Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 507-513
doi:10.1111/j.1745-7270.2006.00189.x
Structural Analysis of Fibroin
Heavy Chain signal peptide of Silkworm Bombyx mori
Sheng-Peng WANG1,2,
Ting-Qing GUO1,
Xiu-Yang GUO1,
Jun-Ting HUANG2,
and Chang-De LU1*
1 Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200031, China;
2 Sericultural Research Institute, Chinese
Academy of Agricultural Sciences, Zhenjiang 212018, China
Received: March 14, 2006
Accepted: April 6, 2006This work was supported by the grants from the National Natural Science
Foundation of China (No. 30370326 and No. 30470350)*Corresponding author: Tel, 86-21-54921234; Fax, 86-21-54921011;
E-mail, [email protected]
Abstract to study the
minimal length required for the secretion of recombinant proteins and silk proteins
in posterior silk gland, the signal peptide (SP) of the fibroin heavy chain
(FibH) of silkworm Bombyx mori was systematically shortened from the
C-terminal. Its effect on the secretion of protein was observed using enhanced
green fluorescent protein (EGFP) as a reporter. Secretion of EGFP fusion
proteins was examined under fluorescence microscope. FibH SPs with lengths of
20, 18, 16 and 12 a.a. can direct the secretion of the reporter, yet those with
lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP was shortened to
12 a.a., the secretion efficiency was decreased slightly and the cleavage
happened within EGFP. When 16 a.a. of the FibH SP were used, the secretion of
fusion protein was normal and the cleavage site was between the Gly-Ser linker
and Met, the starting amino acid of EGFP. These findings are applicable for the
expression of foreign proteins in silkworm silk gland. The cleavage site of the
SP is discussed and compared with the predictive results of the SignalP 3.0
online prediction program.
Key words signal peptide; fibroin heavy chain; silkworm, Bombyx
mori; rAcMNPV
Most secreted and membrane proteins produced in eukaryotic cells are
targeted to or translocated across the endoplasmic reticulum (ER) membrane by a
short peptide termed the signal peptide (SP). The SPs of nascent preproteins
are recognized by signal recognition particles (SRPs) that help them bind and
pass through the receptor on ER, then protein is synthesized continually into
ER [1]. The SP is cleaved by highly specific signal peptidase during or after
protein translocation [2]. A model SP has a canonical structure with three
regions: an N-terminal domain (n-region) which contains a net positive charge,
a hydrophobic core domain (h-region) and a polar C-terminal domain (c-region)
[3,4]. A net positive charge in the n-region is required for efficient
translocation across the inner membrane. The hydrophobic h-region is the most
essential part required for targeting and membrane insertion [4]. The c-region
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 [5]. The ?3,1 rule states that residues in position 3 and 1 relative to the
cleavage site must be small and uncharged, and that large, bulky residues might
reside in position 2. The tendency to conserve a distance of four to five
residues from the h/c boundary to the cleavage site might reflect interaction
between the c-region and the active site of signal peptidase [3,4].The silk gland is a secretory organ of silkworm. Three kinds of silk
proteins, fibroin heavy chain (FibH), fibroin light chain and
fibrohexamerin/P25 are synthesized and secreted by the posterior silk gland
(PSG) [6,7]. The SP of FibH was not clear for a long period. The N-terminal
amino acids of FibH has been studied by traditional chromatography methods, but
gave indefinite results [8]. A potential SP of FibH indicated by EMBL (P05790)
is the first 21 amino acid residues according to the nucleotide sequence of the
FibH gene (GenBank accession No. AF226688) [9]. This was confirmed
experimentally in our laboratory recently using recombinant baculovirus as
vector [10].To study the essential region required for secretion of silk
proteins and foreign proteins in the silk gland of silkworm, the SP sequence of
FibH was systematically shortened from the C-terminal. fusion genes of SP of different length and enhanced green
fluorescent protein (SP-EGFPs) were delivered into silkworm silk gland using
recombinant AcMNPV as a transfer vector. The SP cleavage sites were determined
by N-terminal sequencing for fusion proteins.
Materials and Methods
Plasmids and gene sequences
Plasmid p5L was cloned previously in our laboratory. It contains the
FibH sequence from –874 to +1484 bp including the promoter sequence, exon 1, intron sequence
and partial exon 2 sequence, encoding N-terminal 163 a.a. residues of FibH.
Plasmid p5LEGFPhis was constructed from p5L fused with the Egfp gene and
a His-tag coding fragment. Plasmid pEGFPhis containing Egfp gene,
His-tag coding fragment and SV40 polyA site sequence was constructed in our
previous work [10].
Predictive analysis of SP
The N-terminal 70 a.a. of FibH and different fusion proteins,
SP-EGFPs, were analyzed using the SignalP 3.0 online prediction program (http://www.cbs.dtu.dk/services/SignalP/)
[11,12].
SP shortening by PCR and
construction of recombinant genes
Promoter of FibH and SP encoding sequence of different lengths (1,
8–12,
16, 18 and 20 a.a.) were cloned from plasmid p5L by PCR using primers listed in
table 1. BamHI site
was introduced downstream from the SP coding sequences. All PCR products were
cloned into pGEM-T vector (Promega, Madison, USA) and verified by nucleotide
sequencing (Shanghai BioAisa Biotechnology, Shanghai, China). EGFPhis coding
fragment cut from plasmid pEGFPhis was fused to the signal sequence by BglII
site to form the expression cassette (Fig. 1). The blocked cut site of BamHI/BglII
formed a Gly-Ser linker between FibH SP and EGFP. The deduced amino acid
sequences of different SP-EGFPs are shown in Fig. 1.
Production of recombinant
baculovirus
Recombinant baculovirus was generated using the Bac-to-Bac system
(Invitrogen, Carlsbad, USA). Plasmid pFFa2 was derived from pFastBacHTa
(Invitrogen) with its polyhedrin promoter deleted in our previous work [13].
Expression cassettes of EGFP with FibH SP of different lengths were cloned into
donor vector pFFa2, then transferred into Escherichia coli DH10BacDEGT component
cells to make recombinant bacmids. Purified bacmids were used to transfect Sf9
cultured cells with Cellfectin (Invitrogen) to produce recombinant virus. All
of these procedures referred to our previous works [10,13,14] and Invitrogen? instruction manual. Virus
titer was determined by the Tissue Culture Infectious Dose 50 method (Adeno
Vator Vector System Applications Manual; Qbiogene, Carlsbad, USA), and Sf9
cells were maintained in Grace? medium (Invitrogen) supplemented with 10% fetal bovine serum
(Invitrogen) at 27 ?.
Silkworm inoculation and
dissection
Silkworm larvae (54A bivotine, Japanese strain) provided by
the Sericultural Research Institute, Chinese Academy of Agricultural Sciences (Zhenjiang,
China) were reared on mulberry leaves at 25 ?. The recombinant baculovirus was injected into the hemocoele of
newly ecdysed fifth instar silkworm larvae with a syringe at the amount of 106 pfu per larva. Approximately 5 d post-injection, the fluorescence
of EGFP in silk gland of the silkworm was observed and photographed with
fluorescence microscope (model MZ FL III; Leica, Heerbrugg, Switzerland) after
dissection.
Protein purification by Ni-NTA
system
Silk glands dissected from silkworm larvae were rinsed in cold
sterile distilled water several times to remove adhesive plasma. The PSGs were
homogenized with ddH2O, insoluble materials were removed by
centrifugation at 16,000 g for 10 min; and the supernatant was
lyophilized to a small volume and stored at 4 ? for a few hours and then centrifuged again. This cycle was repeated
several times until no insoluble materials appeared. The ultimate supernatant
was purified through the Ni-NTA Purification System (Invitrogen). All
operations were according to the instruction manual. Two milliliters of resin
was washed with ddH2O several times and balanced with 1?ative Purification Buffer
(50 mM NaH2PO4, pH 8.0, 0.5 M NaCl) before use. Sample for
purification was mixed with 1/4 volume of 5?ative Purification Buffer, centrifuged and bound in the column for
30–60
min; the column was washed with 8 ml Native Wash Buffer (50 mM NaH2PO4, pH 8.0, 0.5 M NaCl, 20 mM imidazole) four times;
and the column was eluted for the target protein by using 12 ml of Native
Elution Buffer (50 mM NaH2PO4, pH
8.0, 0.5 M NaCl, 250 mM imidazole); the fractions were detected for
fluorescence of EGFP with a fluorescence spectrophotometer (F-4010; Hitachi,
Tokyo, Japan) using 490 nm as the excitation wavelength and 510 nm as the
emission wavelength. The fractions with fluorescent peak were collected and
dialyzed against ddH2O at 4 ? overnight, then concentrated by lyophilizing, and stored at –20 ? for further analysis.
SDS-PAGE and Western blot
analysis
Silk gland homogenates or column purified protein samples were
subjected to SDS-PAGE as described by Laemmli [15]. Proteins were transferred onto
polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore, Billerica,
USA). The membrane was stained by Coomassie brilliant blue R-250, destained in
50% MeOH, and fully destained in 100% MeOH after photography. Western blot
analysis was carried out using horseradish peroxidase-linked mouse monoclonal
antibody GFP (B-2) (Santa Cruz Biotechnology, Santa Cruz, USA) and developed
using 3,3‘-diaminobenzidine-tetrachloride (DAB) reagent. After
photography the membrane was re-stained using Coomassie brilliant blue R-250.
N-terminal sequencing
For N-terminal sequencing, proteins were transferred onto PVDF
membranes from polyacrylamide gel in CAPS buffer [10 mM
3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 10% MeOH, pH 11.0] as described
by Matsudaira [16] with a setting of 90 V and 300 mA for 3 h in a transfer tank
(VE-186; Tanon Science & Technology, Shanghai, China). The band of EGFP
fusion protein on PVDF membranes was characterized with Western blotting of
the control lane and excised from the membrane for sequencing on a protein
N-terminal sequencer (PE491A; Applied Biosystems, Foster, USA) at the Research
Center for Proteome Analysis, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences (Shanghai, China).
Results
Construction of recombinant
AcMNPVs for expression of EGFP with different lengths of FibH SP
The SP of FibH is a typical kind of eukaryotic signal peptide and
composed of 21 amino acid residues; it contains two positive amio acid residues
in its n-region (R and K), 11 hydrophobic amino acid residues in its h-region,
and five amino acid residues in its c-region. It obeys the ?3,1 rule with small and
polar amino acid residues, Gly and Thr, at its 1 and 3 positions. The
recombinant AcMNPVs for expression of EGFP with FibH SP of different lengths
were constructed as described in ?aterials and methods. Their structures are shown in Fig.
1.
Expression of EGFP with
different lengths of FibH SP in PSG of silkworm
Fusion protein of EGFP with full length FibH SP (5LEGFPhis) could be
secreted to the lumen of PSG of silkworm normally. Fluorescence of EGFP was
observed in the PSG lumen but not in the cells under fluorescence microscope [Fig.
2(A)]. When the SP of FibH was shortened stepwise to 20, 18 and 16 a.a.
long, the secretion of the fusion proteins was not affected; and the fluorescence
profiles were the same as the full length FibH SP fusion protein (photograph
not shown). when the SP of FibH
was shortened to 12 a.a., fluorescence was seen in both the lumen and PSG cells
[Fig. 2(B)]. Fusion proteins with 8–11 a.a. long SP of FibH
could not be secreted into the lumen, and fluorescence could only be observed
in the cells [Fig. 2(C), SP11EGFP]. It was the same as the protein
SP1EGFP without the FibH SP [Fig. 2(D)].
SDS-PAGE and western
blot analysis showed that the secreted proteins (SP20EGFP, SP18EGFP and
SP16EGFP) had a molecular weight of approximately 28 kDa; and only SP12EGFP had
two bands, one of which was approximately 2 kDa larger than the other. The
fusion protein with a 163 a.a. long N-terminal (5LEGFPhis) had a molecular
weight of approximately 53 kDa (Fig. 3) as previously reported [10].
Protein purification and
N-terminal sequencing
Two fusion proteins (SP12EGFP and SP16EGFP) were purified using the
Ni-NTA system. SDS-PAGE and western
blot analysis showed the results of purification (Fig. 4). SP12EGFP had
two bands near the calculated molecular weight and some small bands that might be
the products of degradation. The purified SP16EGFP protein had only one main
band near the prospective place (Fig. 4). The major bands of SP16EGFP
and SP12EGFP were cut for N-terminal sequencing. N-terminal sequences of these
two proteins were MVSKGEELFT and EELFTGVVPI, respectively. The cleavage site
was between amino acid residues 18 and 19, and behind two linker amino acid
residues (Gly and Ser) in protein SP16EGFP; and the cleavage site was between
Gly19 and Glu20 in protein SP12EGFP, which were Gly5 and Glu6 in EGFP. It
seemed that the amino acid residues in the linker and EGFP were used as part of
the SP in this case, which indicated that the cleavage site moved behind when
the SP of FibH was shortened.
Discussion
SPs have conserved features, and different signal peptide sequences
through common secretory pathways can be interchanged between different
proteins or even proteins of different organisms [17,18]. This work showed that
the SP of FibH of silkworm shares the same structural feature with other
eukaryotic secretory proteins. The h-region of the SP is essential for
recognition by SRPs. When the h-domain was shortened to a limit, the SRPs could
not recognize it and the fusion protein failed to secrete as observed in this
work with SP8EGFP–SP11EGFP. The cleavage of the SP from nascent preprotein is
catalyzed by signal peptidase, and the c-region of the SP is important for
cleavage by signal peptidase, so a change in this region might alter the
cleavage site of the SP. The h-region is also very important for the binding
of signal peptidase. The distance between the h- and c-region boundaries to the
cleavage site is required for catalyzing proper cleavage by signal peptidase.
When the h-region is shortened and becomes unsuitable, signal peptidase will
find a vicarious cleavage site behind if possible. In this work, with SP12EGFP,
for example, the cleavage site moved to between Gly5 and Glu6 of EGFP .Several programs have been developed for the prediction of SPs but
with varying accuracy [12,19]. A comparison of the experimental results of this
work and the predictive results is shown in table 2. We found that the accuracy of cleavage site
prediction has been improved notably in the new version of SignalP 3.0 HMM. As
predicted by SignalP 3.0 HMM, when the SP is shortened from the C-terminal then
linked to EGFP through the two amino acid linker (Gly-Ser), the secretory
machinery might find a suitable sequence as the h-region of the SP for binding
of SRP, then cleave at several a.a. behind by signal peptidase. results in this experiment showed that
SP20EGFP, SP18EGFP, SP16EGFP and SP12EGFP could be secreted into PSG lumen like
5LEGFPhis, but SP11EGFP, SP10EGFP, SP9EGFP and SP8EGFP could not. The
probabilities of an SP for SP12EGFP and SP11EGFP predicted by SignalP 3.0 HMM
were 0.875 and 0.719, respectively. The only difference between SP12EGFP and
SP11EGFP is the lack of one Ala residue in the h-region of the SP, and the
predicted h-region is changed from 8 (FVILCCAG) to 7 (FVILCCG). According to
these results, it seems that FVILCCG is not long enough for the h-region of
this SP. This comparison shows that SignalP 3.0 HMM can provide accurate
predictions with SP20EGFP to SP12EGFP, but not with SP11EGFP. Perhaps the score
of probability for SPs should be as high as with SP12EGFP. The cleavage site of
signal peptide predicted by SignalP 3.0 HMM is showed by the maximal cleavage
site probability (Cmax). The cleavage sites and values of Cmax for SP16EGFP and
SP12EGFP predicted by SignalP 3.0 HMM are S18/M19 with a Cmax of 0.833 and
G19/E20 with a Cmax of 0.686, respectively. The results of N-terminal
sequencing of SP16EGFP and SP12EGFP agree with this prediction. study of
SPs and SP prediction is provoked by the increasing biotechnological interest
in finding a suitable expression system for large-scale production of
commercially interested proteins. Silk gland of silkworm Bombyx mori has
long been of interest for the production of large amounts of silk protein in a
tissue- and stage-specific manner. Its potential use as a bioreactor has gained
more attention [20–22]. FibH makes up 70% of the total silk, so its promoter and SP are
preferred for this kind of use. With the purpose of controlling the N-terminal
amino acid residues of mature foreign protein expressed in silk gland using a
modified FibH SP, and to characterize the functional essential sequence of the
FibH SP, the SP sequence was shortened and linked with EGFP reporter. The
c-region was mutated to two small and neutral linker amino acids, Gly and Ser,
the h-region was shortened stepwise and the n-region kept compact. For
SP16EGFP, the cleavage site is after Ser, just before the first amino acid
residue Met of EGFP. This SP sequence might be used in bioreactors for
expressing and secreting mature recombinant protein without any extra amino
acid residues at the N-terminal.
Acknowledgements
We thank Dr. Yuan Zhao
from the Sericultural Research Institute, Chinese Academy of Agricultural
Sciences for kindly providing silkworm eggs and silkworm for this work.
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