Functional Characterization of Single Nucleotide Polymorphisms in the TMEM163 Gene
Authors: Sanchez Vanessa , Ali Saima , Cuajungco Math P. 
Published: June 2021

Mucolipidosis type IV (MLIV) is a human lysosomal storage disorder caused by a loss or dysfunctional Mucolipin‐1 (TRPML1) ion channel protein. Abnormal levels of zinc accumulation have been found in the brain tissues of MLIV mouse model and cells from MLIV patients. We previously identified a putative zinc transporter called Transmembrane 163 protein (TMEM163) through a yeast two‐hybrid screen in search of interaction partners for TRPML1. We found both proteins to co‐localize on the plasma membrane and lysosomal compartments, and we proposed that both proteins play a role in intracellular zinc homeostasis. The function of TMEM163 has yet to be fully elucidated; however, it is predicted to have six transmembrane domains (TMD) and recent evidence suggests that it transports zinc. To further study TMEM163's function and how it might be involved in certain human diseases like MLIV, we used the single nucleotide polymorphism database (dbSNP) from the National Center for Biotechnology Information (NCBI) website to investigate specific nucleotide sequence variations within the TMEM163 gene. We identified SNPs that produce amino acid substitution when compared with the wild‐type (WT) TMEM163 protein sequence. We then used bioinformatics to predict the topology of TMEM163 protein, and found that some SNP‐associated amino acid substitutions are located in areas of the protein that could affect post‐translational modification and change its structure and function. Thus, we hypothesize that SNPs that result in amino acid substitution dramatically alters the zinc transport function of TMEM163. To accomplish this project, we performed site‐directed mutagenesis using In‐Fusion homologous recombination cloning technique to systematically replace specific nucleotides corresponding to the SNPs located within or in proximity of N‐ and C‐termini, as well as TMD‐1 to TMD‐6. Upon sequence verification, we tested the effect of each SNP‐associated mutant by transfecting them into cultured HeLa cells. Twenty‐four hours post transfection, the cells were exposed to zinc chloride (100 μM) and assayed for intracellular zinc transport using the zinc specific FluoZin‐3 AM dye. We observed that the SNP‐associated mutants have marked decrease in intracellular zinc fluorescence following exposure to exogenous zinc. These observations indicate that certain SNP‐associated amino acid substitution located within critical areas of TMEM163 protein results in abnormal zinc transport. Our preliminary data suggest that TMEM163 is indeed a zinc transporter. We are currently conducting additional experiments using different zinc specific dyes to support this result. Understanding the nature of TMEM163‐TRPML1 interaction is necessary to further support our findings of cellular zinc dyshomeostasis in MLIV cells.Support or Funding InformationThis work was funded by CSUPERB Research and Development Grant 2016, and partly funded by NIH R15 NS101594This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.