Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 3846
doi:10.1111/j.1745-7270.2008.00376.x
Recognition of signal peptide
by protein translocation machinery in middle silk gland of silkworm Bombyx
mori
Xiuyang Guo, Yi Zhang, Xue Zhang,
Shengpeng Wang, and Changde Lu*
State Key
Laboratory of Molecular Biochemistry, Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200031, China
Received: September
8, 2007
Accepted: October
8, 2007
This work was
supported by a grant from the National Natural Science Foundation of China (No.
30470350)
*Corresponding
author: Tel, 86-21-54921234; Fax, 86-21-54921011; E-mail, [email protected]
To investigate
the functions of signal peptide in protein secretion in the middle silk gland
of silkworm Bombyx mori, a series of recombinant Autographa
californica multiple nucleopolyhedroviruses containing enhanced green
fluorescent protein (egfp) gene, led by sericin-1 promoter and
mutated signal peptide coding sequences, were constructed by region-deletions
or single amino acid residue deletions. The recombinant Autographa
californica multiple nucleopolyhedroviruses were injected into the hemocoele
of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of
EGFP in the middle silk gland were examined by fluorescence microscopy and
Western blot analysis. Results showed that even with a large part (up to 14
amino acid residues) of the ser-1 signal peptide deleted, the expressed EGFP
could still be secreted into the cavity of the silk gland. Western blot
analysis showed that shortening of the signal peptide from the C-terminal
suppressed the maturation of pro-EGFP to EGFP. When 8 amino acid residues were
deleted from the C-terminal of the signal peptide (mutant 13 aa), the secretion
of EGFP was incomplete, implicating the importance of proper coupling of the
h-region and c-region. The deletion of amino acid residue(s) in the h-region
did not affect the secretion of EGFP, indicating that the recognition of
signal peptide by translocation machinery was mainly by a structural domain,
but not by special amino acid residue(s). Furthermore, the deletion of Arg2 or replacement with Asp in the n-region of the signal
peptide did not influence secretion of EGFP, suggesting that a positive charge
is not crucial.
Keywords signal
peptide recognition; sericin-1; silkworm; Bombyx mori; recombinant
AcMNPV
The recognition of signal peptide by cytoplasmic signal peptide
recognition particle (SRP) is the first step in protein secretion [1–3]. The signal
peptide-SRP complex is anchored to the endoplasmic reticulum (ER) membrane,
and the signal peptide is subsequently transferred from SRP to the integral
membrane glycoprotein, a signal sequence receptor (SR) located on the ER
membrane close to the translocon, the first gate to the secretory pathway
[4,5]. After being directed to the translocon, the nascent protein will be
translocated through the translocon co-translationally or
post-translationally, most often the former [6–8]. The signal peptide will
then be cleaved from the pro-protein by signal peptidase during the
co-translational translocation [9] to form mature secretory proteins that are
released into ER lumen and the signal peptide will be further cleaved by signal
peptide peptidase [10]. The signal peptide has a canonical structure with positively-charged
amino acid residues at the N-terminal (n-region, 1–5 aa), a hydrophobic core
in the middle (h-region, 7–15 aa) and a more polar region with non-polar small amino acid
residues at positions –1 and –3 at the C-terminal (c-region, 3–7 aa) [11–14]. It has been
reported that properties of residues at the h/c boundary and +1 position of
mature protein can influence the translocation and cleavage of signal peptide
[15,16]. The canonical structure of a signal peptide is conserved throughout
evolution. Based on the common structural features, several prediction
software programs have been developed, such as the SignalP 3.0 server from the
Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP-3.0/#submission)
[17]. The silk gland of silkworm is a typical exocrine gland. It is
a tubular organ consisting of a single layer of huge polyploid cells that can
synthesize and secrete 0.2 g protein (dry weight) in 3–5 d. It is an attractive
model for studying the mechanism of protein translocation and secretion.
Sericin (ser) takes up approximately 30% of the silkworm cocoon. It mainly
consists of six kinds of protein molecules, expressed specifically in the
middle part of the silk gland by two genes, ser1 and ser2,
through alternative splicing [18,19]. The ser1 gene codes for four major
constituents of sericin. It was thought that the first 19 aa constituted the
ser-1 signal peptide [20]. The prediction of ser-1 by SignalP 3.0 indicated
that the most probable cleavage site is between positions 21 and 22, with a
probability of 0.591, and another less probable cleavage site is between
positions 19 and 20. The function of different regions of ser-1 signal peptide
on the secretion of ser-1 remains unclear. Understanding the recognition of a
signal peptide by protein translocation machinery will facilitate the design of
a signal peptide for the secretory expression of foreign genes in the silk
gland of Bombyx mori.In our recent works, we found that some strains of silkworm are
permissive to recombinant Autographa californica multiple nucleopolyhedrovirus
(rAcMNPV) [21]. Using rAcMNPV vector, silk gland-specific secretory expression
of the enhanced green fluorescent protein (EGFP) gene in silkworm
was achieved [22]. Using EGFP as reporter, the secretion of EGFP can be easily
observed with fluorescence microscope. The secretion of fibroin heavy chain of
silkworm and the cleavage site of the signal peptide have been studied
[23,24].In this study, we report the role of different regions of ser-1
signal peptide as recognized by the protein translocation machinery through
region-deletions or single amino acid residue deletions in the ser-1 signal
peptide.
Materials and
Methods
Mutagenesis of ser-1 signal
peptide
The pSerPEGFP plasmid was constructed previously in our laboratory
[22], composed of ser-1 promoter, the coding sequence for the first 21
aa residues of ser-1, the restricted enzyme sites linker (CTGCAGGCATGC, coding
Leu, Gln, Ala, and Cys), the egfp gene sequence (from ATG to TAA), and
the 3‘-terminal of ser-1. To construct the plasmids with signal
peptide deleted from its C-terminal, a fragment from a single restricted
endonuclease site in pSerPEGFP (ie, ClaI or SacI) to the
mutated signal peptide coding sequence was amplified by polymerase chain
reaction (PCR) and used to replace the corresponding normal fragment in
pSerPEGFP. The primers and amino acid sequences for the mutated peptides are
listed in Table 1. The mutants deleted from the C-terminal of ser-1
signal peptide included the first 21, 20, 19, 18, 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, 5, and 1 amino acid residues of ser-1. Two mutants with the first 21
aa and only the first 1 aa of the ser-1 signal peptide were used as positive
and negative controls, respectively.As shown in Fig. 1, fusion PCR strategy was applied to
construct plasmids with signal peptide mutated by region-deletions and single
amino acid residue deletions within the h-region of signal peptide sequence.
The 3‘-half of P2 contained the upstream antisense sequence of the
deleted region (or residue), and the 5‘-half of P2 contained the
downstream antisense sequence of the deleted region (or residue). The 3‘-half
of P3 contained the downstream sense sequence of the deleted region (or
residue), and the 5‘-half of P3 contained the upstream sense sequence of
the deleted region (or residue). The 5‘ sequence of P3 complemented the
5‘ sequence of P2. Two fragments that amplified with primer pairs P1/P2
and P3/P4, respectively, were mixed together. After denaturing and annealing,
the mixtures were used as templates, then amplified with P1 and P4 to produce
the fusion fragment. This fragment was used to replace the corresponding
fragment in pSerPEGFP through two restricted sites. The primers used in fusion PCR are listed in Table 2. Mutants
constructed by fusion PCR were named as follows: 9– means the 9th aa was
deleted; 9–15 means amino acid residues from 9th to 15th of the original signal
peptide were deleted. The amino acid sequences of the mutated signal peptides
are also listed in Table 2. These mutants were 9–, 10–, 3–5, 6–8, 6–11, and 9–15. But when the
Arg2 was deleted, the mutant was named r–, and when this Arg was
replaced with Asp, the resulting mutant was named r/d. All PCR products were verified with DNA sequencing, and all cloning
processes were identified with restriction analysis.
Construction of rAcMNPVs with
ser-1 signal peptide mutants
We constructed a series of rAcMNPVs containing egfp led by
sericin-1 promoter and coding sequences for signal peptide mutants using the
Bac-to-Bac system (Invitrogen, Carlsbad, USA), as described previously [22].
Plasmid pFFa2, modified from pFastBacHTa (Invitrogen) [21], was used to
construct the donor plasmid. The ser-1 promoter-controlled egfp
expression cassette with signal peptide mutant was cut by EcoRI and BglII
from pSerPEGFP derivatives and ligated into pFFa2 digested by EcoRI and BamHI.
The resulting donor plasmids were transformed into Escherichia coli
DH10BacDEGT [21] competent cells to produce recombinant bacmids. The bacmids
were identified by PCR as previously described [22]. Verified rAcMNPV bacmids
were used for sf-9 cell transfection. Generation and large-scale production of
the recombinant baculovirus was achieved according to the instructions of the
Bac-to-Bac baculovirus expression systems manual (Invitrogen) using the sf-9
cell line. Viruses released into the culture medium from infected cells were
collected by ultra-centrifugation at 35,000 g for 60 min. The viruses
were resuspended in phosphate-buffered saline (pH 7.5) supplemented with 1% (V/V)
fetal bovine serum (Gibco BRL, Gaithersburg, USA) and stored at –70 ?C [22].
Titers were determined by a Tissue Culture Infectious Dose 50 method as
described previously [21]. The sf-9 cells were maintained in Grace’s medium
(Gibco BRL) supplemented with 10% fetal bovine serum at 27 ?C.
Silkworm larvae inoculation
and fluorescence observation of silk gland
B. mori larvae (bivoltine race, 54A)
were reared on mulberry leaves at 25 ?C. The recombinant baculovirus was
injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae with
a syringe at 106 pfu/larva. At approximately 5 d
post-injection, the green fluorescence in the middle silk gland of the silkworm
produced by EGFP were observed and photographed with a fluorescence microscope
(Leica MZ FL III; Leica, Switzerland) after dissection.B. mori larvae (bivoltine race, 54A)
were reared on mulberry leaves at 25 ?C. The recombinant baculovirus was
injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae with
a syringe at 106 pfu/larva. At approximately 5 d
post-injection, the green fluorescence in the middle silk gland of the silkworm
produced by EGFP were observed and photographed with a fluorescence microscope
(Leica MZ FL III; Leica, Switzerland) after dissection.In the presence of intact ser-1 signal peptide (21 aa), secreted
EGFP was located within the silk gland cavity, and green fluorescence could be
seen inside the silk gland; when cutting the silk gland or making an opening in
the wall of the middle silk gland dipped in water, secreted green fluorescent
protein gradually flowed out to the water, along with silk proteins [Fig.
2(A,B)]. In the absence of signal peptide, the EGFP was located in the
single layer of huge cells and no green fluorescence could been seen in the
outflow [Fig. 2(C,D)].
Crude EGFP extraction, sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot
analysis
As described previously [22], the silk glands dissected from silkworm
larvae were rinsed with cold double-distilled H2O several times to get rid of adhesive plasma and cells. The middle
part of the silk glands were then cut off and put into ice-cold double-distilled
H2O.
The secreted green fluorescent protein in the silk gland cavity gradually
flowed out into the water, along with silk proteins. The silk gland wall was
then separated carefully and the mixtures of EGFP and silk protein were pound
in water. The soluble part, mainly sericin and EGFP, was collected. The
insoluble part, mainly fibroins, was discarded. The soluble part was then
treated with several cycles of freezing, thawing, and concentration [freezing
at –20 ?C, thawing under room temperature; the insoluble sericin was separated
immediately by centrifugation, and the supernatant was then concentrated by
lyophilization (SpeedVac Savant, Farmingdale, USA)]. The crude EGFP extracts
from several silkworms were finally concentrated to the appropriate volume,
then subjected to 15% SDS-PAGE as described by Laemmli [25], and transferred
onto an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford,
USA) as described previously [26]. Western blot analysis was carried out using
the monoclonal anti-GFP antibody GFP (B-2), sc-9996 (Santa Cruz Biotechnology,
Santa Cruz, USA) and horseradish peroxidase-labeled sheep anti-mouse secondary
antibody, A-6782 (Sigma, St. Louis, USA).
Results
Recognition of signal peptide by
protein translocation machinery in middle silk gland of B. mori
The ser-1 signal peptide predicted by SignalP 3.0-hidden Markov
models (HMM) is the first 21 or 19 aa (Fig. 3). Analysis indicates that
the n-region is the first two amino acid residues “mr”; the h-region
is aa 3–14, “lvlcctlialaa”; and the c-region is aa 15–21,
“lsvkafg” or aa 15–19, “lsvka”. A previous study has shown that amino acid mutation of position –1 might affect
the secretion of protein [27]. To validate the importance of the c-region of
the signal peptide, we constructed rAcMNPVs to express EGFP that was led by
ser-1 signal peptides mutated at the c-region. These mutants encode the first
21, 20, 19, 18, and 15 aa of ser-1, and the ability of these mutants in
directing secretion of EGFP was observed. To our surprise, all of these signal
peptide mutants, even the one with 15 aa, directed secretion of EGFP reporter
normally as judged by fluorescence observation on silk gland (Fig. 4).Then further deletions stepwise from the C-terminal of the h-region,
or region-deletions and single residue deletions within the h-region of signal
peptide, were carried out. It was found that, when deleting from the C-terminal
of the h-region, EGFP could secrete into the silk gland cavity, even if only
the first 7 aa of the ser-1 signal peptide remained, whereas EGFP was not
secreted with mutants 6 aa* and 5 aa*. These results showed that a large part
of the signal peptide of sericin-1 could be deleted with its function in
directing secretion remaining intact, although a hydrophobic region is
indispensable. In studying the role of amino acid residues in the hydrophobic
region of ser-1 signal peptide, that is, 3–15 aa
“fvlcctlialaal”, region-deletions and single residue deletions
showed that almost all signal peptide mutants could direct secretion normally,
as seen from the green fluorescent protein distribution profile in the middle
silk gland, including mutant 9–15 (Fig. 4), in which the hydrophobic region was largely
deleted. These results indicated that the SRP recognizes the signal peptide
mainly by a structural domain, but not by special amino acid.
Positively-charged amino acid
residue at n-region of signal peptide is not crucial
The canonical structure of a signal peptide has a positively-charged
amino acid residue at the n-region. The importance of a positively-charged
amino acid at the n-region of ser-1 signal peptide was studied in this work.
The Arg2 was deleted or replaced by a negatively-charged amino acid residue
Asp in mutants r and r/d, respectively. When the rAcMNPVs containing the
relative EGFP expression cassettes were injected into the hemocoele of silkworm
larvae, the EGFP could be secreted into the silk gland cavity (Fig. 4).
It showed that the deletion of Arg or its replacement with Asp in the n-region
of ser-1 signal peptide also did not affect the secretion. These results are
consistent with those of Nothwehr et al [27].
Shortening of signal peptide
influences its cleavage by signal peptidase
Secreted fluorescent protein of different mutants was extracted and
detected by Western blot analysis. The secreted EGFP in the silk gland cavity
was released into water at the first step of preparation. Equal amounts of the
crude extracts of fluorescent protein sample from different mutants were run
on 15% SDS-PAGE, then transferred onto a polyvinylidene difluoride membrane
and detected with EGFP primary antibody and a horseradish peroxidase-linked
secondary antibody. The Western blot profile of the secreted EGFP, directed by
ser-1 signal peptide mutants shortening from the C-terminal, is shown in Fig.
5. When the first 20 or 21 aa remained, EGFP was secreted with the signal
peptide cleaved, as judged by the 27 kDa single band of the Western blot
profile. When the signal peptide was shortened to the first 19 aa, a very weak
band appeared with larger molecular weight, supposed to be the signal peptide
uncleaved pro-EGFP. The amount of pro-EGFP gradually increased along with the
shortening of the signal peptide from its C-terminal, and turned into the major
band when only the first 9 aa remained, whereas the mature protein, m-EGFP,
decreased gradually and turned into a trace band. These results indicated that
coupling of the cleavage of the signal peptide with the translocation process
under physiological conditions could also be broken. This phenomenon was also
found when the signal peptidase activity was interfered with [17]. Along with
the shortening of the signal peptide from its C-terminal, it might be possible
that pro-EGFP left the ER membrane before the cleavage of signal peptide, then
was released to the ER lumen.
Incomplete secretion of EGFP
reporter led by signal peptide mutant 13 aa
In this investigation, we noticed an incomplete secretion of EGFP
led by mutant 13 aa. Part of EGFP was retained somehow and aggregated as
irregular spots in the middle silk gland cells (Fig. 6). Those signal
peptide mutants with just one amino acid residue difference from 13 aa, that
is, 13 aa*, 14 aa, and 14 aa*, could direct secretion of EGFP normally, as seen
from the distribution of fluorescent protein in the middle silk gland. The
analysis of signal peptide mutants by SignalP 3.0 showed that the most probable
cleavage site in mutant 13 aa was different from any of the other three
mutants, and its probability was only 0.385, whereas it was 0.675 for 14 aa,
0.761 for 14 aa*, and 0.859 for 13 aa* (Table 3). It was proposed that
the lower cleavage rate caused the accumulation of EGFP in the cells. This
result implied that the proper coupling of different regions of the signal
peptide is important for secretion.
Discussion
The c-region of signal peptide has the least length variability and consists
of relatively small and neutral polar residues. This region is very important
for recognition and cleavage by signal peptidase [29]. The “–3, –1″ rule
states that residues in positions –3 and –1 relative to the cleavage
site must be small and uncharged, and large, bulky residues may reside in
position –2. We analyzed functional regions for all the mutants by SignalP
3.0, and the results are shown in Table 3. There are alternative sequences
that can be recognized and cleaved by signal peptidase in those mutants. These
sites locate either within the ser-1 signal peptide mutants, or in the
restriction linker “lqac”, or in the EGFP coding sequence. So,
deletion at the c-region from 21 to 15 aa did not destroy the secretion of
EGFP. When the original c-region and even a large part of the h-region of
ser-1 signal peptide were deleted, a new c-region and h-region could
functionally fill in. The three amino acid residues, taken as aa –3 to –1, were
“vka” or “afg” in ser-1, “lqa” (or
“aqa”) in linker, and “skg” in EGFP. These all suit the
“–3, –1″ rule. Three functional regions of ser-1 signal peptide were investigated
using the rAcMNPV-EGFP system. All results from this work revealed that a
large part of the hydrophobic region could be deleted, the N-terminal
positively-charged amino acid residue could be turned into a negative one, and
3 aa from –1 to –3 positions could be altered to other suitable amino acid residue.
This means that the recognition of the signal peptide by SRP and the whole
subsequent translocation and secretion process is highly flexible. Investigations on the impact of systematic mutation of a signal
peptide on its interaction with the protein translocation apparatuses are no
doubt critical for understanding their interaction mechanism. Mutation
research on eukaryotic signal peptides for clarification of the importance of
the properties of amino acid residue at certain positions have largely been
done with an in vitro transcription-translation system together with an
extracted canine pancreas microsome system [30]. Our results showed that the
effect of subtle changes on the signal peptide on its interaction with
translocation apparatuses could be studied in vivo, in the middle silk
gland of silkworm. The exocrine gland is made up of a single layer of huge
polyploid cells with a tubular shape, it is expedient in secretory condition
judgment by using EGFP reporter and it facilitates the preparation and further
analysis of secreted protein.We have constructed a mutant in which the ser-1 signal peptide was
replaced by the signal peptide of BmcecB, an antibacterial peptide of silkworm
that expresses in fat body and is secreted into the hemocoele [31]. The secretion
of EGFP directed by BmcecB signal peptide was as normal as that of the ser-1
signal peptide (data not shown). The result indicated that the protein
translocation machinery of the middle silk gland can recognize BmcecB signal
peptide, and it shares common characteristics with that of other tissues. Thus,
this system can also be used as a general system for protein translocation
research.
Acknowledgement
We thank Dr. Yuan ZHAO from the
Sericultural Research Institute, Chinese Academy of Agricultural Sciences
(Zhenjiang, China) for kindly providing silkworm eggs and silkworms in this
work.
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