Manganese (Mn) is an essential trace element required for various biological processes. Thus, the utilization of DT40 cells enabled us to clarify the different functions of each residue of hZIP8 on metal transport. Replacing EEXXH with HEXXH, which exists in most ZIP transporters, abolished the Mn- and Cd-transporting activity of hZIP8, indicating that glutamic acid in this motif plays a critical role in the unique affinity of ZIP8 for Mn and Cd. Finally, we examined the effects of artificial mutations in the metal-binding motif EEXXH in the TMD5. We also examined the A391T mutation, which slightly reduced metal transporting activity. Among the four mutations observed in the ZIP8-mutated CDG patients, the S335T and I340 N mutations in the predicted transmembrane domain 5 (TMD5) completely abolished Mn- and Cd-transporting activity, while V33 M or G35R mutations at the N-terminus did not. We established DT40 cells respectively expressing the four mutant hZIP8s and compared the Mn- and Cd-transporting activity between the mutants and wild-type hZIP8. However, the roles of individual mutations of hZIP8 on metal-transporting activity remain elusive. Many genome-wide association studies have reported that the A391T mutation of hZIP8 caused by rs13107325 is associated with a wide range of diseases. Four amino acid residues, V33, G38, S335, and I340, of human ZIP8 (hZIP8) are mutated in patients with congenital disorders of glycosylation (CDG), whose blood Mn levels are extremely low. Recently, associations between various human diseases and variant forms of ZIP8 have been reported. Zinc (Zn) transporter ZIP8, encoded by SLC39A8, is a unique transporter that can transport divalent manganese (Mn) and cadmium (Cd) in addition to Zn. Here we present a collection of these proteins and discuss the functional aspects of BIGM103, based on our results and current findings on two members of the family, Drosophila Catsup and Arabidopsis IAR1. However, the identified proteins are structurally unique compared to known ZIP members and they also possess the hallmark of Zn-metalloproteases, suggesting a new class of multi-TM protein with dual features. In fact, increased accumulation of intracellular Zn 2+ was observed due to the expression of BIGM103 in CHO cells. Further analysis revealed that all of these proteins have detectable similarity with the ZIP family of metal transporters.

The main open reading frame of the transcript encoded a putative seven-transmembrane (TM) protein that showed homology with a number of functionally unknown proteins in the database. The entire gene consisted of eight major exons and was localized on chromosome 4q22–q24, spanning ∼84 kb. Expression of this gene is very low or undetectable in unstimulated monocytes, while a steady expression level was observed during differentiation of monocytes to dendritic cells and macrophages.

Live and heat-killed Mycobacterium, bacterial cell wall, and inflammatory cytokines like TNFα were found to be potent inducers of the transcript. A novel transcript with three-protein coding potential was identified, and the expressed proteins from individual frames showed distinct intracellular localization. To identify novel genes induced during innate immune activation, we screened a cDNA library prepared from monocytes stimulated with Mycobacterium bovis BCG cell wall.

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