Molecular Cloning of AZIN 2 and its Expression Profiling in Goose Tissues and Follicles

Ornithine decarboxylase antizyme inhibitor 2 (AZIN2) plays key roles in regulating the biosynthesis and content of polyamines and other amines such as serotonin and histamine. The AZIN2 coding sequence in the goose was cloned and analysed in this experiment. AZIN2 expression levels in goose tissues and follicles were also measured. A full-length AZIN2 coding sequence (GenBank accession no. MF939648) encoding a 457-amino acid protein was 1374 bp in length. The molecular weight of putative AZIN2 protein was 49.10 kDa. The mRNA expression of AZIN2 was not observed in heart, adrenal gland, breast muscle, thigh muscle and pineal gland tissues in the goose. The highest and lowest expression levels of AZIN2 in all examined tissues were observed in cerebrum and kidney tissues, respectively. The mRNA expression levels of AZIN2 in the cerebrum, cerebellum and hypothalamus were 61.95-, 15.87and 15.04-fold higher compared to the liver, respectively. The mRNA expression of AZIN2 was observed in all examined follicles. The mRNA expression level of AZIN2 was 6.48-fold higher in the F1 follicle compared to the SWF. Taken together, AZIN2 mRNA has a restricted pattern of expression in the goose. AZIN2 may play important roles in modulating physiological functions of the brain and regulating follicular development and ovulation of goose ovaries.

Up to date, the AZIN family was made up of AZIN1 and AZIN2 proteins (KAHANA 2009;MA et al. 2015).Both AZIN1 and AZIN2 bind to OAZ with the same affinity.AZINs are highly homologous to ODC but lack decarboxylase activity (MURAKAMI et al. 1996).Recently, several studies demonstrated that AZIN2 enhanced the stability and activity of ODC and polyamine influx through counteracting the inhibition of OAZ (LOPEZ-CON- TRERAS et al. 2006; KANERVA et al. 2008).
AZIN2 has a significant role in maintaining polyamine homeostasis and regulating cell proliferation, similar to that found for AZIN1 (KEREN-PAZ et al. 2006;SILVA et al. 2015).High expression of AZIN2 is found in the testis and brain and differentiated resting cells (PITKANEN et al. 2001;MAKITIE et al. 2010;LOPEZ-GARCIA et al. 2013).The expression level of AZIN2 is positively correlated to the polyamine level in the brain of Alzheimer's patients (MAKITIE et al. 2010;INOUE et al. 2013).Recently, several studies have suggested that AZIN2 plays a role in regulating the biosynthesis of serotonin and histamine and may have a role in the endocrine function of adrenal glands and pancreas (KANERVA et al. 2009;LOPEZ-GARCIA et al. 2013;ACOSTA-ANDRADE et al. 2016).These reports suggest that AZIN2 has multiple biological functions.Our previous study cloned and characterized the AZIN1 gene of the Sichuan white goose, and implied that AZIN1 played important roles in follicular development (MA et al. 2015).Thus far, the goose AZIN2 gene and its expression profiles remain to be determined.To characterize the goose AZIN2 gene, we cloned the AZIN2 cDNA sequence and measured mRNA expression levels of AZIN2 in different tissues and ovarian follicles.

Preparation of experimental animals and tissue collection
All experimental procedures were performed in accordance with the Institutional Review Board (IRB14044) and the Institutional Animal Care and Use Committee of the Sichuan Agricultural University under permit number DKY-B20140302.The heart, liver, spleen, lungs, kidneys, adrenal glands, breast muscles, thigh muscles, cerebrum, cerebellum, pineal gland, hypothalamus, pituitary gland, uterus, follicles and ovarian stroma in laying Sichuan white geese were collected and prepared on ice as described previously (MA et al. 2015).

Statistical analysis
Statistical analysis was performed by one-way analysis of variance using the SAS 9.2 statistical software for Windows (SAS Institute Inc., NC, USA) followed by Duncan's multiple range test.All values are presented as the mean ±SEM.A P<0.05 was considered to be statistically significant.

AZIN2 protein sequence analysis
The theoretical pI of the putative 49.10 kDa AZIN2 protein was 5.28 in the goose.Putative AZIN2 instability index was computed as 48.57.The grand average of hydropathicity was 0.026.AZIN2 secondary structure was predicted to consist of 39.82% alpha helix, 18.60% extended strand, 7.44% beta turn and 34.14% random coil.The predicted subcellular location of goose AZIN2 protein was 47.8% cytoplasmic, 26.1% nuclear, 17.4% mitochondrial, 4.3% cytoskeletal, and 4.3% plasma membrane.The putative goose AZIN2 protein contained a 238-amino acid pyridoxaldependent decarboxylase (PDX) pyridoxal binding domain, a 123-amino acid PDX C-terminal sheet domain, a 19-amino acid Orn/DAP/Arg decarboxylases family 2 pyridoxal-P attachment site and an 18-amino acid Orn/DAP/Arg decarboxylase family 2 signature 2 (Fig. 1).Based on the putative AZIN2 amino acid sequence, a phylogenetic tree was constructed, and goose AZIN2 was most similar to that found in Meleagris gallopavo and Gallus gallus (Fig. 2).

AZIN2 expression profiling in goose tissues
The mRNA expression of AZIN2 was not observed in heart, adrenal gland, breast muscle, thigh muscle and pineal gland tissues in the goose using qRT-PCR.These results were also confirmed by semi-quantitative reverse transcription PCR (data not shown).The highest and lowest expression levels of AZIN2 in all examined tissues were observed in cerebrum and kidney tissues, respectively (Fig. 3).The mRNA expression levels of AZIN2 were significantly higher in cerebrum, cerebellum and hypothalamus tissues than in other examined tissues (P<0.05).AZIN2 expression levels in the cerebrum, cerebellum and hypothalamus were 61.95-, 15.87-and 15.04-fold higher compared to the liver (P<0.05),respectively.We did not find significant differences in expression levels of AZIN2 in the liver, spleen, lungs, kidneys, pituitary gland, uterus and ovary in the goose (P>0.05).

AZIN2 expression profiles in goose ovarian follicles
The mRNA expression of AZIN2 was observed in all examined follicles (Fig. 4).During follicular development, significant difference in the mRNA expression levels of AZIN2 was not observed among the SWF, SYF and F5-F2 follicles (P>0.05).The mRNA expression level of AZIN2 in the F1 follicle was the highest in all examined follicles and was 6.48-fold higher compared to in the SWF (P<0.05).Significant difference in AZIN2 expression levels were not detected among any POF follicles (P>0.05).

Discussion
The goose AZIN2 coding sequence was cloned and characterized for the first time in this study.The putative AZIN2 protein in the goose had a molecular mass of 49.10 kDa, similar to that of mammal AZIN2 (50 kDa) and goose AZIN1 (50 kDa) (LOPEZ-CONTRERAS et al. 2010;MA et al. 2015).As in the case of AZIN1, goose AZIN2 was also a labile protein (PITKANEN et al. 2001;RAMOS-MOLINA et al. 2014;MA et al. 2015).Human AZIN2 retains 45% identity and 66% similarity to AZIN1 at the amino acid level (PITKANEN et al. 2001;OLSEN & ZETTER 2011).In this study, our data showed that the identity and the similarity between goose AZIN2 and AZIN1 were 41 and 64%, respectively.The theoretical pI of the putative AZIN1, AZIN2 and ODC protein of the goose was 4.79, 5.28 and 5.00, respectively (MA et al. 2015).In the goose, the putative ODC secondary structure consisted of 35.87% alpha helix, 23.7% extended strand, 8.91% beta turn and 31.52%random coil (our unpublished data).MA et al. (2015) reported that goose AZIN1 secondary structure was predicted to consist of 36.00%alpha helix, 16.89% extended strand and 47.11% random coil (MA et al. 2015).In this study, goose AZIN2 secondary structure consisted of 39.82% alpha helix, 18.60% extended strand, 7.44% beta turn and 34.14% random coil.In geese, the subcellular location of putative AZIN1, AZIN2 and ODC proteins was different.The subcellular distribution of the AZIN1 protein was predicted to be 73.9% in cytoplasmic, 8.7% in nuclear, 13.0% in mitochondrial and 4.3% in secretory vesicles in geese (MA et al. 2015).The predicted subcellular location of goose ODC protein was 65.2% cytoplasmic, 21.7% nuclear and 13.0% mitochondrial.Our data showed that the subcellular location of goose AZIN2 protein was 47.8% cytoplasmic, 26.1% nuclear, 17.4% mitochondrial, 4.3% cytoskeletal and 4.3% plasma membrane.
A previous study showed that goose AZIN1 mRNA was expressed in all examined tissues (MA et al. 2015).In this study, the observed mRNA expression levels of AZIN2 in the heart, adrenal glands, breast muscles, thigh muscles and pineal gland in geese were lower than the detection limits of qRT-PCR used.This suggests that AZIN1 is ubiquitously expressed and AZIN2 shows restricted expression in goose tissues.In mammals, studies have revealed that AZIN2 was mainly expressed in testis and brain (LOPEZ-CONTRERAS et al. 2010), but not evenly expressed in all types of cells.AZIN2 mRNA expression is mainly observed in the testicular germinal haploid cells (LOPEZ-CONTRERAS et al. 2009a), whereas in the murine brain AZIN2 appears to be mainly localized in motor and sensory nucleus, hippocampus and some cerebellar areas (LOPEZ-CONTRERAS et al. 2010;RAMOS-MOLINA et al. 2012).These results suggest that AZIN2 has a more restricted pattern of expression than AZIN1 in both bird and mammalian tissues (LOPEZ-CONTRERAS et al. 2010).AZIN2 is exclusively expressed in adrenal medulla of adrenal glands that plays a role in the bio- synthesis and secretion of catecholamines (LOPEZ-CONTRERAS et al. 2009b;LOPEZ-GARCIA et al. 2013).However, in present study, the mRNA expression of AZIN2 was not found in adrenal glands in the goose.The mRNA expression of AZIN2 in different cells of adrenal glands remains to be measured in birds.Studies have suggested that robust expression of AZIN2 in the brain is distributed along neural axons and dendrites in a granular or vesicular pattern (MAKITIE et al. 2010;RASILA et al. 2016).High expression of AZIN2 was also found in human cerebellum (RASILA et al. 2016).In this study, high mRNA expression levels of AZIN2 were observed in cerebrum, cerebellum and hypothalamus tissues in the goose.It is well known that AZIN2 is an inhibitor of OAZ.Thus, it is conceivable that AZIN2 is involved in regulating polyamine homeostasis in the brain.Changes in the expression and activity of different polyamine metabolic enzymes, as well as alternations in polyamine levels, have been associated to different brain insults such as cerebral ischemia and some mental disorders (LI et al. 2007;FIORI & TURECKI 2008;LOPEZ-CONTRERAS et al. 2010).
Taken together, strong AZIN2 expression in the brain suggests that this protein may play important roles in modulating physiological functions of the brain in both mammals and birds, although the actions and mechanisms need to be clarified.
Polyamine synthesis, under endocrine influence, appears necessary for function and differentiation of the somatic cell component of the ovary (THYS- SEN et al. 2002; FERNANDES et al. 2017; QIU et al.  2017).Treatment of immature female mice with DFMO (an irreversible inhibitor of ODC) inhibits ovarian growth, antral follicle formation, and the onset of puberty (BASTIDA et al. 2005;QIU et al. 2017).As commented before, AZIN can restore ODC activity by forming a tight complex with OAZ, thereby releasing ODC from the ODC-OAZ complex (QIU et al. 2017).Taking into consideration polyamines and ODC action as a regulator of ovarian function, it is possible that AZINs should play important roles in regulating follicular development.AZIN1 mRNA expression gradually increased during follicular development and was significantly higher in POF1 follicle than other ovarian follicles.This suggests that AZIN1 plays a key role in follicular development (MA et al. 2015).In the present study, significant differences were not detected among the SWF, SYF and F5-F2 follicles, whereas the mRNA expression level of AZIN2 was significantly higher in the F1 follicle than other follicles.Recent studies suggest that AZINs might be important molecules for modulating cell proliferation and oncogenesis through both the polyamine pathway and additional mediators (LOPEZ-CONTRERAS et al. 2010;LOPEZ-GARCIA et al. 2013;QIU et al. 2017).Taken together, high expression of AZIN1 in the POF1 follicle and of AZIN2 in the F1 follicle indicate that AZINs may play an important role in modulating follicular development and ovulation.The actions and molecular mechanisms responsible for these functions remain to be determined.

Fig. 4 .
Fig. 4. Expression levels of the AZIN2 gene in goose follicles and ovary.Expression levels of the AZIN2 gene were normalized to GAPDH.Expression levels calculated by the 2 -ÄÄCt method were presented in arbitrary units (AU).Values are expressed as the mean ±SEM.Bars without a common letter are significantly different (P<0.05).