TRUNCATED HS3ST4 EXPRESSED DOWNSTREAM OF CHRONIC PAIN- ASSOCIATED SINGLE NUCLEOTIDE POLYMORPHISMS REGULATES TRANSCRIPTIONAL PROMOTER ACTIVITY

The pathogenic mechanism of post-herpetic neuralgia (PHN), a chronic pain that occurs after herpes zoster caused by reactivation of varicella-zoster virus (VZV), has not been fully understood. By statistical analysis of the genome-wide association study (GWAS) data of patients with chronic pain including PHN patients and healthy subjects, we previously found that single nucleotide polymorphisms (SNPs) located in heparan sulfate 3-O-sulfotransferase 4 (HS3ST4) gene were significantly associated with PHN or chronic pain (Nishizawa et al., 2021), and full-length HS3ST4 induces cell fusion in the presence of VZV glycoproteins (Ohka et al., 2021). The chronic pain-associated HS3ST4 SNPs exist in the intronic region of HS3ST4 gene. However, the impact of this intronic region on the expression/function of the HS3ST4 remains unclear.

We aim to clarify the functions of the intronic region where chronic pain-associated SNPs reside.

Public cap-analysis gene expression (CAGE) and poly-A RNA fragment databases were searched. 5’ rapid amplification of cDNA end (RACE) of HS3ST4 mRNA derived from the human primitive neuroectodermal tumor cell line FU-RPNT-1 was performed. The plasmids expressing truncated HS3ST4 and GFP or NanoLuc luciferase under the cytomegalovirus (CMV) promoter or human elongation factor 1-α (EF1α) promoter were introduced in the human neuroblastoma cell line SK-N-SH. Expression of the GFP or luciferase was monitored by the fluorescent microscope or the luciferase assay, respectively.

Public CAGE databases showed transcription start site (TSS) downstream of the chronic pain-associated HS3ST4 SNPs in FU-RPNT-1 cells and the adult human brain. Moreover, a poly-A RNA fragment had been reported from over 10 kbp downstream of the SNPs. 5’ end of a novel mRNA just downstream of the SNPs was identified by 5’ RACE using mRNA from FU-RPNT-1 cells. The novel mRNA had an open reading frame of a truncated traditional HS3ST4 protein. Plasmid transfection experiments revealed that the truncated HS3ST4 protein enhanced the GFP and luciferase expression in neural SK-N-SH cells. These data suggest that the truncated HS3ST4 protein enhanced the transcriptional activity of CMV and EF1α promoters.

Novel mRNA from downstream of the chronic pain-associated SNPs encoded the open reading frame of the truncated traditional HS3ST4. The truncated HS3ST4 could manipulate the promoter activities. It is assumed that the truncated HS3ST4 upregulates pain-related protein transcriptions, contributing to chronic pain expression. These facts may contribute to a novel target of therapeutic agents.

Nishizawa, D. et al. (2021) Genome-wide association study identifies candidate loci associated with chronic pain and postherpetic neuralgia, Mol Pain, 17: 1–21. Available at: DOI: 10.1177/1744806921999924

Ohka, S. et al. (2021) Heparan sulfate 3-O-sulfotransferase 4 is genetically associated with herpes zoster and enhances varicella-zoster virus– mediated fusogenic activity, Mol Pain, 17: 1–10. Available at: DOI: 10.1177/17448069211052171