Cloning, Expression Patterns And Functional Characterization Of Porcine ApoER2 And VLDLR

The well-known family of low-density lipoprotein receptors represents a collection of ancient membrane receptors that have been remarkably conserved throughout evolution. These multifunctional receptors, known to regulate cholesterol transport, are becoming increasingly interesting to the neuroscience community due to their ability to transduce a diversity of extracellular signals across the membrane in the central nervous system.In this research on porcine ApoER2 and VLDLR, which are two members of LDLR family, we first reported the cDNA cloning, chromosome mapping and expression analysis of them in Meishan pigs. In order to reveal different functions of ApoER2 and VLDLR in response to reelin signal, reelin was employed to stimulate the cultured IBRS-2 cell line to investigate expression changes of their mRNA. The results are as follows:1. The full coding sequence of VLDLR and partial sequence of ApoER2 were cloned in Meishan pig, and then their DNA and amino acid sequences were analyzed. Sequence analysis shows that porcine VLDLR gene contains 2538bp open reading frame, which encodes 845-amino acids, while the fragment of porcine ApoER2 we cloned contains 1179 bp which encodes 391-amino acids. There is a 91.3% identity between human (NP₀01018064) and porcine ApoER2 in amino acid level, while the deduced amino acid sequence of porcine VLDLR shows a similarity to human ( NP₀03374, 94.0%; NP₀01018066, 97.3%), to chimp (XP520460, 82.7%), to cattle (NP₇76914, 96.0%), to rat (P98166, 92.9%), to mouse (NP038731, 93.1%), to rabbit (P35953, 93.6%), to chicken (P98165, 79.9%) and to zebrafish (NP₉57217, 63.5%). Obviously, the ApoER2 and VLDLR show an amazing degree of conservation among different species, and it suggested that they play important roles in organisms.2. A BLAST search was performed against the High Throughput Genomic Sequences database (HTGS) of pig with porcine VLDLR cDNA to find homologues of this gene. Compared with genomic and mRNA sequences of mouse and human VLDLR, the results from BLAST interpreted that the gene structure of porcine VLDLR consisted of 18 exons and 17 introns. Localization primers designed according to the genomic sequences of porcine ApoER2 and VLDLR were used to map the two genes by radio hybridization mapping method. ApoER2 was localized to porcine chromosome 6q31-35, and VLDLR to chromosome 1q21-27 by radio hybridization mapping method. Moreover the positions of these two genes are corresponding to that in human.3. Bioinformatics analysis of the core promoter, transcription initiation site (TSS) and transcription factor response elements in 5' -UTR of porcine VLDLR was performed. The BLAST results against the Human Promoter Database (HPD) and Eukaryotic Promoter Database (EPD) shows the potential positions of core promoter, TSS of porcine VLDLR and some transcription factor response elements such as SER-1, SP1, ERE, inverted CAAT box and Pou2fl in the upstream of TSS of porcine VLDLR gene, but typical TATA box is absent.4. Real-time quantitative PCR (qRT-PCR) was performed to investigate the mRNA expression profiles of porcine ApoER2 and VLDLR in diverse tissues of Meishan pig, including heart, liver, spleen, lung, kidney, adipose tissue, muscle, stomach, intestine, cerebrum, cerebellum and testicle. The results suggested that the expression pattern of porcine ApoER2 was tissue-specific while porcine VLDLR was relatively ubiquitously expressed. The procine ApoER2 was highly expressed in cerebrum, cerebellum and testicle, showed a low level in heart, spleen, lung, adipose tissue and intestine at lower level, and almost absent in liver, kidney, muscle and stomach. While porcine VLDLR was expressed in all tissues except liver and muscle.5. After the acute treatment with reelin-containing supernatants, the expression profiles of ApoER2 and VLDLR in cultured IBRS-2 cells were detected by qRT-PCR. The expression level of ApoER2 did not change significantly in 30 minutes, and significantly increased (P<0.005) after 1 hour treatment followed by a decrease to nomal level at the second hour. The expression pofile of VLDLR was similar to ApoER2, except for the highest level (P<0.005) after 2 hours treament. The expression pattern of ApoER2 and VLDLR in response to reelin signal was not consistent, which suggested that their functions and mechanisms in reelin signaling pathway may be different.