Cloning, chromosome mapping and expression pattern of porcine PLIN and M6PRBP1 genes

The PAT proteins, named after the three PLIN/ADRP/TIP47 (PAT) proteins, PLIN for perilipin, ADRP for adipose differentiation-related protein and TIP47 for tail-interacting protein of 47 kDa, now officially named M6PRBP1 for mannose-6-phosphate receptor binding protein 1, is a set of intracellular lipid droplet binding proteins. They are localized in the outer membrane monolayer enveloping lipid droplets and are involved in the metabolism of intracellular lipid. This work describes the cloning and sequencing of porcine PLIN and M6PRBP1 cDNAs, the chromosome mapping of these two genes, as well as the expression pattern of porcine PAT genes. Sequence analysis shows that the porcine PLIN cDNA contains an open reading frame of 1551 bp encoding 516 amino acids and that the porcine M6PRBP1 cDNA contains a coding region of 1320 bp encoding 439 amino acids. Comparison of PLIN and M6PRBP1 amino-acid sequences among various species reveals that porcine and bovine proteins are the most conserved. Porcine PLIN and M6PRBP1 genes have been mapped to pig chromosomes 7 and 2, respectively, by radiation hybrid analysis using the IMpRH panel. Expression analyses in pig showed a high expression of PLIN mRNA in adipose tissue, M6PRBP1 mRNA in small intestine, kidney and spleen and ADRP mRNA in adipose tissue, lung and spleen.


INTRODUCTION
Intracellular neutral lipid storage droplets, essential organelles of eukaryotic cells, are required for energy balance, membrane biosynthesis, cholesterol metabolism and lipid trafficking. Animal lipid storage droplets contain dissected and frozen in liquid nitrogen and then stored at -70 • C until extraction for total RNA. Three pigs were used for the expression analyses.

Total RNA isolation and reverse transcription
Total RNA was extracted and purified from the frozen tissue with Trizol Reagent (Sangon, Shanghai) according to the manufacturer's protocol, and subsequently treated with DnaseI (Takara, Dalian, RNase-free) to degrade possible genomic DNA. Total RNA concentrations were calculated from the optical density (OD) value at wavelength 260 nm and the ratios OD260/OD280 were determined. The quality of the preparations was also checked by denaturing agarose gel electrophoresis. Total RNA was used as template for oligo (dT)18 primed reverse transcription using 200 U of M-MLV reverse transcriptase (Promega), 0.5 mM of each dNTP and 3 mM MgCl 2 .

Cloning of pig PLIN and M6PRBP1 cDNAs
All primers used in this work are included in Table I. Primers P1 and P2 designed from the consensus sequence between human and mouse PLIN cDNAs (GenBank, NM_002666 and NM_175640), were used to amplify the central region of porcine PLIN cDNA, while primers (P3, P4) and (P5, P6) were used to amplify the 5' and the 3'-end coding sequences, respectively. The complete porcine PLIN cDNA sequence was obtained by assembling these three fragments.
Similarly, primers P12 and P13, designed from the consensus sequence between human and mouse M6PRBP1 cDNAs (GenBank, AF057140 and AK004970), were used to amplify the central region of porcine M6PRBP1 cDNA and primers (P14, P15) and (P16, P17) were used to amplify the 5' and the 3'-end coding sequences, respectively.

Radiation hybrid mapping
PLIN and M6PRBP1 genes were mapped by radiation hybrid analysis, which was carried out using DNA samples isolated from the hybrid clones included in the INRA-University of Minnesota porcine Radiation Hybrid (IMpRH) panel [10,28]. The presence or absence of the porcine genes in each of the DNA samples was determined by PCR amplification of a fragment of each gene. For PLIN, primers P9 and P10 (Tab. I) were designed from the sequence of intron 6 amplified by primers P7 and P8. PCR consisted in an initial  [17]. Chromosomal regional localizations were deduced either from the position of the closest linked marker located on the cytogenetic map for PLIN or from the interval on the cytogenetic map of the linkage group carrying the closest linked marker for M6PRBP1.

Semi-quantitative RT-PCR
Total RNA samples prepared from eleven different tissues from Meishan pigs were reverse transcribed by a standard procedure (Promega) using M-MLV reverse transcriptase and an oligo(dT) 18 primer. The reverse transcription reaction products were used as templates in PCR amplifications carried out with the following pig-specific primer pairs, P9 and P11 for PLIN, P22 and P23 for ADRP (designed from pig ADRP, AY550037), P20 and P21 for M6PRBP1 and P24 and P25 for β-actin (designed from pig β-actin, SSU07786). To avoid the PCR entering plateau stages, the number of cycles was adapted in each case. To assess relative mRNA levels of PLIN, ADRP and M6PRBP1 in porcine tissues, the pig housekeeping gene β-actin was used as the internal standard. We performed controls with different dilutions of cDNA template to ascertain the linearity of the amplification (data not shown). Quantitative analyses of relative mRNAs levels were carried out with the Quantity One V 4.313 image analyzing system. The experiment was carried out on three pigs (n = 3) and each value was expressed as the mean value ± SD.

Cloning of porcine PLIN and M6PRBP1 cDNAs
Using adipose tissue and small intestine total RNA, we cloned the porcine PLIN and M6PRBP1 cDNAs, respectively. Porcine PLIN cDNA (GeneBank, AY973170) has an ORF of 1551 nucleotides, encoding a 516 amino-acid peptide and porcine M6PRBP1 cDNA (GeneBank, AY939831) contains a 1320 bp coding region encoding a 439 amino-acid protein. Table II shows the percentages of sequence similarity of porcine PLIN and M6PRBP1 protein sequences 220 X. Tao et al. with those of cattle, dog, man, mouse, rat, monkey, chicken and frog. The highest sequence similarity is observed with the bovine proteins.
The porcine PLIN protein contains six consensus sites for phosphorylation by cAMP-dependent protein kinase A (PKA), serines 81, 277, 436, 491, 516, and threonine 431 (Tab. III). It has been shown that phosphorylation of PLIN is a key process in lipolysis [3,26] and that the three carboxyl-terminal sites (Ser 433, Ser 492, Ser 517) are critical to protect the lipid droplets from lipases [26]. Furthermore, protein kinase A-mediated phosphorylation of PLIN serine 492 promotes the fragmentation and dispersion of lipid droplets [15]. Pig and mouse PLIN each have a unique phosphorylation site i.e. threonine 431 and serine 222, respectively.
A previous study has indicated that the mouse PLIN gene has four mRNA variants that yield four proteins, perilipin A (516 amino-acids), B (421 aminoacids), C (347 amino-acids), and D (244 amino-acids), with the same N-termini sequence and distinct C-termini sequences [14]. The human PLIN gene has two mRNA variants that yield two proteins, perilipin A and B. Perilipin A is the predominant protein isoform and plays key roles in facilitating both the storage and hydrolysis of triacylglycerol (TGA) [7]. Based on the different splicing sites present in the mouse and human genes, we designed different primers to try to amplify porcine PLIN mRNA variants in different tissues, but failed to find other variants.
Porcine PLIN, ADRP and M6PRBP1 proteins share a typical PAT-1 domain, a 33-mer motif, and a PAT-C domain (Tab. IV). The PAT-1 domain is a highly conserved region at the N-terminus of the PAT family proteins [13]. The PAT-1 domain of ADRP protein shares 60.9% and 40.2% sequence similarity with that of M6PRBP1 and PLIN proteins (Tab. IV). Sequence analyses based upon neighbor-joining comparisons confirm that PLIN, ADRP, and M6PRBP1 proteins form separate groups (Fig. 1).

Chromosomal mapping of porcine PLIN and M6PRBP1 genes
Porcine PLIN and M6PRBP1 genes were mapped by radiation hybrid analysis using the 118 clones of the IMpRH panel. PCR results were submitted to the INRA-Minnesota Porcine Radiation Hybrid (IMpRH) Server and retention frequencies were calculated. PLIN and M6PRBP1 genes showed a retention frequency of 13% and 11%, respectively. Thus, the porcine PLIN gene is mapped to SSC7q at a distance of 68 cR from the most significantly linked marker SWR1210 (LOD score threshold 5.21). Since SWR1210 has been previously localized by FISH on SSC7q [22], we could deduce    database http://www.threakdb.org). Since both SW0170 [6] and ADM [12] have previously been FISH-mapped to SSC2p11, we could deduce that M6PRBP1 is localised in this region i.e. SSC2p11. Human PLIN and M6PRBP1 genes are located on chromosomes 15q26 [23] and 19p13.3 (NM_005817), respectively. Thus, our result is consistent with the pig/human comparative map and supports the conservation of synteny between SSC7 and HSA15 and between SSC2 and HSA19 [8].

Tissue expression patterns of porcine PLIN, ADRP and M6PRBP1 genes
The relative transcription levels of porcine PLIN, ADRP and M6PRBP1 are shown in Figures 2a and 2b. Porcine PLIN mRNA is ubiquitously expressed,  with the highest level found in adipose tissue and a low level in all other tissues. ADRP mRNA is highly expressed in adipose tissue, lung and spleen and less in liver, kidney, intestine and stomach. M6PRBP1 mRNA is highly expressed in intestine and at decreasing levels in kidney, spleen, liver, lung, brain, adipose tissue, heart and stomach.
It has been suggested that the expression of PLIN is restricted to adipocytes and steroidogenic cells [5]. These cell classes have a mechanism for the lipolysis of stored triacylglycerol (TAG) that is mediated by cAMPdependent protein kinase A (PKA) and hormone-sensitive lipase (HSL). In our analysis, porcine PLIN mRNA was found to be highly expressed in adipose tissue, but also ubiquitously expressed in the other tissues at a very low level. This may be explained by the fact that these tissues contain few adipocytes and steroidogenic cells (for example liver and kidney). In fact, we have also found that the porcine HSL is ubiquitously expressed in different tissues (data not shown), which indicates that the lipolysis of stored TAG, which is mediated by PKA and HSL, is not restricted to adipose tissue.
Porcine M6PRBP1 mRNA is highly expressed in intestine, kidney, spleen, liver and lung, but much less in adipose tissue. Although the content of lipid droplets in intestine is quite high, it does not exceed that in the adipose tissue. This may contribute to the fact that M6PRBP1 associates not only with lipid droplets, but also with the mannose 6-phosphate receptor [2].
Although the levels of PLIN, ADRP and M6PRBP1 mRNA are different in various tissues, total levels are higher in tissues with a high lipid content (adipose tissue, liver, kidney, lung, spleen, intestine) than in tissues with a low lipid content (heart, muscle, pancreas and stomach). The distribution of PAT mRNAs is consistent with lipid levels in different tissues, thus they can be considered as a marker of lipid accumulation.