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A novel complement factor I involving in the complement system immune response from Lampetra morii
Journal Pre-proof A novel complement factor I involving in the complement system immune response from Lampetra morii Wanrong Lv, Anqi Ma, Xiaoyuan Chi, Qingwei Li, Yue Pang, Peng Su PII:
S1050-4648(19)31060-5
DOI:
https://doi.org/10.1016/j.fsi.2019.11.017
Reference:
YFSIM 6586
To appear in:
Fish and Shellfish Immunology
Received Date: 8 August 2019 Revised Date:
8 October 2019
Accepted Date: 6 November 2019
Please cite this article as: Lv W, Ma A, Chi X, Li Q, Pang Y, Su P, A novel complement factor I involving in the complement system immune response from Lampetra morii, Fish and Shellfish Immunology (2019), doi: https://doi.org/10.1016/j.fsi.2019.11.017. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Short Article
2
A novel Complement Factor I involving in the complement system
3
immune response from Lampetra morii
4 5
Wanrong Lv1, 2, 3#, Anqi Ma 1, 2, 3#, Xiaoyuan Chi1, 2, 3, Qingwei Li1, 2, 3,Yue Pang1, 2,, 3 *,
6
Peng Su1, 2, 3 **
7
1.
College of Life Sciences, Liaoning Normal University, Dalian 116081, China
8
2.
Lamprey Research Center, Liaoning Normal University, Dalian 116081, China
9
3.
Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic
10
University, Dalian 116081, China # These authors contributed equally to this work.
11 12
*
13
116081, China. Email addresses: [email protected] (Yue Pang).
14
**
15
Dalian 116081, China. Email addresses: [email protected] (Peng Su).
Corresponding author. College of Life Sciences, Liaoning Normal University, Dalian
Corresponding author. College of Life Sciences, Liaoning Normal University,
16 17
Abstract:
18
Complement factor I (CFI) is a serine protease which plays a key role in the
19
modulation of complement system and the induced-fit factor responsible for
20
controlling the complement-mediated processes. In this study, a CFI gene was cloned
21
and characterized from Lampetra morii (designated as L-CFI) at molecular and
22
cellular levels. The L-CFI protein included a factor I membrane attack complex
23
domain (FIMAC), a scavenger receptor cysteine-rich domain (SRCR), a trypsin-like
24
serine protease domain (Tryp_SPc) and 2 low-density lipoprotein receptor class A
25
domains (LDLa) which would exhibit functional similarities to CFI superfamily
26
proteins. Tissue expression profile analysis showed that L-CFI mRNA constitutively
27
expressed in all tested tissues except erythrocytes, with the predominant expression in
28
liver. The mRNA expression level of L-CFI increased significantly after Vibrio
29
anguillarum and Staphylocccus aureus stimulation. It is demonstrated that L-CFI
30
interacted with L-C3 protein and affected the deposition of L-C3 on the cell surface.
31
Furthermore, lamprey serum after deplete L-CFI and L-C3 reduced the cytotoxic
32
activity against HeLa cells. These findings suggest that L-CFI plays an important role
33
in lamprey immunity and involved in the lamprey complement system.
34
Key words: Lampetra morii, Complement factor I, C3, Complement system
35 36
1. Introduction
37
The complement system of vertebrates plays a key role in immunity，involving
38
multiple molecules such as pattern-recognition molecules (PRM), protein components,
39
pro-teases, regulators, and cell surface receptors, is essential for resisting pathogens
40
[1-4]
41
the third component, C3. In vertebrates, the complement factor I (CFI) is a regulatory
42
serine protease which degrades C3b and C4b through proteolytic cleavage to
43
complement activation cascades[5-7]. CFI usually contains 5 domains : a factor I
44
membrane attack complex (FIMAC) domain, a scavenger receptor cysteine-rich
45
domain(SRCR), a trypsin-like serine protease domain (Tryp_SPc) and 2 low-density
46
lipoprotein receptor class A domains (LDLa)[8]. It could prevent the assembly of the
47
C3 and C5 convertase enzymes to avoiding inappropriate amplification of the host
48
immune response[9]. CFI molecular structures have been characterized for many
49
species such as Homo sapiens, Chiloscyllium plagiosum, Mus musculus,
50
Oncorhynchus mykiss, Ictalurus punctatus, Cynoglossus semilaevis and Pelteobagrus
51
vachellii[10-15]. However, the information of the CFI gene, especially in lower
52
vertebrates, has been still limited.
. The critical step in the complement activation cascade relies to the activation of
53
Lamprey, one of the most primitive vertebrates, has unique phylogenetic position
54
that intermediate between urochordates and jawed vertebrates[16]. Thus, the
55
complement system of lampreys presents great complexity. Although some certain
56
key complement components are identified such as C3, mannose-binding lectin
57
(MBL), C1q, the later components (C5, C6, C7, C8, and C9) involved in the lytic
58
pathway have not been founded in lamprey. And L-C1q (Lamprey C1q) is complexed
59
with MASP-A in a Ca2+-dependent manner which differs from the C1q protein
60
previously identified in mammals[17,18]. Up to now, the information of complement
61
pathway of lamprey has not been clarified. Here, the molecular cloning and
62
characterization of a complement factor I homolog (L-CFI) from lamprey are first
63
reported, which will help us to understand how L-CFI functioned in the complement
64
system of lamprey.
65
2. Materials and methods
66
2.1 Experimental Animals and cell culture
67
Adult lamprey specimens (length: 48-60 cm, weight: 112-274 g) were collected
68
from the Songhua River in Heilongjiang Province, China, and kept in fiber-reinforced
69
plastic (FRP) tanks with running freshwater at Liaoning Normal University. Lampreys
70
were intraperitoneally injected with 1×107 of Staphylococcus aureus or Vibrio
71
anguillarum each individual. The control lampreys were injected with normal 0.01M
72
PBS. At 2 h, 8 h, 24 h, 48 h and 72 h post injection (hpi), in each group, peripheral
73
blood was collected from the caudal subcutaneous sinus of lampreys and diluted 1:1
74
with 0.01M PBS, 30 mM EDTA. In brief, ficollPaque is a well-referenced media for
75
density gradient centrifugation of blood, and buffy coat leukocytes were extracted
76
after 10 min centrifugation at 160 g by Ficoll-Paque gradient centrifugation using the
77
Ficoll-Paque medium (1.092 g/ml)[19].
78
HeLa cells were maintained in RPMI 1640 medium (Sigma-Aldrich). The media
79
was supplemented with 10% fetus bovine serum (Sigma-Aldrich), 100 U/mL
80
penicillin (Sigma- Aldrich) and 100 mg/mL streptomycin (Sigma-Aldrich). Cells were
81
cultured in an incubator humidified with 5% CO2 and 95% air at 37 °C.
82
2.2 Cloning L-CFI gene and bioinformatics analysis
83
Total RNA was extracted from lamprey liver and converted to cDNA with reverse
84
transcriptase (TaKaRa, Japan). PCR was carried out with the setting: 94 °C for 5 min,
85
followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min and final
86
extension
at
72
°C
for
10
min.
The
L-CFI
forward:
87
5’-GCTCTTTCCAGCCTCTCCTCCT-
88
5’-CGCCTCTGATTAGCACGGTTTGG- 3’. The PCR product was purified and
89
attached to the pMD 19-T vector using a DNA ligation kit (TaKaRa, Japan) prior to
90
DNA sequencing.
91
3’,
reverse:
Homology sequences of CFI were obtained from NCBI. Functional domain
92
analyses
of
CFI
were
conducted
by
using
online
tools
at
93
http://smart.embl-heidelberg.de and aligned by using BioEdit sequence alignment
94
editor. The results of BioEdit were also converted into FASTA format and imported
95
into MEGA 7.0 to construct a phylogenetic tree using the Neighbor-Joining method.
96
The numbers on the branches were bootstrap probabilities in per cent. The conserved
97
motifs were analyzed on-line using MEME version 5.0.5 (http://meme-suite.org/).
98
2.3 Semi-quantitative
99
For the tissue-specific spatial mRNA expression analysis, healthy mullets were
100
dissected and the kidney, supraneural body, liver, gill, intestine, heart, leukocytes and
101
erythrocytes were carefully removed. The cDNA was synthesized using a High
102
Fidelity PrimeScript™ RT-PCR Kit (TaKaRa, Japan) with Oligo dT Primer. L-CFI
103
and GAPDH are amplified in these tissues by PCR. PCR was carried out with the
104
setting: 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s and
105
72 °C for 2 min and final extension at 72 °C for 10 min. The L-CFI forward:
106
5’-GCTCTTTCCAGCCTCTCCTCCT-
107
5’-CGCCTCTGATTAGCACGGTTTGG- 3’. L-GAPDH was amplified as an internal
108
control. The L-GAPDH forward: 5’-AACCAACTGCCTGGCTCCT- 3’, reverse:
109
5’-GTCTTCTGCGTTGCCGTGT- 3’ .
110
2.4 Quantitative real-time PCR
3’,
reverse:
111
Total RNA was extracted from lamprey leukocytes stimulated. Reverse
112
transcription was then performed as previously described. PCR was performed in a 20
113
µL reaction volume, containing 2 µL of 1:10 diluted cDNA, 0.4 µL of 10 mM F
114
primer, 0.4 µL of 10 mM R primer, 10 µL of 2×SYBR Premix Ex Taq (TaKaRa, Japan)
115
and 7.2 µL of ddH2O. Amplification was performed in a PCR Thermal Cycler Dice
116
Real Time System (TaKaRa, Japan). Realtime PCR was performed in triplicate under
117
following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for
118
30 s, 72 °C for 30 s. The L-CFI forward: 5’-GCTCTTTCCAGCCTCTCCTCCT- 3’,
119
reverse: 5’-CGCCTCTGATTAGCACGGTTTGG- 3’. The L-GAPDH forward:
120
5’-AACCAACTGCCTGGCTCCT- 3’, reverse: 5’-GTCTTCTGCGTTGCCGTGT-
121
3’ .
122
2.5 Expression and purification of the L-CFI protein
123
E.coli Rosettablue containing the L-CFI plasmid was cultured in LB broth
124
containing
30
mg/mL
ampicillin
(Sangon,
China)
at
37
°C,
and
125
isopropyl-b-D-thiogalactopyranoside (IPTG) (Sangon, China) was added to induce
126
protein expression. Cells were resuspended and crushed in binding buffer ( 20 mM
127
Tris-HCl, 150 mM NaCl, 20 mM imidazole, 8 M Urea, pH 8.0). The soluble
128
supernatant was collected and subjected to a Ni-NTA His-Bind resin column
129
(Novagen, USA); the recombinant L-CFI protein was collected in elution buffer ( 20
130
mM Tris-HCl, 150 mM NaCl, 8 M Urea and 400 mM imidazole pH 8.0). The purified
131
L-CFI protein was then analyzed by 12% sodium dodecyl sulfate polyacrylamide gel
132
electrophoresis (SDS-PAGE) and stained with coomassie brilliant blue (Sangon,
133
China).
134
2.6 Purification of anti-L-CFI polyclonal antibody and western blot analysis
135
Rabbits were injected with L-CFI recombinant protein after emulsification on ice
136
with an equal volume of Freund's complete adjuvant and Freund's incomplete
137
adjuvant.Blood was collected from the ear vein of the immunized rabbit, and the titer
138
was detected by ELISA. Antigen administration and ELISA were performed as
139
previously described[20]. After the titer reached the requirement, a large amount of
140
blood was taken for antibody purification. The serum was diluted 10-fold with 0.01M
141
PBS and mixed with the Protein G SefinoseTM purification column (Sangon, China).
142
The antibody was purified from the purification column with an elution buffer (0.1 M
143
Gly, pH 2.7).
144
Samples were analyzed via 12% SDS-PAGE and transferred to nitrocellulose filter
145
membrane (Millipore). Following incubation with anti-L-CFI antibody and anti-L-C3
146
antibody, the protein was detected by HRP-labeled goat anti-rabbit antibody and was
147
revealed via ECL (Pierce, USA). Anti-L-C3 antibody was prepared by our laboratory
148
previously and used in published literature[21].
149
2.7 Co-Immunoprecipitation
150
Lamprey serum and IgG antibody with Protein G were incubated to remove
151
non-specific binding. The supernatant was incubated with L-CFI or L-C3 antibody
152
overnight at 4 °C and then incubated with Protein G for 6 h. After centrifugation,
153
supernatant and precipitate were taken to prepare samples. Western blot detected the
154
presence of L-C3 or L-CFI in the samples.
155
2.8 Cell deposition experiment
156
The rabbit erythrocytes separated by gradient centrifugation were incubated with
157
lamprey serum or L-CFI protein and lamprey serum at room temperature for 5 h.
158
Cells were subjected to protein extraction from the cell membrane (Beyotime
159
Company, China) and the samples were prepared. Western blot detected the
160
deposition of L-C3 on the cell surface.
161
2.9 High content imaging assay
162
Untreated HeLa cells were used as a negative control group. HeLa cells were
163
incubated with naive serum as a positive control group. For depletion of IgG, L-CFI
164
or L-C3, serum was incubated with IgG, L-CFI or L-C3 antibody at 4°C overnight.
165
Protein G agarose was then added to the sample which was incubated at 4°C for 4 h.
166
The supernatant was collected after centrifuged as IgG, L-CFI or L-C3 depleted
167
serum. In addition, IgG, L-CFI or L-C3 were added to IgG, L-CFI or L-C3-depleted
168
serum and the mixture was incubated with HeLa cells. HeLa cells were incubated
169
with the serum treated and untreated in a 37 °C cell culture incubator for 30 min and
170
stained with propidium iodide (Beyotime, China) and hoechst (Thermo, USA) for 10
171
min at room temperature protected from light. The excitation and emission
172
wavelengths were 346 nm and 460 nm.
173
2.10 Laser scanning confocal microscopy and flow cytometry analysis
174
Lamprey’s leukocytes and liver cells were blocked with 10% donkey serum for 3 h
175
and incubation with anti-L-CFI antibody at 4 °C overnight. After overnight incubation,
176
cells were washed with 0.01M PBS. Cells were incubated with Alexa Fluor
177
488-labeled donkey anti-rabbit antibody and then stained with 4’, 6-diamidino-
178
2-phenylindole (DAPI). HeLa cells were incubated with the normal serum of lamprey
179
and the serum cleared L-CFI or L-C3 at 37 °C for 3 h, and fixed by using 4%
180
paraformaldehyde, followed by blocking with normal 10% donkey serum for 2 h and
181
incubation with anti-L-C3 antibody overnight. Cells were incubated with Alexa Fluor
182
488-labeled donkey anti-rabbit antibody and stained with DAPI. Samples were
183
captured with a Zeiss LSM 780 inverted microscope (Carl Zeiss, Germany) and
184
analyzed by using Zeiss ZEN software. Samples were also analyzed on a FACSAria
185
flow cytometer (BD Biosciences, USA).
186
2.11 Mass spectrometry
187
The purity and molecular mass of the protein were determined by 12% SDS-PAGE
188
under reduced conditions. Proteins were visualized with 0.25% Coomassie Brilliant
189
Blue R-250 in 50% methanol containing 10% acetic acid. In-gel tryptic digestion was
190
done according to the manufacturer’s protocol. The protocol of LC-MS was
191
performed as previously described[22]. All MS/MS data were analyzed using Compass
192
1.4, Data Analysis 4.1 bulid 335(Bruker, USA), and Proteinscape 3.0 (Bruker, USA)
193
for protein search. Ensembl lamprey protein database (www.ensembl.org) and NCBI
194
protein database (www.ncbi.nlm.nih.gov) were used separately to replenish each other
195
for searching.
196
2.12 Statistical analysis
197
All statistical analyses were using GraphPad Prism 7.00 software. Differences
198
between treatment groups were determined by Student’s t-test. P <0.05 was set as the
199
threshold for significance (*P<0.05, **P<0.01). Bar charts show the means ± SDs of
200
three independent experiments.
201
202
3. Results
203
3.1 Characterization of L-CFI and phylogeny analysis
204
The ORF sequence of L-CFI encoded a protein of 618 amino acids with a predicted
205
molecular mass of 68.3 kDa, as shown in Fig. S1A. The L-CFI shares 30% to 38%
206
sequence with the homolog protein group of zebrafish to human. The domain analysis
207
indicated that L-CFI contained a factor I membrane attack complex domain (FIMAC),
208
a scavenger receptor cysteine-rich domain(SRCR), a trypsin-like serine protease
209
domain (Tryp_SPc) and 2 low density lipoprotein receptor class A domains (LDLa)
210
(Fig.1A). The CFI phylogenetic tree was constructed using the full-length amino acid
211
sequences of 14 species. Through phylogenetic tree, the lamprey was at the bottom of
212
the vertebrate branch (Fig.1B). Multiple sequence alignment of the L-CFI and CFIs
213
from vertebrates, indicating that L-CFI has strongly conserved Tryp_SPc domain (Fig.
214
1C). The comparison of CFI of vertebrates using the MEME system indicated a high
215
level of conservation in the motif composition (Fig. S1B and Table S2). The
216
vertebrate CFI all contain thirteen motifs (motif 1,2,3,4,5,6,7,8,9,10,11,12 and 14 ),
217
whereas motif 15 are not found in L-CFI and Dr-CFI, and motif 13 are not found in
218
L-CFI. The detailed conserved sequences of each motif are shown in Table. S2.
219
3.2 Purification of recombinant L-CFI and the subcellular location of L-CFI
220
The recombinant plasmid (pCold I-L-CFI) was transformed into Rosetta blue and
221
analyzed by SDS-PAGE, a distinct band was revealed with a molecular mass of
222
68kDa (Fig. S2A), which was in accordance with the predicted molecular mass of
223
L-CFI. A rabbit anti-L-CFI polyclonal antibody was generated and antibody titer was
224
detected by ELISA. Plasma L-CFI antibody concentrations were increased by
225
640,000-fold over preimmunization levels, and pre-immunized rabbit IgG was used as
226
a negative control (Fig. S2A). Western blotting showed that the anti-L-CFI antibody
227
recognized native CFI protein from lamprey leukocytes and supraneural body tissues
228
(Fig. S2B). The endogenous localization of L-CFI in leukocytes and liver cells was
229
detected by immunofluorescence and confocal laser-scanning microscopy. The
230
nucleus were stained with DAPI and observed in blue, while the positive signals of
231
L-CFI in green were distributed in nucleus and cytoplasm (Fig. 2B).
232
3. 3 Bacterial infection induces L-CFI expression in leukocytes
233
In the present study, the mRNA expression of L-CFI in kidney, gill, intestine, heart,
234
liver,
supraneural
body,
leukocytes
and
erythrocytes
were
analyzed
by
235
semi-quantitative RT-PCR, the L-GAPDH gene was used as an internal control. As the
236
results showed that the L-CFI was detected with high expression in liver, kidney,
237
intestine and leukocytes, and not examined in erythrocytes (Fig. 2A).
238
To further investigate the immune response of L-CFI to pathogenic infection,
239
lampreys were challenged with Vibrio anguillarum and Staphylocccus aureus,
240
respectively. As shown in Fig. 2C, a significant decrease mRNA level of L-CFI at 2 h
241
S. aureus and V. anguillarum injection (hpi) was detected (P < 0.05). Later on, from 8
242
hpi, the expression of L-CFI at both 2 challenged groups were remarkably elevated (P
243
< 0.05). The results showed that the expression of L-CFI increased after challenged
244
with S. aureus and V. anguillarum in the adult lamprey leukocytes, when compared
245
with 0 h normal controls (Fig. 2C).
246
3. 4 Interaction between L-CFI and L-C3
247
Co-immunoprecipitation and mass spectrometry were used to detect the interaction
248
between L-C3 and L-CFI. As shown in Fig.3A, the results confirmed that L-CFI
249
interacted directly with L-C3 in lamprey serum and mass spectrometry analysis
250
further confirmed the interaction between L-C3 and L-CFI (Table. S1). In order to
251
investigate whether L-CFI can affect the function of L-C3, western blot assay of
252
L-CFI and L-C3 were performed. L-CFI and lamprey serum were incubated with
253
rabbit erythrocytes, the deposition of L-C3 on rabbit erythrocytes was detected by
254
L-C3 polyclonal antibody. The result showed that the expression of L-C3 in lamprey
255
serum were decreased after treated with recombinant L-CFI protein, suggesting L-CFI
256
affected the activity of L-C3 depositing on the cell surface (Fig. 3B).
257
3.5 Lamprey serum reduced the cytotoxic activity after depleting L-CFI and
258
L-C3
259
Firstly, L-CFI and L-C3 were respectively depleted in lamprey serum to test the
260
killing effect on HeLa cells. The results showed that compared with the untreated
261
group, the killing degree of the L-CFI and L-C3 depleted groups was significantly
262
reduced, and recovered after adding L-CFI back (Fig. 3C). In addition, fluorescent
263
confocal and flow cytometry analysis confirmed that the C3 fluorescence intensity of
264
eliminating L-CFI serum was reduced (Fig. 3D and Fig. S3). These results revealed
265
that L-CFI could be associated with the function of L-C3 to kill HeLa cells and reduce
266
the biological activity of L-C3.
267
4. Discussion
268
In this study, L-CFI was fully identified and characterized from Lampetra morii for
269
the first time. Complement Factor I (CFI) is a family of soluble serine protease which
270
plays a crucial role in the modulation of complement cascades. Previous studies
271
showed high conservation of CFI genes in different vertebrates, including the teleost,
272
cartilaginous fish and other species[14,23].The CFI protein always contains FIMAC ,
273
SRCR, Tryp_SPc, LDLr1 and LDLr2 domain.
274
shows 10 Cys residues of the FIMAC module are also conserved in CFIs (Fig. S1B).
275
The 10 Cys residues characterized in this module may contribute to disulfide bridge
276
formation[24]. Thus, we speculate that FIMAC may perform similar functions in L-CFI
277
and FIMAC in Hs-CFI. However, the species-specific sequence repeats between the
278
signal peptide and FIMAC domain reported in cartilaginous fish, such as three repeat
279
sequences
280
residues(VSPTGESNETMSSTQA) in Triakis scyllium did not exist in L-CFI[25]. It
281
might be a reflection of structural and functional diversity of L-CFI in lower jawless
282
vertebrate. As shown in Fig. S1A, L-CFI glycosylation pattern contains 3 N-linked
283
glycosylation sites less than other teleost fish such as 7 of Cynoglossus semilaevis and
284
5 of Danio rerio[14,26]. The homologous sequences in mammals such as human, mouse,
285
and rat have 6, 6 and 7 N-linked glycosylation sites, respectively[11,27,28]. However,
286
cartilaginous fish G. cirratum contains 13 N-linked glycosylation sites in its CFI
287
pre-protein[13]. These results imply that the number of N-linked glycosylation sites in
288
CFI of vertebrates is significantly different.
(RS
1-
3)
in
Nurse
The alignment of 7 vertebrates
shark[13],
a
direct
repeat
of
16
289
Phylogenetic analysis of the sequences indicates that L-CFI shows higher
290
similarity with homologous CFI sequences from cartilaginous fish than those from
291
teleosts, amphibians and mammals (Fig. 1B). Moreover, the phylogenetic trees
292
confirmed that the L-CFI was located in the outer group of vertebrates, indicating that
293
the L-CFI was the original gene of vertebrates. The analysis implies the evolutionary
294
position of L-CFI and the cluster is roughly consistent with the evolution relationships
295
of the species[29].
296
The detection of L-CFI in tissues reveals its wide and constitutive expression in
297
Lampetra morii except erythrocytes. Specially, the highest expression is detected to be
298
in liver, which is consistent with the report that CFI gene also mainly expresses in the
299
liver of human, shark and other teleosts as it is mainly synthesized in the liver [14,30-31].
300
Therefore, these results suggest that the liver may be a major part of the immune
301
system in lamprey. However, results showed that L-CFI did not express in
302
erythrocytes, so we speculated that L-CFI could not play an important function in
303
erythrocytes. In addition, we intended to confirm whether the L-CFI was involved in
304
the immune response against Vibrio anguillarum and Staphylococcus aureus. After
305
stimulation with Vibrio anguillarum and Staphylococcus aureus, L-CFI in leukocytes
306
showed a significant immune response (Fig. 2C). Similar to the results previously
307
observed in Cynoglossus semilaevis, the CFI mRNA expression was strongly
308
up-regulated throughout the challenge period in Cynoglossus semilaevis. Therefore,
309
these results suggest that L-CFI plays an important role in resisting Gram-positive
310
bacteria and Gram-negative bacteria[32-34].
311 312 313
The complement is a multi-functional complex system comprising more than 30
314
proteins present mainly in serum and cell membranes[35]. However, the later
315
components (C5, C6, C7, C8, and C9) involved in the lytic pathway have not been
316
founded in lamprey, even not in the genome of Petromyrzon marinus. Therefore, it
317
appears that the complement system of lamprey is very unique and may have
318
developed independently from jawed vertebrates. Taken together, the current study
319
provided the evidence for the possible involvement of L-CFI in lamprey complement
320
system through interacting and affecting the deposition of L-C3 on the cell surface.
321
The findings suggest potentially productive and intriguing avenues for future research.
322
Herein, the regulatory complement mechanism of lamprey remains to be fully
323
elucidated.
324 325 326
Acknowledgement
327
This work was funded by the Chinese National Natural Science Foundation Grants
328
(No.31772884 and No.31801973); Chinese Major State Basic Research Development
329
Program (973 Program; Grant 2013CB835304); the Marine Public Welfare Project of
330
the State Oceanic Administration (No. 201305016). The project of Department of
331
Ocean and Fisheries of Liaoning Province (No. 201805), and Science and Technology
332
Innovation Fund Research Project (No. 2018J12SN079).
333 334
References
335
[1] Ricklin D, Lambris JD, Complement in immune and inflammatory disorders:
336
pathophysiological mechanisms, J Immunol. 190 (8) (2013) 3831-3838.
337
[2] Carroll MC, The complement system in regulation of adaptive immunity, Nat
338
Immunol. 5(10) (2004) 981-986.
339
[3] Botto M, Kirschfink M, Macor P, et al, Complement in human diseases: lessons
340
from complement deficiencies, Mol Immunol. 46 (14) (2009) 2774-2783.
341
[4] Alper CA, Rosen FS, Lachmann PJ, Inactivator of the third component of
342
complement as an inhibitor in the properdin pathway, Proc Natl Acad Sci USA. 69 (10)
343
(1972) 2910-2913.
344
[5] Shiang RI, Murray JC, Morton CC, et al, Mapping of the human complement
345
factor I gene to 4q25, Genomics. 4 (1) (1989) 82-86.
346
[6] Ullman CG, Perkins SJ, et al, The Factor I and follistatin domain families: the
347
return of a prodigal son, Biochem J. 326 ( Pt 3) (1997) 939-941.
348
[7] Catterall CF, Lyons A, Sim RB, et al, Characterization of primary amino acid
349
sequence of human complement control protein factor I from an analysis of cDNA
350
clones, Biochem J. 242 (3) (1987) 849-856.
351
[8] Nakao M, Hisamatsu S, Nakahara M, et al, Molecular cloning of the complement
352
regulatory factor
353
Immunogenetics. 54 (11) (2003) 801-806.
354
[9] Gros P, Milder FJ, Janssen BJ, Complement driven by conformational changes,
355
Nat Rev Immunol. 8 (1) (2008) 48-58.
356
[10] Tsiftsoglou SA, Arnold JN, Roversi P, et al, Human complement factor I
357
glycosylation:
358
oligosaccharides, Biochim Biophys Acta. 1764 (11) (2006) 1757-1766.
359
[11] Schlaf G, Rothermel E, Oppermann M, et al, Rat complement factor I: molecular
360
cloning, sequencing and expression in tissues and isolated cells, Immunology. 98 (3)
361
(1999) 464-474.
362
[12] Anastasiou V, Mikrou A, Papanastasiou AD, et al, The molecular identification of
363
factor H and factor I molecules in rainbow trout provides insights into complement
364
C3 regulation, Fish Shellfish Immunol. 31(3) (2011) 491-499.
365
[13] Shin DH, Webb BM, Nakao M, et al, Characterization of shark complement
366
factor I gene(s): genomic analysis of a novel shark-specific sequence, Mol Immunol.
367
46 (11-12) (2009) 2299-2308.
368
[14] Xiang J, Li X, Chen Y, et al, Complement factor I from flatfish half-smooth
369
tongue (Cynoglossus semilaevis) exhibited anti-microbial activities, Dev Comp
370
Immunol. 53 (1) (2015) 199-209.
371
[15] Qin C, Gong Q, Wen Z, et al, Molecular characterization and expression of
372
complement factor I in Pelteobagrus vachellii during Aeromonas hydrophila infection,
373
Dev Comp Immunol. 82 (2018) 66-71.
374
[16] Gustafsson OS, Collin SP, Kröger RH, Early evolution of multifocal optics for
375
well-focused colour vision in vertebrates, J Exp Biol. 211 (Pt 10) (2008) 1559-1564.
376
[17] Matsushita M, The Complement System of Agnathans, Front Immunol. 9 (2018)
377
1405.
378
[18] Boehm T, McCurley N, Sutoh Y, et al, VLR based adaptive immunity, Annu Rev
379
Immunol. 30 (2012) 203–20.
380
[19] Wang Z, Lu J, Li C, et al, Characterization of the LECT2 gene and its protective
I isotypes
structural
and
from
the
functional
common
carp
characterisation
(Cyprinus
of
the
carpio),
N-linked
381
effects against microbial infection via large lymphocytes in Lampetra japonica, Dev
382
Comp Immunol. 79 (2018) 75-85.
383
[20] Wang D, Gou M, Hou J, et,al. The role of serpin protein on the natural immune
384
defense against pathogen infection in Lampetra japonica, Fish Shellfish Immunol. 92
385
(2019):196-208.]
386
[21] Wu F, Chen L, Liu X, et,al. Lamprey Variable Lymphocyte Receptors Mediate
387
Complement-Dependent Cytotoxicity, J Immunol . 190(2013): 922-930
388
[22] Li B, Gou M, Han J, et al, Proteomic analysis of buccal gland secretion from
389
fasting and feeding lampreys (Lampetra morii), Proteome Sci. 22 (2018) 16-19.
390
[23] Sjöberg AP, Trouw LA, Blom AM, Complement activation and inhibition: a
391
delicate balance, Trends Immunol. 30 (2) (2009) 83-90.
392
[24] Haefliger JA, Tschopp J, Vial N, et al, Complete primary structure and functional
393
characterization of the sixth component of the human complement system.
394
Identification of the C5b-binding domain in complement C6, J Biol Chem. 264 (30)
395
(1989) 18041-18051.
396
[25] Terado T, Nonaka MI, Nonaka M, et al, Conservation of the modular structure of
397
complement factor I through vertebrate evolution, Dev Comp Immunol. 26 (5) (2002)
398
403-413.
399
[26] Strausberg RL, Feingold EA, Grouse LH, et al, Generation and initial analysis of
400
more than 15,000 full-length human and mouse cDNA sequences, Proc Natl Acad Sci
401
U S A. 99 (26) (2002) 16899-16903.
402
[27] Catterall CF, Lyons A, Sim RB, et al, Characterization of primary amino acid
403
sequence of human complement control protein factor I from an analysis of cDNA
404
clones, Biochem J. 242 (3) (1987) 849-56.
405
[28] Minta JO, Wong MJ, Kozak CA, et al, cDNA cloning, sequencing and
406
chromosomal assignment of the gene for mouse complement factor I (C3b/C4b
407
inactivator): identification of a species specific divergent segment in factor I, Mol
408
Immunol. 33 (1) (1996) 101-112.
409
[29] Huang S, Yuan S, Guo L, et al, Genomic analysis of the immune gene repertoire
410
of amphioxus reveals extraordinary innate complexity and diversity, Genome Res. 18
411
(7) (2008) 1112-1126.
412
[30] Nilsson SC, Nita I, Månsson L, et al, Analysis of binding sites on complement
413
factor I that are required for its activity, J Biol Chem. 285 (9) (2010) 6235-6245.
414
[31] Morris KM, Aden DP, Knowles BB, et al, Complement biosynthesis by the
415
human hepatoma-derived cell line HepG2, J Clin Invest. 70 (4) (1982) 906-913.
416
[32] Xiang J, Li X, Chen Y, et, al. Complement factor I from flatfish half-smooth
417
tongue (Cynoglossus semilaevis) exhibited anti-microbial activities, Dev. Comp.
418
Immunol. 53(2015), 199-209.
419
[33] Wang Y, Chen B, Ke Y, et, al. Molecular characterization and expression analysis
420
of the complement factor I (CpFI) in the white spotted bamboo shark (Chiloscyllium
421
plagiosum), Fish Shellfish Immunol. 40(2014): 414-423.
422
[34] Qin C, Gong Q, Wen Z, et, al. Molecular characterization and expression of
423
complement factor I in Pelteobagrus vachellii during Aeromonas hydrophila infection,
424
Dev Comp Immunol. 82(2018):66-71.
425
[35] Marina B, Michael K, Paolo M, Complement in human diseases: Lessons from
426
complement deficiencies, Mol. Immunol. 46 (14) (2009): 2774-2783.
427 428
Supplementary Information
429
Supplementary Information includes Fig. S1-Fig. S3, Table S1.
430 431
Figure legends
432
Fig.1 Sequence analysis of CFI. (A) The functional domains of the L-CFI and
433
Hs-CFI according to http://smart.embl-heidelberg.de. (B) Phylogenetic tree for CFI
434
among different species by Neighbor-Joining method. Scale bar (0.1) indicates
435
genetic distance. (C) Conserved domains and motifs of CFI from different
436
species.The FIMAC domain was marked by a solid orange line, SRCR domain was
437
marked by a solid green line, LDLa domain was marked by a solid blue line and
438
Tryp_SPc domain was marked by a solid yellow line. Due to the lack of motif 13 and
439
motif 15 in lamprey and the the lack of motif 13 in zebrafish, these motifs were
440
marked with different dotted lines: motif 13 was marked by a dotted pink line and
441
motif 15 was marked by a dotted red line. Hs: Homo sapiens ; Mm: Mus musculus;
442
Tct: Terrapene carolina triunguis; Xl: Xenopus laevis; Dr: Danio rerio; Gg: Gallus
443
gallus; Cs: Cynoglossus semilaevis; Sf: Scleropages formosus; IP: Ictalurus punctatus;
444
Ac: Antrostomus carolinensis; Cp: Chiloscyllium plagiosum; Gc: Ginglymostoma
445
cirratum; Cm: Callorhinchus milii; L: Lampetra morii.
446
Fig.2 Distribution of L-CFI in lamprey tissues and cells and the immune response
447
of L-CFI by Staphylococcus aureus and Vibrio anguillarum. (A) RNA was
448
extracted from the kidney, liver, gill, intestine, heart, leukocytes and erythrocytes.
449
RNA was reverse-transcribed to cDNA and subjected to PCR. GAPDH was amplified
450
as an internal control.M: DNA marker .(B) Immunofluorescence detected L-CFI
451
localization in liver cells and leukocytes by confocal microscope (Carl Zeiss, Inc).
452
L-CFI was visualized by L-CFI antibody and Alexa Fluor 488 donkey anti-rabbit IgG
453
antibody, and the nucleus were stained with DAPI. Scale bar: 5 µm. (C) Q-PCR
454
detected the changes of L-CFI under the stimulation of Staphylococcus aureus and
455
Vibrio anguillarum. All experiments were repeated at least three times with similar
456
results (*P < 0.05, **P < 0.01).
457
Fig.3 L-CFI interacted with L-C3 and affected the deposition of L-C3 on the cell
458
membrane surface. (A) Co-Immunoprecipitation detected the interaction of L-CFI
459
and L-C3. S: supernatant; P: precipitation. (B) Western blot assay showed that L-CFI
460
protein affected the deposition on the rabbit erythrocytes surface. S: supernatant; P:
461
precipitation. (C) High content imaging assay showed that the remove of L-CFI and
462
L-C3 inhibited HeLa cells death. Serum was omitted in the positive control (*P<0.05).
463
(D) The deposition of L-C3 on HeLa cells, as determined via laser scanning confocal
464
microscopy. The primary antibody consisted of rabbit anti-L-C3 and anti-L-CFI
465
antibody, and the secondary antibody consisted of Alexa 488-labeled donkey
466
anti-rabbit antibody. The nucleus were stained with DAPI. Scale bar:10 µm.
467
Table legends
468
Table S1. Identification of L-C3 by LC-MS/MS
469
Table S2. Conserved motifs discovered in vertebrates using the MEME system
470
Supplementary figure legends
471
Fig. S1 (A) Nucleotide and amino acid sequences of L-CFI. Amino acid sequences
472
are shown below the coding regions. Three N-linked glycosylation sites were marked
473
in red circles. The FIMAC domain, SRCR domain, two LDLa domains and Tryp-SPc
474
domain were shaded in different colors. (B) The motif composition of CFI by MEME
475
version 5.0.5.
476
Fig. S2 (A) SDS-PAGE analysis of L-CFI protein expressed in E.coli Rosettablue. M:
477
molecular weight protein marker; 1: flowthrough sample; 2: equilibrium sample; 3:
478
induced expression of Rosetta blue/pCold I-L-CFI; 4: elution-1; 5: elution-2; 6:
479
elution-3; 7: elution-4. (B) Titer detection of L-CFI polyclonoal antibody by ELISA.
480
The X-axis was the concentration of the antiserum after dilution, the negative control
481
was the normal rabbit serum, the control group is the dilution; the y-axis was the
482
absorbance value of OD450 nm. (C) Analysis of specific antibody of Western blot. 1:
483
L-CFI recombinant protein; 2: liver tissue lysate of lamprey; 3: leukocytes lysate of
484
lamprey; 4: erythrocytes lysate of lamprey
485
Fig. S3 The deposition of L-C3 on HeLa cells, as determined via flow cytometry
486
analysis. The primary antibody consisted of rabbit anti-L-C3 and anti-L-CFI antibody,
487
and the secondary antibody consisted of Alexa 488-labeled donkey anti-rabbit
488
antibody.
489
490
491
492
493
494
495
496
497 498
499 500
Table S1.
Identification of L-C3 by LC-MS/MS
m/z means 969.4815 619.8336
sequence K.STDEGIVDGTSFTIPAISK.H R.EYILPTFEVK.I
945.5193 1023.5298 732.3942 642.8462 653.3690 555.8000
K.LVDSSSTTLVAGEGLSILK.K K.ATVLSSQAAETEEAELVGIK.I K.AELPYFIQVEVR.N K.VQVGSNTINPQK.M R.LGETLNVFLTAK.T R.IPITPDMAPR.F
757.8923 765.8894 794.4409
K.AMLTLDLIGEPDAR.V K.AMLTLDLIGEPDAR.V R.VGLLAVDQAVYAVNR.K
501 502
Table S2. Conserved motifs discovered in vertebrates using the MEME system Motif 1
503 504
2
sequence ACKGDSGGPLVCYDANNVAYVWGIVSWGENCGEPGFPGVYTKVAN YFDWI YTHLSCDKVFCQPWQRCVEGTCICKLPYQCPKNGTPVCSTB
3 4 5 6 7 8
ITCGGIYIGGCWVLTAAHCVR YSVPACVPWSPYLFQPGDTCTVSGWGREK DSFPVKQVIIHEKYNAATYQNDIALLELK NSFHCKSGVCIPRQYVCNGEVDCJTGEDE RCVNGKCIPEEKACDGINDCGDLSDELCCKECK NHTLTRRKRIIGGKTAEKGEFPWQVAIKD
9 10 11
MFICDSEWSMAEANVACRHLGFELGA LKWGNVNLIGNCSKFYKERFF EPSQCLHVTCRGLETSLAECTL
12 13
RSFPTYCHLKSFECLHPEKKFLNNGKCAAEEKFNVSLVYGDSESEGV VQV NYDMDAERKLJKSLLPKLSCGV
14
MECAGTYDGSI
15
SRVHRYQIWTGLLDTJKYD
The expression of L-CFI increased after challenged with S. aureus and V. anguillarum in the adult lamprey leukocytes. L-CFI could be associated with the function of L-C3 to kill HeLa cells and reduce the biological activity of L-C3. The expression of L-C3 in lamprey serum were decreased after treated with recombinant L-CFI protein, suggesting L-CFI affected the activity of L-C3 depositing on the cell surface.
S1050-4648(19)31060-5
DOI:
https://doi.org/10.1016/j.fsi.2019.11.017
Reference:
YFSIM 6586
To appear in:
Fish and Shellfish Immunology
Received Date: 8 August 2019 Revised Date:
8 October 2019
Accepted Date: 6 November 2019
Please cite this article as: Lv W, Ma A, Chi X, Li Q, Pang Y, Su P, A novel complement factor I involving in the complement system immune response from Lampetra morii, Fish and Shellfish Immunology (2019), doi: https://doi.org/10.1016/j.fsi.2019.11.017. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Short Article
2
A novel Complement Factor I involving in the complement system
3
immune response from Lampetra morii
4 5
Wanrong Lv1, 2, 3#, Anqi Ma 1, 2, 3#, Xiaoyuan Chi1, 2, 3, Qingwei Li1, 2, 3,Yue Pang1, 2,, 3 *,
6
Peng Su1, 2, 3 **
7
1.
College of Life Sciences, Liaoning Normal University, Dalian 116081, China
8
2.
Lamprey Research Center, Liaoning Normal University, Dalian 116081, China
9
3.
Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic
10
University, Dalian 116081, China # These authors contributed equally to this work.
11 12
*
13
116081, China. Email addresses: [email protected] (Yue Pang).
14
**
15
Dalian 116081, China. Email addresses: [email protected] (Peng Su).
Corresponding author. College of Life Sciences, Liaoning Normal University, Dalian
Corresponding author. College of Life Sciences, Liaoning Normal University,
16 17
Abstract:
18
Complement factor I (CFI) is a serine protease which plays a key role in the
19
modulation of complement system and the induced-fit factor responsible for
20
controlling the complement-mediated processes. In this study, a CFI gene was cloned
21
and characterized from Lampetra morii (designated as L-CFI) at molecular and
22
cellular levels. The L-CFI protein included a factor I membrane attack complex
23
domain (FIMAC), a scavenger receptor cysteine-rich domain (SRCR), a trypsin-like
24
serine protease domain (Tryp_SPc) and 2 low-density lipoprotein receptor class A
25
domains (LDLa) which would exhibit functional similarities to CFI superfamily
26
proteins. Tissue expression profile analysis showed that L-CFI mRNA constitutively
27
expressed in all tested tissues except erythrocytes, with the predominant expression in
28
liver. The mRNA expression level of L-CFI increased significantly after Vibrio
29
anguillarum and Staphylocccus aureus stimulation. It is demonstrated that L-CFI
30
interacted with L-C3 protein and affected the deposition of L-C3 on the cell surface.
31
Furthermore, lamprey serum after deplete L-CFI and L-C3 reduced the cytotoxic
32
activity against HeLa cells. These findings suggest that L-CFI plays an important role
33
in lamprey immunity and involved in the lamprey complement system.
34
Key words: Lampetra morii, Complement factor I, C3, Complement system
35 36
1. Introduction
37
The complement system of vertebrates plays a key role in immunity，involving
38
multiple molecules such as pattern-recognition molecules (PRM), protein components,
39
pro-teases, regulators, and cell surface receptors, is essential for resisting pathogens
40
[1-4]
41
the third component, C3. In vertebrates, the complement factor I (CFI) is a regulatory
42
serine protease which degrades C3b and C4b through proteolytic cleavage to
43
complement activation cascades[5-7]. CFI usually contains 5 domains : a factor I
44
membrane attack complex (FIMAC) domain, a scavenger receptor cysteine-rich
45
domain(SRCR), a trypsin-like serine protease domain (Tryp_SPc) and 2 low-density
46
lipoprotein receptor class A domains (LDLa)[8]. It could prevent the assembly of the
47
C3 and C5 convertase enzymes to avoiding inappropriate amplification of the host
48
immune response[9]. CFI molecular structures have been characterized for many
49
species such as Homo sapiens, Chiloscyllium plagiosum, Mus musculus,
50
Oncorhynchus mykiss, Ictalurus punctatus, Cynoglossus semilaevis and Pelteobagrus
51
vachellii[10-15]. However, the information of the CFI gene, especially in lower
52
vertebrates, has been still limited.
. The critical step in the complement activation cascade relies to the activation of
53
Lamprey, one of the most primitive vertebrates, has unique phylogenetic position
54
that intermediate between urochordates and jawed vertebrates[16]. Thus, the
55
complement system of lampreys presents great complexity. Although some certain
56
key complement components are identified such as C3, mannose-binding lectin
57
(MBL), C1q, the later components (C5, C6, C7, C8, and C9) involved in the lytic
58
pathway have not been founded in lamprey. And L-C1q (Lamprey C1q) is complexed
59
with MASP-A in a Ca2+-dependent manner which differs from the C1q protein
60
previously identified in mammals[17,18]. Up to now, the information of complement
61
pathway of lamprey has not been clarified. Here, the molecular cloning and
62
characterization of a complement factor I homolog (L-CFI) from lamprey are first
63
reported, which will help us to understand how L-CFI functioned in the complement
64
system of lamprey.
65
2. Materials and methods
66
2.1 Experimental Animals and cell culture
67
Adult lamprey specimens (length: 48-60 cm, weight: 112-274 g) were collected
68
from the Songhua River in Heilongjiang Province, China, and kept in fiber-reinforced
69
plastic (FRP) tanks with running freshwater at Liaoning Normal University. Lampreys
70
were intraperitoneally injected with 1×107 of Staphylococcus aureus or Vibrio
71
anguillarum each individual. The control lampreys were injected with normal 0.01M
72
PBS. At 2 h, 8 h, 24 h, 48 h and 72 h post injection (hpi), in each group, peripheral
73
blood was collected from the caudal subcutaneous sinus of lampreys and diluted 1:1
74
with 0.01M PBS, 30 mM EDTA. In brief, ficollPaque is a well-referenced media for
75
density gradient centrifugation of blood, and buffy coat leukocytes were extracted
76
after 10 min centrifugation at 160 g by Ficoll-Paque gradient centrifugation using the
77
Ficoll-Paque medium (1.092 g/ml)[19].
78
HeLa cells were maintained in RPMI 1640 medium (Sigma-Aldrich). The media
79
was supplemented with 10% fetus bovine serum (Sigma-Aldrich), 100 U/mL
80
penicillin (Sigma- Aldrich) and 100 mg/mL streptomycin (Sigma-Aldrich). Cells were
81
cultured in an incubator humidified with 5% CO2 and 95% air at 37 °C.
82
2.2 Cloning L-CFI gene and bioinformatics analysis
83
Total RNA was extracted from lamprey liver and converted to cDNA with reverse
84
transcriptase (TaKaRa, Japan). PCR was carried out with the setting: 94 °C for 5 min,
85
followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min and final
86
extension
at
72
°C
for
10
min.
The
L-CFI
forward:
87
5’-GCTCTTTCCAGCCTCTCCTCCT-
88
5’-CGCCTCTGATTAGCACGGTTTGG- 3’. The PCR product was purified and
89
attached to the pMD 19-T vector using a DNA ligation kit (TaKaRa, Japan) prior to
90
DNA sequencing.
91
3’,
reverse:
Homology sequences of CFI were obtained from NCBI. Functional domain
92
analyses
of
CFI
were
conducted
by
using
online
tools
at
93
http://smart.embl-heidelberg.de and aligned by using BioEdit sequence alignment
94
editor. The results of BioEdit were also converted into FASTA format and imported
95
into MEGA 7.0 to construct a phylogenetic tree using the Neighbor-Joining method.
96
The numbers on the branches were bootstrap probabilities in per cent. The conserved
97
motifs were analyzed on-line using MEME version 5.0.5 (http://meme-suite.org/).
98
2.3 Semi-quantitative
99
For the tissue-specific spatial mRNA expression analysis, healthy mullets were
100
dissected and the kidney, supraneural body, liver, gill, intestine, heart, leukocytes and
101
erythrocytes were carefully removed. The cDNA was synthesized using a High
102
Fidelity PrimeScript™ RT-PCR Kit (TaKaRa, Japan) with Oligo dT Primer. L-CFI
103
and GAPDH are amplified in these tissues by PCR. PCR was carried out with the
104
setting: 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s and
105
72 °C for 2 min and final extension at 72 °C for 10 min. The L-CFI forward:
106
5’-GCTCTTTCCAGCCTCTCCTCCT-
107
5’-CGCCTCTGATTAGCACGGTTTGG- 3’. L-GAPDH was amplified as an internal
108
control. The L-GAPDH forward: 5’-AACCAACTGCCTGGCTCCT- 3’, reverse:
109
5’-GTCTTCTGCGTTGCCGTGT- 3’ .
110
2.4 Quantitative real-time PCR
3’,
reverse:
111
Total RNA was extracted from lamprey leukocytes stimulated. Reverse
112
transcription was then performed as previously described. PCR was performed in a 20
113
µL reaction volume, containing 2 µL of 1:10 diluted cDNA, 0.4 µL of 10 mM F
114
primer, 0.4 µL of 10 mM R primer, 10 µL of 2×SYBR Premix Ex Taq (TaKaRa, Japan)
115
and 7.2 µL of ddH2O. Amplification was performed in a PCR Thermal Cycler Dice
116
Real Time System (TaKaRa, Japan). Realtime PCR was performed in triplicate under
117
following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for
118
30 s, 72 °C for 30 s. The L-CFI forward: 5’-GCTCTTTCCAGCCTCTCCTCCT- 3’,
119
reverse: 5’-CGCCTCTGATTAGCACGGTTTGG- 3’. The L-GAPDH forward:
120
5’-AACCAACTGCCTGGCTCCT- 3’, reverse: 5’-GTCTTCTGCGTTGCCGTGT-
121
3’ .
122
2.5 Expression and purification of the L-CFI protein
123
E.coli Rosettablue containing the L-CFI plasmid was cultured in LB broth
124
containing
30
mg/mL
ampicillin
(Sangon,
China)
at
37
°C,
and
125
isopropyl-b-D-thiogalactopyranoside (IPTG) (Sangon, China) was added to induce
126
protein expression. Cells were resuspended and crushed in binding buffer ( 20 mM
127
Tris-HCl, 150 mM NaCl, 20 mM imidazole, 8 M Urea, pH 8.0). The soluble
128
supernatant was collected and subjected to a Ni-NTA His-Bind resin column
129
(Novagen, USA); the recombinant L-CFI protein was collected in elution buffer ( 20
130
mM Tris-HCl, 150 mM NaCl, 8 M Urea and 400 mM imidazole pH 8.0). The purified
131
L-CFI protein was then analyzed by 12% sodium dodecyl sulfate polyacrylamide gel
132
electrophoresis (SDS-PAGE) and stained with coomassie brilliant blue (Sangon,
133
China).
134
2.6 Purification of anti-L-CFI polyclonal antibody and western blot analysis
135
Rabbits were injected with L-CFI recombinant protein after emulsification on ice
136
with an equal volume of Freund's complete adjuvant and Freund's incomplete
137
adjuvant.Blood was collected from the ear vein of the immunized rabbit, and the titer
138
was detected by ELISA. Antigen administration and ELISA were performed as
139
previously described[20]. After the titer reached the requirement, a large amount of
140
blood was taken for antibody purification. The serum was diluted 10-fold with 0.01M
141
PBS and mixed with the Protein G SefinoseTM purification column (Sangon, China).
142
The antibody was purified from the purification column with an elution buffer (0.1 M
143
Gly, pH 2.7).
144
Samples were analyzed via 12% SDS-PAGE and transferred to nitrocellulose filter
145
membrane (Millipore). Following incubation with anti-L-CFI antibody and anti-L-C3
146
antibody, the protein was detected by HRP-labeled goat anti-rabbit antibody and was
147
revealed via ECL (Pierce, USA). Anti-L-C3 antibody was prepared by our laboratory
148
previously and used in published literature[21].
149
2.7 Co-Immunoprecipitation
150
Lamprey serum and IgG antibody with Protein G were incubated to remove
151
non-specific binding. The supernatant was incubated with L-CFI or L-C3 antibody
152
overnight at 4 °C and then incubated with Protein G for 6 h. After centrifugation,
153
supernatant and precipitate were taken to prepare samples. Western blot detected the
154
presence of L-C3 or L-CFI in the samples.
155
2.8 Cell deposition experiment
156
The rabbit erythrocytes separated by gradient centrifugation were incubated with
157
lamprey serum or L-CFI protein and lamprey serum at room temperature for 5 h.
158
Cells were subjected to protein extraction from the cell membrane (Beyotime
159
Company, China) and the samples were prepared. Western blot detected the
160
deposition of L-C3 on the cell surface.
161
2.9 High content imaging assay
162
Untreated HeLa cells were used as a negative control group. HeLa cells were
163
incubated with naive serum as a positive control group. For depletion of IgG, L-CFI
164
or L-C3, serum was incubated with IgG, L-CFI or L-C3 antibody at 4°C overnight.
165
Protein G agarose was then added to the sample which was incubated at 4°C for 4 h.
166
The supernatant was collected after centrifuged as IgG, L-CFI or L-C3 depleted
167
serum. In addition, IgG, L-CFI or L-C3 were added to IgG, L-CFI or L-C3-depleted
168
serum and the mixture was incubated with HeLa cells. HeLa cells were incubated
169
with the serum treated and untreated in a 37 °C cell culture incubator for 30 min and
170
stained with propidium iodide (Beyotime, China) and hoechst (Thermo, USA) for 10
171
min at room temperature protected from light. The excitation and emission
172
wavelengths were 346 nm and 460 nm.
173
2.10 Laser scanning confocal microscopy and flow cytometry analysis
174
Lamprey’s leukocytes and liver cells were blocked with 10% donkey serum for 3 h
175
and incubation with anti-L-CFI antibody at 4 °C overnight. After overnight incubation,
176
cells were washed with 0.01M PBS. Cells were incubated with Alexa Fluor
177
488-labeled donkey anti-rabbit antibody and then stained with 4’, 6-diamidino-
178
2-phenylindole (DAPI). HeLa cells were incubated with the normal serum of lamprey
179
and the serum cleared L-CFI or L-C3 at 37 °C for 3 h, and fixed by using 4%
180
paraformaldehyde, followed by blocking with normal 10% donkey serum for 2 h and
181
incubation with anti-L-C3 antibody overnight. Cells were incubated with Alexa Fluor
182
488-labeled donkey anti-rabbit antibody and stained with DAPI. Samples were
183
captured with a Zeiss LSM 780 inverted microscope (Carl Zeiss, Germany) and
184
analyzed by using Zeiss ZEN software. Samples were also analyzed on a FACSAria
185
flow cytometer (BD Biosciences, USA).
186
2.11 Mass spectrometry
187
The purity and molecular mass of the protein were determined by 12% SDS-PAGE
188
under reduced conditions. Proteins were visualized with 0.25% Coomassie Brilliant
189
Blue R-250 in 50% methanol containing 10% acetic acid. In-gel tryptic digestion was
190
done according to the manufacturer’s protocol. The protocol of LC-MS was
191
performed as previously described[22]. All MS/MS data were analyzed using Compass
192
1.4, Data Analysis 4.1 bulid 335(Bruker, USA), and Proteinscape 3.0 (Bruker, USA)
193
for protein search. Ensembl lamprey protein database (www.ensembl.org) and NCBI
194
protein database (www.ncbi.nlm.nih.gov) were used separately to replenish each other
195
for searching.
196
2.12 Statistical analysis
197
All statistical analyses were using GraphPad Prism 7.00 software. Differences
198
between treatment groups were determined by Student’s t-test. P <0.05 was set as the
199
threshold for significance (*P<0.05, **P<0.01). Bar charts show the means ± SDs of
200
three independent experiments.
201
202
3. Results
203
3.1 Characterization of L-CFI and phylogeny analysis
204
The ORF sequence of L-CFI encoded a protein of 618 amino acids with a predicted
205
molecular mass of 68.3 kDa, as shown in Fig. S1A. The L-CFI shares 30% to 38%
206
sequence with the homolog protein group of zebrafish to human. The domain analysis
207
indicated that L-CFI contained a factor I membrane attack complex domain (FIMAC),
208
a scavenger receptor cysteine-rich domain(SRCR), a trypsin-like serine protease
209
domain (Tryp_SPc) and 2 low density lipoprotein receptor class A domains (LDLa)
210
(Fig.1A). The CFI phylogenetic tree was constructed using the full-length amino acid
211
sequences of 14 species. Through phylogenetic tree, the lamprey was at the bottom of
212
the vertebrate branch (Fig.1B). Multiple sequence alignment of the L-CFI and CFIs
213
from vertebrates, indicating that L-CFI has strongly conserved Tryp_SPc domain (Fig.
214
1C). The comparison of CFI of vertebrates using the MEME system indicated a high
215
level of conservation in the motif composition (Fig. S1B and Table S2). The
216
vertebrate CFI all contain thirteen motifs (motif 1,2,3,4,5,6,7,8,9,10,11,12 and 14 ),
217
whereas motif 15 are not found in L-CFI and Dr-CFI, and motif 13 are not found in
218
L-CFI. The detailed conserved sequences of each motif are shown in Table. S2.
219
3.2 Purification of recombinant L-CFI and the subcellular location of L-CFI
220
The recombinant plasmid (pCold I-L-CFI) was transformed into Rosetta blue and
221
analyzed by SDS-PAGE, a distinct band was revealed with a molecular mass of
222
68kDa (Fig. S2A), which was in accordance with the predicted molecular mass of
223
L-CFI. A rabbit anti-L-CFI polyclonal antibody was generated and antibody titer was
224
detected by ELISA. Plasma L-CFI antibody concentrations were increased by
225
640,000-fold over preimmunization levels, and pre-immunized rabbit IgG was used as
226
a negative control (Fig. S2A). Western blotting showed that the anti-L-CFI antibody
227
recognized native CFI protein from lamprey leukocytes and supraneural body tissues
228
(Fig. S2B). The endogenous localization of L-CFI in leukocytes and liver cells was
229
detected by immunofluorescence and confocal laser-scanning microscopy. The
230
nucleus were stained with DAPI and observed in blue, while the positive signals of
231
L-CFI in green were distributed in nucleus and cytoplasm (Fig. 2B).
232
3. 3 Bacterial infection induces L-CFI expression in leukocytes
233
In the present study, the mRNA expression of L-CFI in kidney, gill, intestine, heart,
234
liver,
supraneural
body,
leukocytes
and
erythrocytes
were
analyzed
by
235
semi-quantitative RT-PCR, the L-GAPDH gene was used as an internal control. As the
236
results showed that the L-CFI was detected with high expression in liver, kidney,
237
intestine and leukocytes, and not examined in erythrocytes (Fig. 2A).
238
To further investigate the immune response of L-CFI to pathogenic infection,
239
lampreys were challenged with Vibrio anguillarum and Staphylocccus aureus,
240
respectively. As shown in Fig. 2C, a significant decrease mRNA level of L-CFI at 2 h
241
S. aureus and V. anguillarum injection (hpi) was detected (P < 0.05). Later on, from 8
242
hpi, the expression of L-CFI at both 2 challenged groups were remarkably elevated (P
243
< 0.05). The results showed that the expression of L-CFI increased after challenged
244
with S. aureus and V. anguillarum in the adult lamprey leukocytes, when compared
245
with 0 h normal controls (Fig. 2C).
246
3. 4 Interaction between L-CFI and L-C3
247
Co-immunoprecipitation and mass spectrometry were used to detect the interaction
248
between L-C3 and L-CFI. As shown in Fig.3A, the results confirmed that L-CFI
249
interacted directly with L-C3 in lamprey serum and mass spectrometry analysis
250
further confirmed the interaction between L-C3 and L-CFI (Table. S1). In order to
251
investigate whether L-CFI can affect the function of L-C3, western blot assay of
252
L-CFI and L-C3 were performed. L-CFI and lamprey serum were incubated with
253
rabbit erythrocytes, the deposition of L-C3 on rabbit erythrocytes was detected by
254
L-C3 polyclonal antibody. The result showed that the expression of L-C3 in lamprey
255
serum were decreased after treated with recombinant L-CFI protein, suggesting L-CFI
256
affected the activity of L-C3 depositing on the cell surface (Fig. 3B).
257
3.5 Lamprey serum reduced the cytotoxic activity after depleting L-CFI and
258
L-C3
259
Firstly, L-CFI and L-C3 were respectively depleted in lamprey serum to test the
260
killing effect on HeLa cells. The results showed that compared with the untreated
261
group, the killing degree of the L-CFI and L-C3 depleted groups was significantly
262
reduced, and recovered after adding L-CFI back (Fig. 3C). In addition, fluorescent
263
confocal and flow cytometry analysis confirmed that the C3 fluorescence intensity of
264
eliminating L-CFI serum was reduced (Fig. 3D and Fig. S3). These results revealed
265
that L-CFI could be associated with the function of L-C3 to kill HeLa cells and reduce
266
the biological activity of L-C3.
267
4. Discussion
268
In this study, L-CFI was fully identified and characterized from Lampetra morii for
269
the first time. Complement Factor I (CFI) is a family of soluble serine protease which
270
plays a crucial role in the modulation of complement cascades. Previous studies
271
showed high conservation of CFI genes in different vertebrates, including the teleost,
272
cartilaginous fish and other species[14,23].The CFI protein always contains FIMAC ,
273
SRCR, Tryp_SPc, LDLr1 and LDLr2 domain.
274
shows 10 Cys residues of the FIMAC module are also conserved in CFIs (Fig. S1B).
275
The 10 Cys residues characterized in this module may contribute to disulfide bridge
276
formation[24]. Thus, we speculate that FIMAC may perform similar functions in L-CFI
277
and FIMAC in Hs-CFI. However, the species-specific sequence repeats between the
278
signal peptide and FIMAC domain reported in cartilaginous fish, such as three repeat
279
sequences
280
residues(VSPTGESNETMSSTQA) in Triakis scyllium did not exist in L-CFI[25]. It
281
might be a reflection of structural and functional diversity of L-CFI in lower jawless
282
vertebrate. As shown in Fig. S1A, L-CFI glycosylation pattern contains 3 N-linked
283
glycosylation sites less than other teleost fish such as 7 of Cynoglossus semilaevis and
284
5 of Danio rerio[14,26]. The homologous sequences in mammals such as human, mouse,
285
and rat have 6, 6 and 7 N-linked glycosylation sites, respectively[11,27,28]. However,
286
cartilaginous fish G. cirratum contains 13 N-linked glycosylation sites in its CFI
287
pre-protein[13]. These results imply that the number of N-linked glycosylation sites in
288
CFI of vertebrates is significantly different.
(RS
1-
3)
in
Nurse
The alignment of 7 vertebrates
shark[13],
a
direct
repeat
of
16
289
Phylogenetic analysis of the sequences indicates that L-CFI shows higher
290
similarity with homologous CFI sequences from cartilaginous fish than those from
291
teleosts, amphibians and mammals (Fig. 1B). Moreover, the phylogenetic trees
292
confirmed that the L-CFI was located in the outer group of vertebrates, indicating that
293
the L-CFI was the original gene of vertebrates. The analysis implies the evolutionary
294
position of L-CFI and the cluster is roughly consistent with the evolution relationships
295
of the species[29].
296
The detection of L-CFI in tissues reveals its wide and constitutive expression in
297
Lampetra morii except erythrocytes. Specially, the highest expression is detected to be
298
in liver, which is consistent with the report that CFI gene also mainly expresses in the
299
liver of human, shark and other teleosts as it is mainly synthesized in the liver [14,30-31].
300
Therefore, these results suggest that the liver may be a major part of the immune
301
system in lamprey. However, results showed that L-CFI did not express in
302
erythrocytes, so we speculated that L-CFI could not play an important function in
303
erythrocytes. In addition, we intended to confirm whether the L-CFI was involved in
304
the immune response against Vibrio anguillarum and Staphylococcus aureus. After
305
stimulation with Vibrio anguillarum and Staphylococcus aureus, L-CFI in leukocytes
306
showed a significant immune response (Fig. 2C). Similar to the results previously
307
observed in Cynoglossus semilaevis, the CFI mRNA expression was strongly
308
up-regulated throughout the challenge period in Cynoglossus semilaevis. Therefore,
309
these results suggest that L-CFI plays an important role in resisting Gram-positive
310
bacteria and Gram-negative bacteria[32-34].
311 312 313
The complement is a multi-functional complex system comprising more than 30
314
proteins present mainly in serum and cell membranes[35]. However, the later
315
components (C5, C6, C7, C8, and C9) involved in the lytic pathway have not been
316
founded in lamprey, even not in the genome of Petromyrzon marinus. Therefore, it
317
appears that the complement system of lamprey is very unique and may have
318
developed independently from jawed vertebrates. Taken together, the current study
319
provided the evidence for the possible involvement of L-CFI in lamprey complement
320
system through interacting and affecting the deposition of L-C3 on the cell surface.
321
The findings suggest potentially productive and intriguing avenues for future research.
322
Herein, the regulatory complement mechanism of lamprey remains to be fully
323
elucidated.
324 325 326
Acknowledgement
327
This work was funded by the Chinese National Natural Science Foundation Grants
328
(No.31772884 and No.31801973); Chinese Major State Basic Research Development
329
Program (973 Program; Grant 2013CB835304); the Marine Public Welfare Project of
330
the State Oceanic Administration (No. 201305016). The project of Department of
331
Ocean and Fisheries of Liaoning Province (No. 201805), and Science and Technology
332
Innovation Fund Research Project (No. 2018J12SN079).
333 334
References
335
[1] Ricklin D, Lambris JD, Complement in immune and inflammatory disorders:
336
pathophysiological mechanisms, J Immunol. 190 (8) (2013) 3831-3838.
337
[2] Carroll MC, The complement system in regulation of adaptive immunity, Nat
338
Immunol. 5(10) (2004) 981-986.
339
[3] Botto M, Kirschfink M, Macor P, et al, Complement in human diseases: lessons
340
from complement deficiencies, Mol Immunol. 46 (14) (2009) 2774-2783.
341
[4] Alper CA, Rosen FS, Lachmann PJ, Inactivator of the third component of
342
complement as an inhibitor in the properdin pathway, Proc Natl Acad Sci USA. 69 (10)
343
(1972) 2910-2913.
344
[5] Shiang RI, Murray JC, Morton CC, et al, Mapping of the human complement
345
factor I gene to 4q25, Genomics. 4 (1) (1989) 82-86.
346
[6] Ullman CG, Perkins SJ, et al, The Factor I and follistatin domain families: the
347
return of a prodigal son, Biochem J. 326 ( Pt 3) (1997) 939-941.
348
[7] Catterall CF, Lyons A, Sim RB, et al, Characterization of primary amino acid
349
sequence of human complement control protein factor I from an analysis of cDNA
350
clones, Biochem J. 242 (3) (1987) 849-856.
351
[8] Nakao M, Hisamatsu S, Nakahara M, et al, Molecular cloning of the complement
352
regulatory factor
353
Immunogenetics. 54 (11) (2003) 801-806.
354
[9] Gros P, Milder FJ, Janssen BJ, Complement driven by conformational changes,
355
Nat Rev Immunol. 8 (1) (2008) 48-58.
356
[10] Tsiftsoglou SA, Arnold JN, Roversi P, et al, Human complement factor I
357
glycosylation:
358
oligosaccharides, Biochim Biophys Acta. 1764 (11) (2006) 1757-1766.
359
[11] Schlaf G, Rothermel E, Oppermann M, et al, Rat complement factor I: molecular
360
cloning, sequencing and expression in tissues and isolated cells, Immunology. 98 (3)
361
(1999) 464-474.
362
[12] Anastasiou V, Mikrou A, Papanastasiou AD, et al, The molecular identification of
363
factor H and factor I molecules in rainbow trout provides insights into complement
364
C3 regulation, Fish Shellfish Immunol. 31(3) (2011) 491-499.
365
[13] Shin DH, Webb BM, Nakao M, et al, Characterization of shark complement
366
factor I gene(s): genomic analysis of a novel shark-specific sequence, Mol Immunol.
367
46 (11-12) (2009) 2299-2308.
368
[14] Xiang J, Li X, Chen Y, et al, Complement factor I from flatfish half-smooth
369
tongue (Cynoglossus semilaevis) exhibited anti-microbial activities, Dev Comp
370
Immunol. 53 (1) (2015) 199-209.
371
[15] Qin C, Gong Q, Wen Z, et al, Molecular characterization and expression of
372
complement factor I in Pelteobagrus vachellii during Aeromonas hydrophila infection,
373
Dev Comp Immunol. 82 (2018) 66-71.
374
[16] Gustafsson OS, Collin SP, Kröger RH, Early evolution of multifocal optics for
375
well-focused colour vision in vertebrates, J Exp Biol. 211 (Pt 10) (2008) 1559-1564.
376
[17] Matsushita M, The Complement System of Agnathans, Front Immunol. 9 (2018)
377
1405.
378
[18] Boehm T, McCurley N, Sutoh Y, et al, VLR based adaptive immunity, Annu Rev
379
Immunol. 30 (2012) 203–20.
380
[19] Wang Z, Lu J, Li C, et al, Characterization of the LECT2 gene and its protective
I isotypes
structural
and
from
the
functional
common
carp
characterisation
(Cyprinus
of
the
carpio),
N-linked
381
effects against microbial infection via large lymphocytes in Lampetra japonica, Dev
382
Comp Immunol. 79 (2018) 75-85.
383
[20] Wang D, Gou M, Hou J, et,al. The role of serpin protein on the natural immune
384
defense against pathogen infection in Lampetra japonica, Fish Shellfish Immunol. 92
385
(2019):196-208.]
386
[21] Wu F, Chen L, Liu X, et,al. Lamprey Variable Lymphocyte Receptors Mediate
387
Complement-Dependent Cytotoxicity, J Immunol . 190(2013): 922-930
388
[22] Li B, Gou M, Han J, et al, Proteomic analysis of buccal gland secretion from
389
fasting and feeding lampreys (Lampetra morii), Proteome Sci. 22 (2018) 16-19.
390
[23] Sjöberg AP, Trouw LA, Blom AM, Complement activation and inhibition: a
391
delicate balance, Trends Immunol. 30 (2) (2009) 83-90.
392
[24] Haefliger JA, Tschopp J, Vial N, et al, Complete primary structure and functional
393
characterization of the sixth component of the human complement system.
394
Identification of the C5b-binding domain in complement C6, J Biol Chem. 264 (30)
395
(1989) 18041-18051.
396
[25] Terado T, Nonaka MI, Nonaka M, et al, Conservation of the modular structure of
397
complement factor I through vertebrate evolution, Dev Comp Immunol. 26 (5) (2002)
398
403-413.
399
[26] Strausberg RL, Feingold EA, Grouse LH, et al, Generation and initial analysis of
400
more than 15,000 full-length human and mouse cDNA sequences, Proc Natl Acad Sci
401
U S A. 99 (26) (2002) 16899-16903.
402
[27] Catterall CF, Lyons A, Sim RB, et al, Characterization of primary amino acid
403
sequence of human complement control protein factor I from an analysis of cDNA
404
clones, Biochem J. 242 (3) (1987) 849-56.
405
[28] Minta JO, Wong MJ, Kozak CA, et al, cDNA cloning, sequencing and
406
chromosomal assignment of the gene for mouse complement factor I (C3b/C4b
407
inactivator): identification of a species specific divergent segment in factor I, Mol
408
Immunol. 33 (1) (1996) 101-112.
409
[29] Huang S, Yuan S, Guo L, et al, Genomic analysis of the immune gene repertoire
410
of amphioxus reveals extraordinary innate complexity and diversity, Genome Res. 18
411
(7) (2008) 1112-1126.
412
[30] Nilsson SC, Nita I, Månsson L, et al, Analysis of binding sites on complement
413
factor I that are required for its activity, J Biol Chem. 285 (9) (2010) 6235-6245.
414
[31] Morris KM, Aden DP, Knowles BB, et al, Complement biosynthesis by the
415
human hepatoma-derived cell line HepG2, J Clin Invest. 70 (4) (1982) 906-913.
416
[32] Xiang J, Li X, Chen Y, et, al. Complement factor I from flatfish half-smooth
417
tongue (Cynoglossus semilaevis) exhibited anti-microbial activities, Dev. Comp.
418
Immunol. 53(2015), 199-209.
419
[33] Wang Y, Chen B, Ke Y, et, al. Molecular characterization and expression analysis
420
of the complement factor I (CpFI) in the white spotted bamboo shark (Chiloscyllium
421
plagiosum), Fish Shellfish Immunol. 40(2014): 414-423.
422
[34] Qin C, Gong Q, Wen Z, et, al. Molecular characterization and expression of
423
complement factor I in Pelteobagrus vachellii during Aeromonas hydrophila infection,
424
Dev Comp Immunol. 82(2018):66-71.
425
[35] Marina B, Michael K, Paolo M, Complement in human diseases: Lessons from
426
complement deficiencies, Mol. Immunol. 46 (14) (2009): 2774-2783.
427 428
Supplementary Information
429
Supplementary Information includes Fig. S1-Fig. S3, Table S1.
430 431
Figure legends
432
Fig.1 Sequence analysis of CFI. (A) The functional domains of the L-CFI and
433
Hs-CFI according to http://smart.embl-heidelberg.de. (B) Phylogenetic tree for CFI
434
among different species by Neighbor-Joining method. Scale bar (0.1) indicates
435
genetic distance. (C) Conserved domains and motifs of CFI from different
436
species.The FIMAC domain was marked by a solid orange line, SRCR domain was
437
marked by a solid green line, LDLa domain was marked by a solid blue line and
438
Tryp_SPc domain was marked by a solid yellow line. Due to the lack of motif 13 and
439
motif 15 in lamprey and the the lack of motif 13 in zebrafish, these motifs were
440
marked with different dotted lines: motif 13 was marked by a dotted pink line and
441
motif 15 was marked by a dotted red line. Hs: Homo sapiens ; Mm: Mus musculus;
442
Tct: Terrapene carolina triunguis; Xl: Xenopus laevis; Dr: Danio rerio; Gg: Gallus
443
gallus; Cs: Cynoglossus semilaevis; Sf: Scleropages formosus; IP: Ictalurus punctatus;
444
Ac: Antrostomus carolinensis; Cp: Chiloscyllium plagiosum; Gc: Ginglymostoma
445
cirratum; Cm: Callorhinchus milii; L: Lampetra morii.
446
Fig.2 Distribution of L-CFI in lamprey tissues and cells and the immune response
447
of L-CFI by Staphylococcus aureus and Vibrio anguillarum. (A) RNA was
448
extracted from the kidney, liver, gill, intestine, heart, leukocytes and erythrocytes.
449
RNA was reverse-transcribed to cDNA and subjected to PCR. GAPDH was amplified
450
as an internal control.M: DNA marker .(B) Immunofluorescence detected L-CFI
451
localization in liver cells and leukocytes by confocal microscope (Carl Zeiss, Inc).
452
L-CFI was visualized by L-CFI antibody and Alexa Fluor 488 donkey anti-rabbit IgG
453
antibody, and the nucleus were stained with DAPI. Scale bar: 5 µm. (C) Q-PCR
454
detected the changes of L-CFI under the stimulation of Staphylococcus aureus and
455
Vibrio anguillarum. All experiments were repeated at least three times with similar
456
results (*P < 0.05, **P < 0.01).
457
Fig.3 L-CFI interacted with L-C3 and affected the deposition of L-C3 on the cell
458
membrane surface. (A) Co-Immunoprecipitation detected the interaction of L-CFI
459
and L-C3. S: supernatant; P: precipitation. (B) Western blot assay showed that L-CFI
460
protein affected the deposition on the rabbit erythrocytes surface. S: supernatant; P:
461
precipitation. (C) High content imaging assay showed that the remove of L-CFI and
462
L-C3 inhibited HeLa cells death. Serum was omitted in the positive control (*P<0.05).
463
(D) The deposition of L-C3 on HeLa cells, as determined via laser scanning confocal
464
microscopy. The primary antibody consisted of rabbit anti-L-C3 and anti-L-CFI
465
antibody, and the secondary antibody consisted of Alexa 488-labeled donkey
466
anti-rabbit antibody. The nucleus were stained with DAPI. Scale bar:10 µm.
467
Table legends
468
Table S1. Identification of L-C3 by LC-MS/MS
469
Table S2. Conserved motifs discovered in vertebrates using the MEME system
470
Supplementary figure legends
471
Fig. S1 (A) Nucleotide and amino acid sequences of L-CFI. Amino acid sequences
472
are shown below the coding regions. Three N-linked glycosylation sites were marked
473
in red circles. The FIMAC domain, SRCR domain, two LDLa domains and Tryp-SPc
474
domain were shaded in different colors. (B) The motif composition of CFI by MEME
475
version 5.0.5.
476
Fig. S2 (A) SDS-PAGE analysis of L-CFI protein expressed in E.coli Rosettablue. M:
477
molecular weight protein marker; 1: flowthrough sample; 2: equilibrium sample; 3:
478
induced expression of Rosetta blue/pCold I-L-CFI; 4: elution-1; 5: elution-2; 6:
479
elution-3; 7: elution-4. (B) Titer detection of L-CFI polyclonoal antibody by ELISA.
480
The X-axis was the concentration of the antiserum after dilution, the negative control
481
was the normal rabbit serum, the control group is the dilution; the y-axis was the
482
absorbance value of OD450 nm. (C) Analysis of specific antibody of Western blot. 1:
483
L-CFI recombinant protein; 2: liver tissue lysate of lamprey; 3: leukocytes lysate of
484
lamprey; 4: erythrocytes lysate of lamprey
485
Fig. S3 The deposition of L-C3 on HeLa cells, as determined via flow cytometry
486
analysis. The primary antibody consisted of rabbit anti-L-C3 and anti-L-CFI antibody,
487
and the secondary antibody consisted of Alexa 488-labeled donkey anti-rabbit
488
antibody.
489
490
491
492
493
494
495
496
497 498
499 500
Table S1.
Identification of L-C3 by LC-MS/MS
m/z means 969.4815 619.8336
sequence K.STDEGIVDGTSFTIPAISK.H R.EYILPTFEVK.I
945.5193 1023.5298 732.3942 642.8462 653.3690 555.8000
K.LVDSSSTTLVAGEGLSILK.K K.ATVLSSQAAETEEAELVGIK.I K.AELPYFIQVEVR.N K.VQVGSNTINPQK.M R.LGETLNVFLTAK.T R.IPITPDMAPR.F
757.8923 765.8894 794.4409
K.AMLTLDLIGEPDAR.V K.AMLTLDLIGEPDAR.V R.VGLLAVDQAVYAVNR.K
501 502
Table S2. Conserved motifs discovered in vertebrates using the MEME system Motif 1
503 504
2
sequence ACKGDSGGPLVCYDANNVAYVWGIVSWGENCGEPGFPGVYTKVAN YFDWI YTHLSCDKVFCQPWQRCVEGTCICKLPYQCPKNGTPVCSTB
3 4 5 6 7 8
ITCGGIYIGGCWVLTAAHCVR YSVPACVPWSPYLFQPGDTCTVSGWGREK DSFPVKQVIIHEKYNAATYQNDIALLELK NSFHCKSGVCIPRQYVCNGEVDCJTGEDE RCVNGKCIPEEKACDGINDCGDLSDELCCKECK NHTLTRRKRIIGGKTAEKGEFPWQVAIKD
9 10 11
MFICDSEWSMAEANVACRHLGFELGA LKWGNVNLIGNCSKFYKERFF EPSQCLHVTCRGLETSLAECTL
12 13
RSFPTYCHLKSFECLHPEKKFLNNGKCAAEEKFNVSLVYGDSESEGV VQV NYDMDAERKLJKSLLPKLSCGV
14
MECAGTYDGSI
15
SRVHRYQIWTGLLDTJKYD
The expression of L-CFI increased after challenged with S. aureus and V. anguillarum in the adult lamprey leukocytes. L-CFI could be associated with the function of L-C3 to kill HeLa cells and reduce the biological activity of L-C3. The expression of L-C3 in lamprey serum were decreased after treated with recombinant L-CFI protein, suggesting L-CFI affected the activity of L-C3 depositing on the cell surface.