Molecular cloning, expression and characterization of cDNAA key factor participating in natural rubber biosynthesis Kasem Asawatreratanakul1,2, Yuan-Wei Zhang1,*, Dhirayos Wititsuwannaku
Trang 1Molecular cloning, expression and characterization of cDNA
A key factor participating in natural rubber biosynthesis
Kasem Asawatreratanakul1,2, Yuan-Wei Zhang1,*, Dhirayos Wititsuwannakul3, Rapepun Wititsuwannakul4, Seiji Takahashi1, Atiya Rattanapittayaporn4and Tanetoshi Koyama1
1 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan; 2 Department of Chemistry, Thaksin University, Songkla, Thailand; 3 Department of Biochemistry, Mahidol University, Bangkok, Thailand; 4 Department of Biochemistry, Prince of Songkla University, Hat-Yai, Thailand
Natural rubber from Hevea brasiliensis is a high molecular
mass polymer of isoprene units with cis-configuration The
enzyme responsible for the cis-1,4-polymerization of
iso-prene units has been identified as a particle-bound rubber
transferase, but no gene encoding this enzyme has been
cloned from rubber-producing plants By using sequence
information from the conserved regions of cis-prenyl chain
elongating enzymes that were cloned recently, we have
iso-lated and characterized cDNAs from H brasiliensis for a
functional factor participating in natural rubber
biosyn-thesis Sequence analysis revealed that all of the five highly
conserved regions among cis-prenyl chain elongating
enzymes were found in the protein sequences of the Hevea
cis-prenyltransferase Northern blot analysis indicated that
the transcript(s) of the Hevea cis-prenyltransferase were
expressed predominantly in the latex as compared with other Heveatissues examined In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia colirevealed that the enzyme catalyzed the for-mation of long chain polyprenyl products with approximate sizes of 2· 103)1 · 104Da Moreover, in the presence of washed bottom fraction particles from latex, the rubber transferase activity producing rubber product of high molecular size was increased These results suggest that the Hevea cis-prenyltransferase might require certain activation factors in the washed bottom fraction particles for the pro-duction of high molecular mass rubber
Keywords: prenyltransferase; rubber transferase; Hevea brasiliensis; isoprenoid
Although over 2000 species of higher plants are recognized
for producing latex with polyisoprenes [1], only the rubber
tree (Hevea brasiliensis) has been established as a key
commercial rubber source due to its good yield of rubber
and the excellent physical properties of the rubber products
Hevearubber is a high molecular mass polymer of isoprene
units in cis-configuration Rubber molecules are produced
and aggregated or packaged as rubber particles in latex
vessels of the rubber tree [2] Although natural rubber is
synthesized and made almost entirely of isoprene units derived from isopentenyl diphosphate (IPP), an allylic diphosphate is also required as the priming cosubstrate to initiate the subsequent extensive prenyl chain elongation process for the formation of rubber macromolecules [3–5] Synthesis of the allylic prenyl diphosphates are catalyzed
by IPP isomerase and trans-prenyltransferase enzymes, the enzymatic activities of which were found in both the bottom fraction and the supernatant cytosol (C-serum) of centri-fuged fresh Hevea latex [6–8]
The enzyme responsible for cis-1,4-polymerization of isoprene units from IPP onto the allylic primer has been identified as a particle-bound rubber transferase (EC 2.5.1.20) [3,9,10] The particle-bound rubber transferase activities were demonstrated in various rubber producing plants; guayule [4,5], Ficus elastica [11] and Ficus carica [12]
It has been shown that IPP is incorporated into rubber at the surface of the rubber particles in latex, by reaction with a terminal allylic diphosphate group of the rubber molecules [13–15] This indicates that rubber transferase is bound to the rubber particles and is still present even after the particles are washed repeatedly However, the precise mechanism for the biosynthesis of rubber molecules has not yet been established Moreover, the exact site of the formation of new rubber molecules still remains unknown
It has been suggested that the bottom fraction membrane could possibly serve as the site for initiation of new rubber formation [16,17]
Correspondence toT Koyama, Institute of Multidisciplinary Research
for Advanced Materials, Tohoku University, Katahira 2-1-1,
Aoba-ku, Sendai 980–8577, Japan.
Fax: + 81 22 217 5620, Tel.: + 81 22 217 5621,
E-mail: koyama@tagen.tohoku.ac.jp
Abbreviations: IPP, isopentenyl diphosphate; HRT, Hevea rubber
transferase; WBP, washed bottom fraction particles; IPTG, isopropyl
thio-b- D -galactoside; GPC, gel permeation chromatography;
dedol-PP, dehydrodolichyl diphosphate.
Enzyme: rubber cis-polyprenylcistransferase (EC 2.5.1.20).
*Present address: Department of Pharmacology, School of Medicine,
Yale University, New Haven, CT, USA.
Note: The nucleotide sequences reported in this paper are available in
the DDBJ/GenBankTM/EMBL Data Bank under the accession
numbers AB061234 and AB064661.
(Received 15 July 2003, revised 26 September 2003,
accepted 2 October 2003)
Trang 2In the biosynthesis of polyisoprenoid compounds, the
prenyl chain elongation, catalyzed by prenyltransferases,
proceeds consecutively and terminates precisely at certain
chain lengths according to the specificities of individual
enzymes [18–20] These enzymes have been classified into
two major groups, referred to as trans- or (E)-prenyl
diphosphate synthases (trans-prenyltransferases) and as
cis- or (Z)-prenyl diphosphate synthases
(cis-prenyltrans-ferases), depending on the stereochemistry of the
conden-sation reaction of IPP with the corresponding allylic prenyl
diphosphate initiator During the past 16 years, many
different genes encoding trans-prenyltransferases have been
cloned and characterized [19,20] On the other hand, very
limited information was available on cis-prenyltransferases
until the recent cloning and characterization of the genes
encoding cis-prenyltransferases from Micrococcus luteus
B-P 26, Escherichia coli, Haemophilus influenzae,
Strepto-coccus pneumonica, Saccharomyces cerevisiae and
Arabid-opsis thaliana[21–25]
Natural rubber has been thought to be made almost
entirely of cis-isoprene units derived from IPP, and the
enzyme responsible for polymerization is believed to have
characteristics similar to the cis-prenyl diphosphate
synth-ases However, the genes encoding Hevea
cis-prenyltrans-ferases or rubber transferase (HRT) from H brasiliensis
have not yet been reported It was suggested that rubber
biosynthesis in H brasiliensis is mediated by the association
of a soluble trans-prenyltransferase with a rubber elongation
factor, a 14.6 kDa protein, tightly bound to the rubber
particles in the laticifers [26] However, Cornish [10] has
demonstrated that the soluble trans-prenyltransferase
func-tions as farnesyl diphosphate synthase, and almost certainly
plays no direct role in the cis-1,4-polyisoprene elongation
A detailed understanding of rubber biosynthesis
proces-ses at the molecular level is important for genetic
manipu-lation of the isoprenoid biosynthesis pathway enzymes In
this study, by using the sequence information from the
conserved regions of the cis-prenyl chain elongating
enzymes [20–22], we isolated and characterized two Hevea
cis-prenyltransferases cDNAs designated as HRT1 and
HRT2 from the H brasiliensis latex Sequence analysis
showed that all five regions that are conserved among
cis-prenyl chain elongating enzymes were present in each of
the deduced amino acid sequences encoded by the two
cDNAs In vitro assay of the rubber transferase activity of
the recombinant HRT proteins was carried out in the
presence of washed bottom fraction particles (WBP) of fresh
Hevealatex These results suggest that the HRT2 protein
catalyzes the synthesis of new rubber molecules with the
active involvement of a number of factors in WB P of fresh
Hevealatex
Materials and methods
Plant materials and RNA isolation
Latex and various tissue samples were obtained from
ten-year old rubber plants (H brasiliensis clone RRIM 600)
being grown at the Rubber Research Center of Songkla,
Thailand Latex collection was performed as described by
Kush et al [27] The latex total RNA was extracted by
using RNAgents Total RNA Isolation System (Promega)
Total RNAs of rubber leaf and other tissues were obtained using QuickPrep Total RNA Extraction kit (Amersham Biosciences) and RNA Isolation kit (Qiagen), respectively Poly(A)+RNA was isolated from the total RNA with Oligodex-dT30 mRNA Purification kit (TaKaRa, Ohtsu, Japan)
RT-PCR amplification ofHevea cis-prenyltransferase cDNA fragment
RT-PCR was carried out using Ready-To-GoTMRT-PCR Beads (Amersham Biosciences) First strand cDNA syn-thesis was performed by reverse transcription with 100 ng of poly(A)+ RNA isolated from latex using poly-d(T)12)18 primer Two oligonucleotide degenerate primers were designed to amplify the Hevea cis-prenyltransferase cDNA fragment according to the highly conserved regions among cis-prenyl chain elongating enzymes; sense primer, P1 (AFIMDGN, region I) 5¢-GCTTTTATTATGGAYG GHAA-3¢ and antisense primer, P2 (IRTSGE, region V) 5¢-CTCACCAGAWGTWCKWAT-3¢, where H is A, C or T; K is G or T; W is A or T and Y is C or T PCR was performed in a final volume of 50 lL containing 50 pmol of amplification primer pair for 45 cycles of 30 s at 95C, 30 s
at 45C and 1 min at 72 C with a 5 min preheat and a
10 min final extension at 72C The resulting band of PCR products were extracted from agarose gel and subcloned into pT7Blue T vector (Novagen) for sequencing One
of the resulting clones, which showed homology to cis-prenyltransferases, was termed LT600
3¢- and 5¢-RACE reaction and cloning ofHevea cis-prenyltransferase cDNA
Cloning of the full-length cDNA of Hevea cis-prenyltrans-ferase, used poly(A)+RNA from latex as the template for performing both 3¢- and 5¢-RACE reactions based on the cDNA sequence of LT600 The procedures applied for 3¢- and 5¢-RACE reaction were according to the manufac-turers instructions (3¢-Full RACE Core Set, TaKaRa; 5¢-RACE kit, Roche) The primer, F1, used for 3¢-RACE was 5¢-AGGGCTACTGCCAACAATTCC-3¢ and the primers, R1 and R2, used for 5¢-RACE reaction were 5¢-GCTTCCAGTTGCATTTGCCTCCTCC-3¢ and 5¢-GC TAAAGGCATAGATAGTCGC-3¢ respectively Accord-ing to the sequence information obtained by the 5¢- or 3¢-RACE reaction, the cDNA was amplified by RT-PCR with the latex poly(A)+ RNA as the template and then sequenced Finally, two cDNAs were obtained and desig-nated HRT1 and HRT2, respectively
DNA sequencing analysis Sequencing reactions were performed using Thermo Sequenase Cycle Sequencing kit (Amersham Pharmacia Biotech) with fluorescent labeled primers Nucleotide sequences were determined by the dideoxy chain termin-ation method [28] with a DNA sequencer (LI-COR, model 4200, LI-COR Inc., Lincoln, NE, USA) Compu-ter analysis and comparison of DNA sequences were carried out usingGENETYXgenetic information processing software (Genetyx Corp., Tokyo, Japan)
Trang 3Analysis ofHRT gene expression
For Northern hybridization, total RNAs (15 lg) from
various tissues were subjected to electrophoresis on 0.8%
agarose gel containing 1% formaldehyde, and blotted onto
a positively charged nylon membrane (Roche) The
mem-branes were hybridized with32P-labeled HRT cDNAs for
1 h at 68C in ExpressHyb solution (Clontech) High
stringency washes were performed twice at 50C in
2· NaCl/Cit, 0.05% SDS and twice in 0.1 · NaCl/Cit,
0.1% SDS The hybridized membranes were exposed for
12 h on a Fuji imaging plate and then analyzed with a Fuji
BAS 1000 Mac Bioimage analyzer RT-PCR for the
analysis of HRT expression was performed by using total
RNAs (2 lg) from various Hevea tissues, amplified with
HRT1 or HRT2 specific primers The PCR reaction was
carried out with 25 cycles of programmed temperature
control of 30 s at 95C, 30 s at 50 C and 1 min at 72 C
with a 5 min preheat at 95C and a 10 min final extension
at 72C using primers, S1 (5¢-GCAAATGCAACTGGA
AGCGG-3¢) and A1 (5¢-ACAGCCTGCTAGCAAAGA
GG-3¢) for amplification of HRT1, and primers S2
(5¢-GAAGAATCCTCTAAGGATAA-3¢) and A2 (5¢-TA
CAAGGATTAATCCCTTGC-3¢) for amplification of
HRT2 The PCR products were analyzed by agarose gel
electrophoresis with ethidium bromide staining
Construction of expression vector systems inE coli,
and purification ofHevea cis-prenyltransferase
Expression vector systems for the HRTs were constructed
using pET32b(+) vector (Novagen), which is designed to
express the gene product as a thioredoxin- and His-tagged
fusion protein, suitable for production of soluble protein in
E coli cytoplasm and rapid purification The restriction
enzyme recognition site for NcoI or BamHI was introduced
by PCR at either the 5¢-end or 3¢-end of the coding regions
of the two HRT cDNAs The resulting fragments were
sequenced, digested with NcoI and BamHI, and ligated into
the NcoI–BamHI vector of pET32b(+), yielding the
expression plasmids pETHRT1 and pETHRT2 Each of
the expression plasmids was used for transformation of
E coli BL21(DE3), and 1 mL of an overnight culture
of the transformant in Luria–Bertani medium containing
50 lgÆmL)1 ampicillin was inoculated into 200 mL of
M9YG medium [29] containing 50 lgÆmL)1 ampicillin
The cells were grown at 37C to an A600 value of 0.4
Isopropyl thio-b-D-galactoside (IPTG) was added to a final
concentration of 0.5 mM, and then a further incubation at
30C for 4 h was carried out Overproduction of the
proteins was confirmed by SDS/PAGE according to the
standard method of Laemmli [30]
The cells were harvested by centrifugation (5000 g for
10 min) and then disrupted by sonication The cell
homo-genates were fractionated into soluble and insoluble proteins
by centrifugation at 8000 g for 10 min The expressed
proteins were purified essentially according to the protocol
of Xpress Protein Purification System (Invitrogen), using
a Ni2+ nitrilotriacetic acid-agarose column The soluble
proteins were applied to a Ni2+resin column, and the
His-tagged fusion protein was eluted with a gradient of
50–500 m imidazole in 20 m phosphate buffer, pH 6.0
The insoluble proteins were solubilized with 6Mguanidine hydrochloride lysis buffer, pH 7.8 and subjected to a Ni2+ resin column The column was washed and the tagged protein was eluted with 8M urea in 20 mM phosphate buffer, pH 4.0 The purified protein was renaturated by removal of urea via stepwise dialysis The portion of purified fusion protein was treated with enterokinase to remove the N-terminal fused thioredoxin The digested protein was used for the rubber transferase activity assay and for product analysis Protein concentration was measured by the Bradford method [31]
In vitro rubber transferase activity assay The washed bottom fraction particles (WBP) were prepared
by ultracentrifugation (49 000 g, 45 min, 4C) of fresh Hevea latex followed by repeated washing of the fresh bottom fraction with 50 mM Tris/HCl buffer, pH 7.4 containing 0.9% NaCl (w/v) according to the method of Wititsuwannakul et al [17] The rubber transferase activity assay was performed by the modified method of Tangpak-dee et al [16] The reaction mixture contained, in a final volume of 0.2 mL, 50 mMTris/HCl buffer (pH 7.4), 30 mM
KCl, 2 mM MgCl2, 5 lM ZnCl2, 5 mM dithiothreitol,
20 mMKF, 0.1 mMdeoxycholate, 0.5 mg of WBP, 15 lM
farnesyl diphosphate, 50 lM[1-14C]IPP (2.15 GBqÆmmol)1, Amersham Pharmacia Biotech), and a suitable amount of cell-free homogenate or purified protein After incubation for 4 h at 30C, short and medium chain polyprenyl diphosphate products were extracted with 1-butanol, and then residual radioactive rubber in aqueous phase was extracted three times with 0.6 mL of toluene/hexane mixture (1 : 1 v/v) The extracts were concentrated to a small volume and the radioactivity was measured with an Aloka LSC-1000 liquid scintillation counter (Tokyo, Japan) The rubber transferase activity was determined by measuring the amount of [14C]IPP incorporated into rubber
in the toluene/hexane extracts
Analysis of HRT Reaction Products The radioactive products extracted with the toluene/hexane mixture were treated with potato acid phosphatase accord-ing to the method reported previously [32] The radioactive products were extracted with toluene and analyzed by TLC
on a reversed phase RP-18 plate (Merck) with a solvent system of acetone/water (39 : 1 v/v) The positions of authentic standards were visualized with iodine vapor, and the distribution of radioactive products on the TLC plate was analyzed with a Fuji BAS-1000 Mac Bioimage analyzer
Distribution of molecular size of rubber products were analyzed by gel permeation chromatography (GPC) which was carried out with a Tosoh high performance liquid chromatography system, equipped in tandem with a series
of four TSK gel GPC columns, G7000H, G5000H, G2500H, and G1000H (Tosoh Corp., Tokyo, Japan), each
of which has an exclusion limit of 4· 108, 4· 106, 2· 104 and 1· 103Da, respectively The chromatography was carried out at 35C using tetrahydrofuran as eluent, at a flow rate of 0.5 mLÆmin)1 The eluate was monitored by
UV absorption at 210 nm following collection at 1 min
Trang 4intervals, and assayed for radioactivity The molecular mass
of the reaction products were estimated by comparing them
with the elution volumes of commercially available standard
polystyrenes
Expression of HRT in yeast strain SNH23-7D
To express the HRT cDNA in the yeast mutant strain
SNH23-7D (MATa rer2-2 mfa1::ADE2 mfa2::TRP1
bar1::HIS3 ade2 trp1 his3 leu2 ura3 lys2) according to the
study of Sato et al [23], the BglII–SalI fragments containing
HRT1 or HRT2 cDNA were cloned into the corresponding
sites of plasmid pJR1133, which contain the URA3 marker
gene and yeast glyceraldehyde phosphate dehydrogenase
promoter [25] The resulting plasmids designated pJRHRT1
and pJRHRT2, contained HRT1 and HRT2 respectively
The SNH23-7D yeast strain was transformed with plasmid
pJRHRT1 and pJRHRT2 according to the protocol of
FastTMYeast Transformation kit (Geno Technology Inc.,
St Louis, USA) Ura+ transformants were selected at
23C on agar plates containing minimal medium [0.67%
(w/v) yeast nitrogen base without amino acid, 2% glucose,
supplemented with 60 lgÆmL)1 leucine and 30 lgÆmL)1 lysine] Selected Ura+ colonies and yeast strain SNY9 (MATa mfa1::ADE2 mfa2::TRP1 bar1::HIS3 ade2 trp1 his3 leu2 ura3 lys2), which contains the wild type RER2 gene, were streaked on agar plates containing YPD medium and incubated at 23C or 37 C
Results
Isolation and characterization of HRT cDNAs
A pair of degenerate primers designed from two highly conserved regions (I and V) of the known cis-prenyl chain elongating enzymes [20–22,33–35], were used to amplify of a possible cDNA encoding cis-prenyltransferase(s) in Hevea latex that might be responsible for the prenyl chain elongation of natural rubber RT-PCR with these primers yielded amplified products of 600 bp in length These products were extracted and cloned into pT7Blue T vector One of the cDNA fragments, which contained regions homologous to those of the conserved regions II, III and IV
of cis-prenyltransferase was designated as LT600 The
Fig 1 Nucleotide and deduced amino acid sequences of HRT1 (A) and HRT2 (B) Numbers of nucleotide sequence and amino acid sequence are indicated on the left and right, respectively The underline in HRT1 indicates the sequence corresponding to the LT600 fragments.
Trang 5nucleotide sequence of the fragment was used to design the
primers to amplify unknown 3¢- and 5¢-end sequences of
HRT cDNAs by using 3¢- and 5¢-RACE strategies The
amplified products of the 3¢-end gave two distinct cDNA
fragments which were 689 and 557 bp in length, with a
sequence identity of 85% having identical sequence near the
3¢-end of the possible open reading frame When the
5¢-cDNA was amplified, only a 424 bp cDNA fragment was
obtained To obtain full-length sequences of these ORFs, a
pair of primers was designed according to the sequence
information from the RACE analysis After amplification
by RT-PCR from latex poly(A)+RNA, we sequenced a
number of fragments and obtained two distinct cDNAs that
contain sequences identical to those obtained by 3¢-RACE
General cDNAs containing 5¢- and 3¢-untranslated regions
were also amplified by RT-PCR and sequenced [Fig 1]
These clones were designated as HRT1 and HRT2 The
cDNA of HRT1 was 1282 bp long containing an 870 bp
ORF, flanked by a 155 bp 5¢-UTR and a 254 bp 3¢-UTR
including a poly(A) tail of 15 bp (Fig 1A) The cDNA of
HRT2 was 1051 bp in length containing an 852 bp ORF
(Fig 1B) and with 92% sequence homolgy to that of
HRT1 The complete cDNA sequences of HRT1 and
HRT2 cloned in this study are available from the DDBJ/
GenBankTM/EMBL database under accession numbers
AB061234 and AB064661 respectively The ORFs of
HRT1 and HRT2 encode 290 and 284 amino acid residues
(87.3% identity) with predicted molecular masses of 33.2
and 32.8 kDa respectively Hydropathy and
transmem-brane motif analysis of the deduced amino acid sequences
(TOPPRED2 program; http://bioweb.pasteur.fr/seqanal/ interfaces/toppred.html) predicted that both gene products, HRT1 and HRT2, are hydrophilic proteins having a putative N-terminus membrane-spanning segment (amino acid residues 22–42) The deduced amino acid sequences of HRT1 and HRT2 showed high identities of 31.6, 32.0, 31.6, 29.9 and 30.1% to those of the cis-prenyl chain elongating enzymes from M luteus B-P 26 [21], S cerevisiae (Rer2p [23], Srt1p [36]), E coli [22,37] and A thaliana [24,25] respectively Moreover, all of the five highly conserved regions of cis-prenyl chain elongating enzymes [20–22,34,35] are found in both the HRT1 and HRT2 sequences [Fig 2] These conserved regions are proposed to be important for the catalytic function, in addition to substrate binding, for cis-prenyl chain elongating enzymes [33–35,38,39]
Expression analysis of HRT mRNAs
To examine the HRT expression in various Hevea tissues, Northern blot analysis was carried out by using32P-labeled HRT1 or HRT2 cDNA as the probes As shown in Fig 3 (A1 and A2) both probes gave specific hybridization bands
of 1 kb which are consistent with the sizes of each of the cDNAs The expression patterns of HRT1 and HRT2 mRNAs among the examined Hevea tissues seem predomi-nant in latex, whereas little expression was detected in leaves and shoot tips However, it is possible that the probes of HRT1 and HRT2 cross-hybridize with each other because
of the high level of sequence identity For the study of the specific expression patterns, RT-PCR analyses were carried
Fig 2 Comparison of the deduced amino acid sequences of HRT1 and HRT2 The deduced amino acid sequences of HRT1 and HRT2 are compared with those of cis-prenyltransferases from A thaliana (GenBankTMaccession no AF162441), yeast [Rer2p (Swiss-Prot P35196), Srt1p (Swiss-Prot Q03175)], M luteus B-P 26 (GenBankTMaccession no AB004319) and E coli (Swiss-Prot Q47675) The five conserved regions for cis-prenyl chain elongating enzymes (I to V) are indicated with bars at the top of the sequences The sequences similar to HRT1 are shaded Identical amino acid residues in greater than four of the seven sequences are boxed.
Trang 6out by designing specific primers from the low homology
regions of HRT1 and HRT2 coding sequences and each of
the 3¢-UTRs As shown in Fig 3B, specific bands of the
RT-PCR products with HRT1 and HRT2 specific primers
could be detected only in the reactions with latex mRNA
These results indicate the specific expression of HRT1 and
HRT2 in latex, supporting the proposal that the HRT
mRNAs probably function in Hevea latex where natural
rubber is produced
Overproduction of recombinant HRT proteins
inE coli cells
In order to obtain HRT gene products, the cDNAs were
expressed in E coli by means of a pET32b(+) expression
system that contains a thioredoxin fusion sequence suitable for production of a soluble protein in E coli cytoplasm By induction with IPTG the E coli cells harboring HRT1 or HRT2 produced recombinant proteins at 52 kDa in the pellet fraction (Fig 4, lanes P1 and P2) However, a detectable amount of HRT2 could be found also in the soluble fraction of cell-free extract (Fig 4, lane S2) The soluble and pellet fractions of HRT2 were subjected to a metal affinity column for purification of the His-tagged fusion protein, under native and denatured conditions respectively As shown in Fig 4, the affinity-purified HRT2 protein was the major protein band of 52 kDa (lane P) corresponding to the His-tagged fusion protein, and when the fused N-terminal thioredoxin sequence was removed by enterokinase digestion, a protein of 33 kDa (lane E), was produced which is attributable to the predicted molecular mass of the HRT2 protein
Enzymatic activity of HRT2 proteins The cell-free homogenates of E coli BL21(DE3)/pET-HRT1 and E coli BL21(DE3)/pETHRT2 were examined for in vitro rubber transferase activity Rubber materials were extracted with a solvent mixture of toluene and hexane (1 : 1 v/v) after the extraction of medium chain polyprenyl diphosphates with 1-butanol The crude homogenate from HRT2-overexpressing cells showed a slight increase in the production of polyprenyl diphosphates able to be extracted with butanol compared to the crude homogenate of the host cells (Table 1), whereas the HRT1 protein overproduced in
E coli cells showed no significant increase in enzymatic activity Similarly, a slight increase in the radioactivity of the toluene/hexane extracts was detected after the reaction with
Fig 3 Expression of HRT mRNAs (A) Northern blot analysis of
HRT mRNAs Total RNA samples (15 lg) from: Hevea latex, LT;
leaves, LE; petioles, P; flowers, F; shoot tips, S and roots, R were
separated by agarose gel electrophoresis and transferred onto a nylon
membrane The blot was hybridized with 32 P-labeled HRT1 or HRT2
cDNA probe The 0.8–1.4 kb regions of the autoradiogram of the
hybridized membrane, using32P-labeled HRT1 (1) or HRT2 (2) as a
probe, are shown To illustrate equal loading, ethidium bromide
staining of rRNA under UV light is shown (3) (B) RT-PCR analysis of
HRT mRNAs Two micrograms of total RNA samples from various
Hevea tissues were used as templates PCR was carried out for
25 cycles using HRT1 or HRT2 specific primers RT-PCR products
were separated by 1% agarose gel electrophoresis and stained with
ethidium bromide The 400–600 bp regions of the products, which
were amplified by HRT1 specific primers (1) or HRT2 specific primers
(2), are shown.
Fig 4 Overexpression of HRT1 and HRT2 in E coli and purification
of HRT2 The pETHRT1 and pETHRT2 were constructed and introduced into E coli BL21(DE3) The expression of these genes were induced by addition of 0.5 m M IPTG Overexpression of HRT1 and HRT2 is shown in the left panel: M, molecular mass marker;
SC, supernatant of the homogenate of E coli BL21(DE3)/pET32b without IPTG; S1, supernatant of the homogenate of E coli BL21(DE3)/pETHRT1 with IPTG; S2, supernatant of the homogen-ate of E coli BL21(DE3)/pETHRT2 with IPTG; PC, pellet of the homogenate of E coli BL21(DE3)/pET32b without IPTG; P1, pellet
of the homogenate of E coli BL21(DE3)/pETHRT1 with IPTG; P2, pellet of the homogenate of E coli BL21(DE3)/pETHRT2 with IPTG Purification of HRT2 is shown in the right panel: M, Molecular mass marker; P2, pellet of the homogenate of E coli BL21(DE3)/pET-HRT2 with IPTG; P, purified BL21(DE3)/pET-HRT2 fusion protein; E, purified BL21(DE3)/pET-HRT2 fusion protein after digestion with enterokinase.
Trang 7the crude homogenate of HRT2-overexpressed cells, but not
that of HRT1-overexpressed cells (Table 1)
Because the rubber transferase activities of HRT1 and
HRT2, which were determined by measuring the amount of
rubber material extractable with the toluene/hexane
mix-ture, were not particularly significant, an in vitro rubber
transferase assay was carried out with the addition of fresh
WBP because it has been suggested that these serve as the
site for the initiation of new rubber formation [16,17] The
amount of [1-14C]IPP incorporation in the toluene/hexane
extracts of 2500 dpm that was observed in the control
reaction with WBP is attributable to the endogenous rubber
transferase activity in the WBP When the homogenates of
BL21(DE3)/pETHRT1 were coincubated with WBP, the
amount of IPP incorporation into the toluene/hexane
extracts was similar to that of the control experiment with
WBP alone On the other hand, addition of WBP to the
homogenate of BL21(DE3)/pETHRT2 resulted in a
remarkable increase in the amount of IPP incorporation
in the toluene/hexane extracts (Table 1) The increase in
rubber transferase activity over the additive effect of
endogenous activity in WBP clearly indicates that the
rubber transferase activity of HRT2 was enhanced by
various factors in the WBP The endogenous rubber
transferase activity in the WBP could be denatured most
effectively by heat treatment at 100C for 20 min (Table 1)
However, the rubber transferase activity of HRT2 could be
induced partially, even by the addition of boiled WBP,
implying the presence of heat-stable activator(s) in the
WBP, in addition to heat-sensitive ones
Apparent rubber transferase activity could be obtained
when the purified HRT2 protein was used Figure 5A
shows the effect of the HRT2 protein on rubber transferase
activity in coincubation with WBP The activity increased as
the concentration of HRT2 was raised, until the activity
reached saturation The effect of WBP on the rubber
transferase activity of HRT2, which was assayed in the
presence of 5 lg of HRT2 protein, is shown in Fig 5B
The rubber transferase activity increased proportionally
Table 1 In vitro rubber transferase activities in the cell-free homogenates of HRT transformants The prenyl chain elongating enzyme activity were assayed in the absence of WBP, in the presence of WBP, and in the presence of boiled WBP as described in Materials and methods The amounts of [ 14 C]IPP incorporation are averaged from triplicated measurements.
Cell-free homogenate
[14C]IPP incorporation (d.p.m.)
BuOH extract Toluene/hexane extract
In the absence of WBP
In the presence of WBP
E coli BL21(DE3)/pET32b + WBP 3150 ± 173 2230 ± 267
E coli BL21(DE3)/pETHRT1 + WBP 3700 ± 340 2770 ± 204
E coli BL21(DE3)/pETHRT2 + WBP 5190 ± 414 7120 ± 356
In the presence of boiled WBP
E coli BL21(DE3)/pET32b + boiled WBP 3070 ± 316 980 ± 115
E coli BL21(DE3)/pETHRT1 + boiled WBP 3440 ± 405 1020 ± 161
E coli BL21(DE3)/pETHRT2 + boiled WBP 4080 ± 387 3360 ± 292
Fig 5 Effect of HRT2 and WBP amounts on rubber transferase activity of HRT2 (A) Effect of HRT2 amount on rubber transferase activity In vitro rubber transferase assay was performed in 0.2 mL reaction volume containing 0.5 mg of WBP and the indicated amount
of purified HRT2 (d) or E coli BL21(DE3)/pETHRT2 cell-free homogenate without IPTG (j) After extraction of polyprenyl diphosphate products with 1-butanol, rubber transferase activity was measured as described in Materials and methods (B) Effect of WBP amount on rubber transferase activity The rubber transferase was assayed in the presence of 5 lg of purified HRT2 and the indicated amount of WBP (d) The control reaction (j) was assayed under similar conditions without the addition of HRT2.
Trang 8to the amount of added WBP and the activity became
saturated when the WBP exceeded 1 mg
Product analysis of the rubber materials in the toluene/
hexane extracts of the HRT2 reactions that were
coincu-bated with WBP, by reversed phase TLC, showed a large
spot of radioactivity at the origin of the TLC plate
indicating a high molecular mass rubber product (data
not shown) Conversely, the reaction of WBP or HRT2
alone gave a small spot at the origin of the TLC plate In
order to determine the molecular masses of the rubber
products from the reaction of HRT2 coincubated with
WBP, the radioactive products detected at the origin of the
reversed phase TLC were analyzed by GPC (Fig 6B) The
major radioactivity eluted in the range of molecular mass of
2· 105)1 · 106Da (corresponding to the rubber polymer
of 3000–15 000 isoprene units), with minor peaks of radioactivity at 3· 104, 2· 103and 1· 103Da, equivalent
to 400, 30 and 15 isoprene units respectively, showing similar distribution of endogenous rubber materials in WBP (Fig 6A) When HRT2 was assayed in the absence of WBP, only a small amount of medium chain polyisoprene intermediates of the size 2000 to 104Da were produced (Fig 6C) The products of the control reaction with WBP alone (Fig 6D), show very low quantities of long chain rubber products whose molecular size distributions are similar to those in Fig 6Bindicating the low endogenous rubber transferase activity in WBP These results suggest that the long chain rubber molecules (approximate size of
105)106Da) can be produced in larger amounts when the HRT2 protein is coincubated with WBP
Functional complementation of yeast mutant stain SNH23-7D by HRTs
To determine whether HRT1 and HRT2 cDNAs encode functional enzymes related to the cis-prenyl chain elongating enzyme in vivo, the cDNAs were expressed in the yeast mutant strain SNH23-7D, which is deficient in the activity
of dehydrodolichyl diphosphate (dedol-PP) synthase [24] SNH23-7D shows a temperature-sensitive growth pheno-type at 37C As shown in Fig 7, overexpression of HRT2 suppresses the temperature-sensitive growth phenotype of strain SNH23-7D, whereas the HRT1 showed no effect on this phenotype of the yeast mutant strain
Discussion
In this study, we isolated two cDNA clones termed HRT1 and HRT2 that possibly encode the latex cis-prenyltrans-ferases of the rubber tree H brasiliensis Both of the deduced amino acid sequences, having all of the five highly conserved regions among cis-prenyl chain elongating enzymes, showed high homology (87% identity) These regions have been found to construct the major part of the hydrophobic cleft in the three-dimensional structure [38], and are important for the catalytic function as well as the substrate binding of the enzymes [33–35,39,40] Therefore, it is reasonable to assign both HRT1 and HRT2 to a family of cis-prenyl chain elon-gating enzymes present in the laticifers of H brasiliensis Recently, an A thaliana gene encoding dedol-PP syn-thase, was identified and shown to be highly expressed in
Fig 6 GPC analysis of the reaction products from HRT2 The reaction
products derived from the in vitro rubber transferase assay were
sub-jected to GPC as described in Materials and methods (A) Molecular
mass distribution of endogenous rubber materials in WBP, detected by
UV absorption at 210 nm (B) The molecular mass of 14 C-labeled
products synthesized in vitro by purified HRT2 in the presence of
WBP (C) 14 C-labeled products synthesized by purified HRT2 alone.
(D) 14 C-labeled products synthesized by WBP alone.
Fig 7 Functional complementation of the dedol-PP synthase-deficient yeast strain SNH23-7D by HRT2 Strain SNY9 and SNH23-7D transformed with plasmid pJR1133, pJRHRT1 and pJRHRT2 were streaked onto YPD plates and incubated at 23 C or 37 C for 4 days.
Trang 9roots and leaves [24,25] On the other hand, in the present
study, Northern blot and RT-PCR analyses of the HRT
genes in various Hevea tissues showed predominant
expres-sion of these genes in the latex As it has been suggested
previously that rubber biosynthesis takes place only in the
laticifers, however, the specific expression in the latex
strongly suggests a possible functional role of both HRT1
and HRT2 in the Hevea latex as a cis-prenyl chain elongating
enzyme, i.e rubber transferase
The majority of HRT1 and HRT2 were expressed as
insoluble fusion proteins in E coli, which suggests that these
proteins might each be associated with the membrane as a
particle-bound enzyme in a similar manner to rubber
transferase [3,10,40] Furthermore, hydropathy and
trans-membrane motif analysis revealed that HRT1 and HRT2
sequences have a possible membrane-spanning segment
at amino acid residues 22–42, which is very similar to those
in the dedol-PP synthases cloned from yeast [23] and
A thaliana[24,25]
Although rubber transferase has been reported
previ-ously, to be bound to rubber particles [13–15], the detailed
mechanism of rubber biosynthesis was not clearly
under-stood More recently, it has been found that the WBP of
centrifuged fresh Hevea latex is active for in vitro rubber
biosynthesis [16,17], suggesting that the WBP might have
the necessary enzyme systems that are responsible for
rubber biosynthesis In the current study, we
demonstra-ted that the recombinant HRT2 protein coincubademonstra-ted with
WBP could synthesize medium chain polyprenyl
diphos-phate intermediates as well as long chain rubber, although
HRT2 showed a low cis-prenyl chain elongating enzyme
activity when assayed in the absence of WBP HRT2 may
possibly function as a cis-prenyl chain elongating enzyme
that cooperates with other activation factors in the WBP
for the biosynthesis of long chain rubber molecules
Furthermore, the partial enhancement of HRT2 activity
by the addition of the boiled WBP suggests that some
heat-stable compounds in WBP could act as the activator
of rubber transferase In addition to heat-stable
com-pounds, HRT2 may require other heat-unstable factors in
WBP for complete activation
Suppression of HRT2 on the temperature-sensitive
growth of yeast mutant strain SNH23-7D shows clearly that
HRT2 functions as a cis-prenyltransferase and produces
dedol-PP, whose prenyl chain length is compatible with that
of yeast dolichols (C80–C100; 1.1–1.3 kDa) GPC analysis of
the rubber material synthesized by the action of purified
HRT2 in the absence of WBP indicated the production of a
small amount of medium chain polyisoprene intermediates
of molecular size between 103and 104Da, which are smaller
than those produced by the coincubation of HRT2 with
WBP (2· 105)1 · 106Da) but similar to the molecular size
of dolichols in yeast (Fig 6C) Furthermore, HRT2 showed
a little activity for the production of prenyl diphosphates
extractable with butanol Taken altogether, two probable
mechanisms of natural rubber biosynthesis including HRT2
as well as the other factor(s) in WBP could be deduced as
follows (a) HRT2 synthesizes medium chain polyprenyl
diphosphate whose chain lengths are comparable to those of
dolichols ( 103Da), then various factors in WBP combine
the medium chain polyprenyl products to synthesize high
molecular mass rubber materials, and (b) HRT2 catalyzes
further prenyl chain elongation to synthesize linear high molecular mass polyprenyl products with the cooperation of various factors in WBP that remarkably enhance HRT2 activity
We were not able to detect the rubber transferase activity
in any fraction of HRT1 protein overproduced in E coli These results led us to speculate that the 35 amino acids mismatch between HRT1 and HRT2 may be effective on their native structures and also important for their enzy-matic functions The high similarity between HRT1 and HRT2 suggests the possibility that HRT1 may encode a cis-prenyl chain elongating enzyme such as dedol-PP synthase whose function in the rubber tree is still unknown Tateyama et al analyzed the polyisoprenoid alcohols of
H brasiliensis using a two-plate TLC method [41], and established that polyprenols and dolichols are found with prenyl chain-length distribution of around C50–C105 in several tissues of H brasiliensis In addition, we found many protein sequences that originated from H brasiliensis in protein databases, showing high homology to cis-prenyl-transferase, such as AAM92880 (AAM92889, AAM92890), AAM92881, AAM92879, BAB92023 (AAM92883, AAM92884, AAM92885, AAM92887, AAM92888), BAB92024 and AAM92882 (AAM92886) (submitted to GenbankTMand DDBJ by Coldren et al and Sando et al respectively) However, most of these amino acid sequences, except for that of AAM92882, are similar to that of HRT1 (more than 98% identity) but not to HRT2 None of them have been proved to show rubber transferase activity The precise mechanisms of rubber biosynthesis in
H brasiliensis are not yet well understood The results of our study of Hevea rubber transferase represent an import-ant step in understanding the process of rubber biosynthesis
in rubber-producing plants at the molecular level and provide a basis for further investigation of the molecular mechanism of HRT reaction More biochemical studies with the purified HRT are required to further characterize the involvement of this enzyme in rubber biosynthesis, especially on the molecular analysis of the activator(s) in WBP
Acknowledgements
We are grateful to Dr A Nakano and Dr M Sato (RIKEN, Japan) for kindly providing the yeast strains SNH23-7D and SNY9, and Dr A Ferrer (University of Barcelona, Spain) for kindly providing the yeast expression vector pJR1133 This work was supported in part by Grants-in-Aid for Scientific Research (12480169 to T.K and 13680667
to Y.-W.Z.) from the Ministry of Education, Science and Culture of Japan, and by the Asahi Glass-, Heiwa–Nakajima-, Sumitomo- and Goho-Life Science Foundations.
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