**1. Introduction**

Obese pregnancy has been demonstrated to provoke an adverse intrauterine milieu, and as a result, poor pregnancy outcomes in human beings and some animal species, such as pig [1,2]. Although the connection between fetal development and maternal obesity is confirmed, the underlying mechanisms connecting adverse maternal environment to the fetus remain elusive. As the interface between the fetus and maternal environment, the placenta has become an important source of pathogenic factors affecting fetal metabolism and development [3,4]. Recently, several studies suggested that maternal obesity during pregnancy is associated with elevated maternal circulating levels of fatty acids and inflammatory cytokines, resulting in a lipotoxic milieu within the placenta characterized by increased placental lipid, inflammation and oxidative stress [5–7]. Lipotoxicity has been demonstrated to induce placental dysfunction evidenced by maternal obesity associated dysregulation of lipid transport and metabolism in the human or pig full-term placenta [8,9]. Recent evidences further revealed that maternal obesity contributes to decreased placental efficiency (a ratio of fetal weight to placental

weight) and excessive placental fat accumulation through an aberrant activation of WNT signaling and PPARδ in placenta from an obesity-prone rat model [10], thus leading to compromised fetal development. Furthermore, our studies showed that WNT signaling and inflammatory NF-κB and JNK signaling are activated in term placenta from sows with excessive back-fat [11], suggesting that maternal obesity may induce lipotoxicity in the full-term porcine placenta. However, the precise cellular and molecular mechanisms responsible for maternal obesity associated lipid accumulation in the pig placenta are still barely understood.

Microtubule affinity-regulating kinase 4 (MARK4) is a member of the AMP-activated protein kinase (AMPK)-related family of kinases, which has been reported to expressed in multiple tissues [12]. As the mammalian homologs of nematode Par-1, microtubule affinity regulatory kinases (MARKs) family contains four members, MARK1(Par-1c), MARK2(Par-1b/EMK1), MARK3(Par-1a/C-TAK1) and MARK4 (Par-1d/MARKL-1), and they share a highly conserved structure consisting of three distinct domains: a catalytic kinase domain, a ubiquitin-associated domain and a kinase associated domain [13]. Studies have implicated Mark4 in diverse physiological processes, including regulation of programmed cell death [14], cell proliferation [15], and glucose homeostasis and energy metabolism [16]. Recent evidences demonstrate that MARK4 promotes adipogenesis and triggers adipocytes apoptosis along with increased adipose inflammation and oxidative stress [17,18]. Moreover, our findings indicated that excessive back-fat is associated with increased activation of MARK4 in pig term placenta, suggesting a potential mechanism for increased activation of JNK mediated mitochondrial apoptotic pathway [19]. All these findings suggest that MARK4 is a versatile protein involved in large number of metabolic processes. However, the regulatory role of MARK4 on placental lipid accumulation, especially in maternal obese condition, is still unknown in porcine. To date, MARK4 gene has been characterized molecularly in several vertebrate species, including pigs [13,20], while the knowledge of molecular structure of MARK4 in *Sus scrofa* (Pig) is still limited, as warrants further studies.

Given the regulatory role played by MARK4 in adipogenesis and energy metabolism, we aimed to evaluate whether MARK4 expression is correlated with lipid accumulation in pig placental trophoblast cells *in vitro*. In addition, we cloned the full-length cDNA of the MARK4 gene from the placenta of porcine using 5 and 3 RACE amplification and employed bioinformatics analysis to identify the molecular characterization and structure of MARK4 from *Sus scrofa*. In this study, we demonstrated that, through activating the WNT/β-catenin and inhibiting the PPARγ pathways, MARK4 promoted lipogenesis in pig placental trophoblasts, implicating MARK4 as a potential regulator of lipid accumulation associated with maternal obesity in the pig placenta.

#### **2. Results**

#### *2.1. Molecular Characterization of MARK4 Gene*

After performing core fragment amplification and 5 and 3 RACE, the full-length cDNA of MARK4 gene (GenBank accession number: MH926032) from *Sus scrofa* was obtained (Figure S1). The full-length cDNA covered 3216 bp with an ORF of 2259 bp encoding 752 amino acids. The MARK4 protein had a calculated molecular weight (Mw) of 82535.70 Da and isoelectric point (PI) of 9.70. This amino acid (AA) sequence contained several conserved functional sites, including one proton acceptor (Asp181), one protein kinase ATP-binding region signature (IIe65-Lys88), one serine/threonine protein kinase active-site signature (IIe177-Leu189) and one protein kinase domain (Tyr59-IIe310). Based on the results predicted by the online SABLE program, the secondary structure of this MARK4 protein consisted of 13 α-helices, 13 β-strands and 26 coils (Figure S2).

Additionally, conserved motifs were identified in the amino acid sequence of the MARK4 protein, including the activation loop, the catalytic kinase domain (KD), the ubiquitin-associated domain (UBA), the kinase associated domain1 (KA1) and three conserved functional sites (lysine 88 ATP binding site, aspartic 181 active site and threonine 214 phosphorylation site; Figure 1). This MARK4 protein sequence had a high similarity, and showed similar structural features to the MARK4 protein of other species (Figure S3).

**Figure 1.** The tertiary protein structures of MARK4 protein in Pig (*Sus scrofa*) modeled by the ProModII program. Kinase domain (KD) colored red, ubiquitin-associated domain (UBA) is blue and kinase associated domain1 (KA1) in pink.

#### *2.2. Phylogenetic Analysis*

The phylogenetic tree among 11 species based on the amino acid (AA) sequences of MARK4 protein was presented in Figure S4. MARK4 of pig (*Sus scrofa*) showed a close phylogenetic relationship with that of human (*Homo sapiens*) and chimpanzee (*Pan troglodytes*). Conservation of MARK4 was also evident from similarity comparisons in NCBI, as the MARK4 protein of *Sus scrofa* showed a high identity (95%–99%) to that of David's myotis (*Myotis davidii*), Chimpanzee (*Pan troglodytes*), American beaver (*Castor canadensis*), Domestic guinea pig (*Cavia porcellus*), Norway rat (*Rattus norvegicus*), House mouse (*Mus musculus*), Dingo (*Canis lupus dingo*), Horse (*Equus caballus*) and Human (*Homo sapiens*).
