects,
O-methyltransferase activity in the corpora allata of the
D punctatahas been reported to be high during the early
stages of development suggesting a possible regulatory role
for O-methyltransferase on JH biosynthesis at these stages
[10] Similarly, the presence of FAMeT in these juvenile
tissues suggests that this protein could be an important
component in the regulation of MF synthesis during the
early developmental stages of the shrimp
FAMeT was localized in the neurosecretory cells of the
X-organ sinus gland complex by immunocytochemical
detection (Fig 3A) The X-organ-sinus gland complex, a
neurohemal organ of decapod crustaceans, is the site of
synthesis, storage and release of the neuropeptides of the
CHH/MIH/GIH family [19,28] We have previously shown
that expression of the FAMeT in the eyestalkand ventral
nerve cord coincides with the expression of some of these
eyestalkneuropeptides [18,19] Eyestalkneuropeptides of
the CHH/MIH/GIH family are also located in the
neu-rosecretory cells of the X-organ-sinus gland [19,23]
More-over the results from previous studies indicate that in the
crab, the biosynthesis of MF in the mandibular organ is
regulated by an eyestalkneuropeptide, mandibular organ
inhibiting hormone [9,16,17] Interestingly, a protein of a
larger molecular mass ( 44 kDa) was also detected in the
eyestalkof both the adult and juvenile M ensis (Fig 2A–
C) Analysis of FAMeT for protein modification signatures
(PROSITE) suggest that modification is unlikely to be
responsible for the shift in protein size observed Therefore,
the occurrence of the larger protein in the eyestalkmay be
attributable to binding of FAMeT with the eyestalkfactors
related to MF biosynthesis in the shrimp Another
alterna-tive is that the antiserum is simply recognizing in eyestalk
tissue, another protein with similar antigenic determinants
However, further studies are required to ascertain the
significance of this larger protein
Molting, a prerequisite for growth in arthropods, is under
direct control of neurohormones in the Crustacea [29,30]
There is also evidence supporting a regulatory role of MF in
the molting process of the crustaceans [31–33] Therefore, to
ascertain the expression of FAMeT in relation to the
molting process of the shrimp, we determined the level of
expression of this gene during the molt cycle In general, the
circulating hemolymph ecdysteroid titer remains low during
intermolt, increases rapidly during premolt and drops precipitously prior to ecdysis in crustaceans [20,34,35] According to our observations, FAMeT mRNA is expressed in the ventral nerve cord and eyestalkthroughout the molt cycle in both the adult and juvenile shrimp (Fig 4A–D) Furthermore, analysis of FAMeT mRNA in the eyestalkof the adult shrimp from both sexes gives similar results (Fig 4B) Because MF appears to be involved in the regulation of molting, the expression of FAMeT mRNA throughout the molting cycle is required for the synthesis of MF We have observed that FAMeT is expressed in a sex-specific manner in the juvenile shrimp [18] Therefore the differential expression of FAMeT during the premolt stage in juveniles (Fig 4A) is likely to be related
to sex-specific expression in juvenile shrimps
Our data from the assay of rFAMeT demonstrates that the enzyme is able to produce a significant quantity of MF (Fig 5) This indicates that the FAMeT gene codes for a functional farnesoic acid O-methyltransferase in M ensis and is the first demonstration of the biological function of this gene The refolding of the protein during renaturation is important for the function of the enzyme When we used recombinant protein renatured following purification under denaturing conditions, a very low enzyme activity was observed This is presumably a consequence of the incom-plete refolding of the fusion protein However, when we use
a partially purified rFAMeT extract in the subsequent functional assays a significantly higher level of O-methyl-transferase activity was observed (Fig 5) Because the enzyme activity attributable to the bacterial proteins or the vector proteins (background very low and insignificant) were subtracted from the final data of the rFAMeT enzyme activity, the data shows the absolute level of O-methyl-transferase activity of the recombinant protein Although the activity of rFAMeT is comparatively low when com-pared to the native O-methyltransferase activity of crusta-ceans and insects, the actual amounts of FAMeT in these studies were unknown [10,25,26] Moreover, it is well known that bacterial expression systems commonly fail to process the disulfide bonds and the correct refolding as a consequence of the lackof post-translational modifications
in prokaryotes Therefore, recombinant proteins from bacterial systems usually have much lower activity than the native proteins This has been observed in other recombinant proteins of the shrimp and also in other recombinant proteins expressed in bacteria [19,36,37] Hence, only a small fraction of the rFAMeT was expected
to fold in the correct conformation and consequently low FAMeT activity in the functional assay
In conclusion, the wide tissue distribution of the FAMeT
in shrimp implies that this gene may also be involved in other physiological processes apart from its role in repro-duction and metamorphosis Further, the specific cellular localization of FAMeT in the X-organ-sinus gland complex suggests that FAMeT may interact with the eyestalk neuropeptides and thereby regulate the synthesis and role
of MF All this suggests that FAMeT is a key regulatory enzyme in the MF biosynthetic pathway of the shrimp In addition, the occurrence of FAMeT mRNA throughout the molting cycle suggests that FAMeT may be involved in molting or related processes in the shrimp Finally, the present workprovides a frameworkfor study of the regulation of biosynthesis and function of MF by FAMeT
Trang 8and its mode of interaction with eyestalkneuropeptides to
establish definitive roles of MF in shrimp and all
crusta-ceans
A C K N O W L E D G E M E N T S
This workwas supported by a RGC earmarked grant (# HKU 7227/
00M) awarded to S M C by the Hong Kong SAR Government and
by operating grants from the Natural Sciences and Engineering
Research Council of Canada Y S G is the recipient of a Presidential
scholarship from the Sri Lankan Government and the J G Phillips
Memorial Scholarship from the University of Hong Kong.
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