Functional analysis of two single nucleotide polymorphisms in SLC30A2 (ZnT2): implications for mammary gland function and breast disease in women

Abstract

Zinc transporter 2 (ZnT2) plays a major role in zinc (Zn) export from the mammary gland. Recently, we determined that ZnT2 is associated with secretory vesicles reflecting its role in Zn secretion during lactation. Herein, we identified two distinct single nucleotide polymorphisms (SNPs) in SLC30A2, which encodes ZnT2. SNP1 (rs35235055) results in a leucine-to-proline substitution (Leu(23)Pro), while SNP2 (rs35623192) results in an arginine-to-cysteine substitution (Arg(340)Cys). We examined the localization and function of each SNP in cells generated to express these polymorphic variants. SNP1 was mislocalized to lysosomes, while SNP2 was mislocalized to the Golgi apparatus. FluoZin-3 fluorescence illustrated increased lysosomal accumulation of Zn in cells expressing SNP1 concomitant with the abrogation of Zn secretion. In contrast, ectopic expression of SNP2 was associated with the expansion of cytoplasmic Zn pools, elevated reactive oxygen species, and increased Zn efflux. Taken together, our data indicate that polymorphic variants in ZnT2 distinctly alter mammary cell Zn metabolism. We speculate that these SNPs may compromise mammary cell function, which may have important implications in human health and breast disease.

Fig. 1.

Polymorphic variants in zinc transporter 2 (ZnT2) result in mislocalization of the protein. A: representative immunoblot (IB) of crude membrane proteins (50 μg protein/lane) from cells transfected with pcDNA3.1 (Mock), or cells expressing wild-type ZnT2-hemagglutinin (HA) fusion protein (ZnT2), single nucleotide polymorphism (SNP)1-HA fusion protein (SNP1), or SNP2-HA fusion protein (SNP2) detected with HA antibody. *Nonspecific protein recognized by the anti-HA antibody. B: localization of wild-type ZnT2-HA (A–C), SNP1-HA (D–F), or SNP2-HA (G–I) in transfected HC11 cells examined using confocal microscopy. ZnT2 was detected with Alexa 488-conjugated anti-HA antibody (green; A, D, and G), and the nucleus was stained with TOPRO nucleic acid dye (red; B, E, and H).

Fig. 2.

Confocal microscopy identified lysosomes and the Golgi apparatus as the specific subcellular compartments associated with SNP1 and SNP2, respectively. Cells transfected with pcDNA3.1-ZnT2, -SNP1, and -SNP2 to express HA-tagged ZnT2 (ZnT2-HA), SNP1 (SNP1-HA), and SNP2 (SNP2-HA) were detected with HA antibody and visualized with Alexa 488 conjugated anti-mouse IgG. Confocal micrographs illustrate that the wild-type ZnT2 (green) colocalizes (merge, yellow) with mannose-6-phosphate receptor (M6PR, red, endosome marker). SNP1 (green) colocalizes (merge, yellow) with Lamp1 (red, lysosomal marker) [Pearson's coefficient (PC) = 0.9]. SNP2 colocalizes (merge, yellow) with p58 (red, Golgi marker) (PC = 0.8). The value of PC can range from 1 to −1, with 1 standing for complete positive correlation and −1 for a negative correlation, with zero standing for no correlation.

Fig. 3.

Cytoplasmic zinc (Zn) pools were significantly reduced in cells expressing SNP1 but not affected by ectopic expression of SNP2. Cytoplasmic Zn level was measured using a 4X-metal response element (MRE) luciferase reporter construct in cells co-transfected with each SNP and compared with cells transfected to overexpress wild-type ZnT2. Data represent mean luminescence (arbitrary units/μg protein) (n = 3) from 2 independent experiments. Means with different letters are significantly different as analyzed by 1-way analysis of variance followed by a Tukey posttest (P < 0.05).

Fig. 4.

Expression of SNP1 leads to increased Zn accumulation into vesicles. Zn accumulation was assessed using FluoZin-3 fluorescence in cells transfected to express each SNP and compared with cells transfected to overexpress wild-type ZnT2. Fluorescence was quantified by fluorimetry (A) and visualized by confocal microscopy (B). Data represent mean fluorescence ± SD (n = 8 samples/group). Means with different letters are significantly different as analyzed by 1-way analysis of variance followed by a Tukey posttest (p < 0.05). DIC, differential interference contrast.

Fig. 5.

Ectopic expression of SNP1 accumulates Zn into lysosomes. Cells were transfected with pcDNA3.1-SNP1 to express HA-tagged SNP1. Confocal micrographs illustrate that colocalization (yellow) of FluoZin-3 (green) with the lysosomal marker Lysotracker (red) in the absence of chloroquine disphosphate (−CQ). Colocalization is eliminated in chloroquine disphosphate (+CQ)-treated SNP1-expressing cells, indicating lysosomes are a site of Zn accumulation in SNP1-expressing cells.

Fig. 6.

Zn secretion was significantly reduced in cells expressing SNP1 but increased by ectopic expression of SNP2. Cells were preincubated in serum-free medium containing 0.1 μM ZnSO₄ and 0.1 μCi ⁶⁵Zn for 3 h. Medium was replaced, and ⁶⁵Zn was measured after 120 min. Values represent mean pmol Zn/μg protein ± SD (n = 3 samples/genotype). Means with different letters are significantly different as analyzed by 1-way analysis of variance followed by a Tukey posttest (P < 0.05).

Fig. 7.

Reactive oxygen species (ROS) levels were significantly increased in cells ectopically expressing SNP2. ROS level was assessed using dichlorofluorescein (DCF)-HA fluorescence in cells transfected to express each SNP and compared with cells generated to overexpress wild-type ZnT2. To validate our assay, H₂O₂ (100 μM) was used as a positive control. Data represent mean fluorescence ± SD (n = 8 samples/group). Means with different letters are significantly different as analyzed by 1-way analysis of variance followed by a Tukey posttest (P < 0.05).