dLp/HDL-BGBP and MTP Cloning and Expression Profiles During Embryonic Development in the Mud Crab Scylla paramamosain

Abstract

Lipids are the main energy source for embryonic development in oviparous animals. Prior to the utilization and catabolism, lipids are primarily transported from the yolk sac to embryonic tissues. In the present study, cDNA encoding a circulatory large lipid transfer protein (LLTP) superfamily member, the precursor of large discoidal lipoprotein (dLp) and high-density lipoprotein/β-1,3-glucan-binding protein (HDL-BGBP), named dLp/HDL-BGBP of 14,787 bp in length, was cloned from the mud crab Scylla paramamosain. dLp/HDL-BGBP was predicted to encode a 4,831 amino acids (aa) protein that was the precursor of dLp and HDL-BGBP, which were both detected in hemolymph by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. For the intracellular LLTP, three microsomal triglyceride transfer protein (MTP) cDNAs of 2,905, 2,897, and 3,088 bp in length were cloned from the mud crab and were predicted to encode MTP-A of 881 aa, MTP-B of 889 aa, and MTP-C of 919 aa, respectively, which were different merely in the N-terminal region and shared an identical sequence of 866 aa. During embryonic development, the expression level of dLp/HDL-BGBP consecutively increased from the early appendage formation stage to the eye pigment-formation stage, which indicated that HDL-BGBP is probably the scaffolding protein for yolk lipid. For the MTP gene, MTP-C accounted for ~70% of MTP mRNA from the blastocyst stage to the nauplius stage, as well as the pre-hatching stage; MTP-C and MTP-A expression levels were comparable from the early appendage formation stage to the late eye pigment-formation stage; MTP-A was extremely low in blastocyst and gastrula stages; MTP-B was expressed at a relatively low-level throughout embryo development. The variations in the expression profiles among MTP transcripts suggested that MTP might play roles in the lipid droplet maturation and lipoprotein assembly during embryonic development.

Keywords: MTP; dLp/HDL-BGBP; decapod; embryo; lipid transfer; mud crab.

Figure 1

Phylogenetic tree of large lipid transfer proteins in decapodan species, using the maximum likelihood method. The tree topology was based on the alignment of the conserved amino acid sequence of the LPD_N domain. The numbers at each node indicate bootstrap values and bootstrap values < 50 are not provided. apoCr, apolipocrustacein; CP, clotting protein; dLp/HDL-BGBP, precursor of discoidal lipoprotein and beta-1,3-glucan binding protein; MTP, microsomal triglyceride transfer protein. Detailed information of LLTPs is listed in the Supplementary Table.

Figure 2

Expression profile of dLp/HDL-BGBP during embryonic development in Scylla paramamosain. dLp/HDL-BGBP, precursor of discoidal lipoprotein and beta-1,3-glucan binding protein. The relative expression level of dLp/HDL-BGBP was determined in blastocyst (I), gastrula (II), nauplius (III), early and late appendage formation (IV–V), early, middle, and late eye pigment formation (VI–VIII), and prehatching (IX) stages of embryos by qPCR, and EF-1α was the endogenous control. The same letters indicate no significant difference in expression level among embryonic development stages, according to one-way ANOVA in combination with Duncan's test for post-hoc multiple comparisons. A significant difference was identified at P < 0.05.

Figure 3

Expression profile of MTP transcript variants during embryonic development in Scylla paramamosain. MTP, microsomal triglyceride transfer protein. The relative expression level of MTP transcript variants was determined in blastocyst (I), gastrula (II), nauplius (III), early and late appendage formation (IV–V), early, middle, and late eye pigment formation (VI–VIII), and prehatching (IX) stages of embryos by qPCR, and EF-1α was the endogenous control.