Clip domain serine proteins (cSPs) play an important role in the innate immune
response of crustacean. However, few comparative studies have been reported on the
function of this protein in different cSPs of the same species, and there is a lack of
research on the regulation of immune signals. The variable lymphocyte receptor (VLR)
genes are the molecular basis of the acquired immunity in Petromyzon marinus, and it
has been reported that VLR plays a special role in the adaptive immune defense of
invertebrates in the recent years. This study based on molecular cloning, prokaryotic
system protein expression, RNA interference, real-time quantitative PCR etc.,
compared the immune functions of three clip domain serine proteases (PtcSP1,
PtcSP2 and PtcSP3) of Portunus trituberculatus and studied the regulatory
relationships among other immune genes of PtcSPs. Also, the structure of the variable
lymphocyte receptor (EsVLRA) of Eriocheir sinensis was analyzed and the effect of
immune priming was investigated. This research compared the immune functional
diversity based on its recombinant proteins and explained the immune memory of
EsVLRA. The research essays and results are as following:
Sequence alignment of PtcSPs-C showed that PtcSP1 and PtcSP3 had
trypsin-specific Asp189, Gly216, and Gly226 binding site, but PtcSP2 had more
diverse sequences in the binding pocket sites, and phylogenetic tree analysis showed
that PtcSP2 from P. trituberculatus and the chymotrypsins were clustered together.
SP domain (PtcSPs-C) and the clip domain (PtcSPs-N) of PtcSPs of P. trituberculatus
were expressed by the prokaryotic protein expression system separately. Proteinase
activity verification-experiments further proved that the purified PtcSP1 or PtcSP3
had trypsin-like activity, while PtcSP2 recombinant protein had chymotrypsin-like
activity. Moreover, rPtcSP1-N and rPtcSP3-N protein could inhibit all the tested
Gram-negative bacteria (Pseudomonas aeruginosa and Vibrio alginolyticus) and
Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus). Remarkably,
PtcSP1 and PtcSP3 showed different antibacterial MIC: RPtcSP1-N had the highest
antibacterial activity against gram-positive bacteria M. luteus (MIC value less than
0.99 μM), while rPtcSP3-N strongly inhibited growth of the Gram-negative bacteria V.
alginolyticus (MIC value of less than 0.58 μM). However, no obvious antimicrobialactivity was observed against yeast, and no significant antibacterial activity of
PtcSP1-C or PtcSP3-C was detected. In addition, in the bacterial-binding assay,
rPtcSP1-N and rPtcSP3-N were detected against Gram-negative bacteria (P.
aeruginosa and V. alginolyticus), Gram-positive bacteria (S. aureus and M. luteus),
and the fungus (Pichia pastoris) showed bacterial-binding activity. The results of the
bacteria clearance test showed that PtcSP1 has the bacteria clearance activity of V.
alginolyticus and presents a time-dependent pattern, suggesting that PtcSP1 may
participate in the immune response as an opsonin.
Under PtcSP1 or PtcSP2 gene RNAi, all PtALF genes were inhibited except
anti-lipopolysaccharide factor 5 (PtALF5), and the expression of all detected PtALFs
was suppressed after PtcSP3 was silenced, indicated that PtcSPs could regulate the
diverse PtALFs synthesis. Silencing of PtcSP1 or PtcSP3 could up-regulate the
expression of complement-like genes, suggesting an antagonistic relationship between
serine protease genes and complement-like genes. In addition, the PO activity of the
crab was not affected after PtcSP2 gene silencing, and the expression level of serine
protease inhibitor-related genes, which are related to PO activation did not change,
while in the low expression of PtcSP1 or PtcSP3 crab the PO activity was
EsVLRA ORF contains 2400 bp, and encodes 799 amino acids. Including one
LRR_NT domain, thirteen LRRs domain and one LRR_CT domain, meanwhile a
large protruding-structure at the C-terminal. Multi-sequence alignment analysis of
amino acid sequences showed that EsVLRA has a conserved amino acid sequence,
which is consistent with VLRA from other species; phylogenetic analysis showed that
EsVLRA and VLRAs were clustered together, indicating the VLR from E. sinensis is
a new member of VLRAs. The mRNA transcript of EsVLRA was detected in all
examined tissues, including hemocytes, heart, muscle, hepatopancreas, gill, intestine
and gonads. EsVLRA highly expressed in hepatopancreas, gonads and gill. Besides,
the expression level of EsVLRA in hepatopancreas, gonads, gill and brain of male
crabs was significantly higher than that in female crabs.
rEsVLRA could strongly bind to Gram-negative bacteria Vibrio.
parahaemolyticus and P. aeruginosa, Gram-positive bacteria M. luteus and S. aureus
and yeast P. pastoris. However, no significant antimicrobial activity of rEsVLRA was
observed, indicating that EsVLRA is mainly involved in immune recognition as a
receptor. In the immune priming assay, it was found that when healthy crabs werestimulated by formaldehyde-inactivated V. parahaemolyticus firstly, EsVLRA
showed a significant increase at third hour (P < 0.05). After injected with live V.
parahaemolyticus, EsVLRA was significantly up-regulated at the first hour and was
much higher than that in the first injection (approximately 50 times to the first
injection, P < 0.01). The highest expression level appeared at the sixth hour after the
second challenge (approximately 240 times to the first injection, P < 0.01), this high
expression level is lasting to the 48th hour (P < 0.01). It showed that EsVLRA might
be memorable for the pathogen-stimulation.
Studies on the immunological function of the 3 kinds of serine protease of P.
trituberculatus showed that they have diverse immune activities. PtcSPs functioned as
an immune factor in crustacean innate immunity through various functions including
antibacterial activity, pathogen recognation protein, opsonin-mediated cellular
immunity, proPO activation-mediated humoral immunity, and the regulation of other
innate immune genes. Investigations of the variable lymphocyte receptors of mitten
crab immune priming-related genes had shown that EsVLRA is structurally similar to
VLRA in lamprey, and functions as a pattern recognition receptor to participate in the
immunity of crustaceans. In summary, this study enriched the theoretical basis of
serine proteases diverse immune functions, and provided new clues for the study of
crustacean immune priming.