Characterization of the arylsulfatase I (ARSI) gene preferentially expressed in the human retinal pigment epithelium cell line ARPE-19

Abstract

Purpose: The aim of this study was to characterize the arylsulfatase I (ARSI) gene that has been shown to be preferentially expressed in the human retinal pigment epithelium cell line ARPE-19 and to propose it as a candidate gene responsible for inherited eye diseases such as retinitis pigmentosa (RP).

Methods: Full-length cDNA clones encoding ARSI, arylsulfatase A (ARSA), and sulfatase modifying factor 1 (SUMF1) were isolated from ARPE-19 cDNA libraries constructed using the vector-capping method. The expression vectors for their FLAG-tagged proteins were transfected into ARPE-19 cells, and the expression products were characterized by western blot analysis and arylsulfatase assay. The entire region of the ARSI gene locus was sequenced using the genomic DNA samples of 68 RP patients.

Results: Transiently produced ARSI-FLAG was localized to the endoplasmic reticulum and was detected in the cellular fraction and the medium. When ARSI-FLAG and SUMF1-FLAG were coexpressed, the conditioned medium of the transfected cells showed arylsulfatase activity at a range of neutral pH. No mutation was found in the ARSI gene locus of the RP patients examined.

Conclusions: ARSI may be a secreted sulfatase and may function in the extracellular space. Although ARSI may not be a causative gene for lysosomal storage diseases, preferentially expressed in the eye, ARSI would be a candidate gene causing inherited eye diseases for future mutation screening.

Figure 1

Expression profile of arylsulfatase-related genes. A: Thirteen tissues expressing ARSI were described based on the expressed sequence tag (EST) counts of UniGene database. The total EST counts and the total number of tissues expressing each gene are listed in the inset table. B: Arylsulfatase-related gene expressions in human cell lines were analyzed using RT–PCR. A eukaryotic elongation factor 1 alpha 1 (EEF1A1) gene was used as an internal control.

Figure 2

Transient expression of arylsulfatase-related genes. A and B: Endogenous PDI (ER marker, A) and cathepsin D (lysosome marker, B) were immunostained. ARPE-19 cells were transfected with 6 μg of the expression vector ARSI-FLAG. Scale bars, 10 μm. C: The structures of FLAG-tagged protein constructs were depicted. The colored box at the N-terminal represents a signal sequence. The active site of ARSI, Cys (C), was replaced by Ser (S) in a mutant derivative. The size of each secreted mature protein was represented. D: The expression products were analyzed with western blot. ARPE-19 cells were transfected with 1 μg SUMF1-FLAG, 5 μg ARSA-FLAG, or 8 μg ARSI derivatives, and cultured in serum-containing media for 22 h and then in serum-free media for 9 h. Loaded were 20% of the total sample for insoluble fraction, 10% for cell extract, and 25% for medium. The lane number corresponds to the construct number of the FLAG-tagged protein described in C.

Figure 3

Arylsulfatase activity of cellular extracts. A: Cellular extracts were analyzed with western blot. Transfected were 5 μg ARSA-FLAG (lanes 1 and 2), 8 μg ARSI-FLAG (lanes 3 and 4), and 1 μg SUMF1-FLAG (lanes 2 and 4) into ARPE-19 cells .The transfected cells were cultured in media for 72 h. The amount of the loaded sample was 10 μg protein for ARSA and 12.5 μg protein for ARSI. B: The ARS activity of ARSA-FLAG was measured at pH 5.0 using 0.5 mM 4-MUS as a substrate. C: The ARS activity of ARSI-FLAG was measured at pH 5.6, using 5 mM 4-MUS as a substrate. The activity was calculated as the value per mg of cellular protein in each fraction. D: The pH dependence of ARS activities was shown. The ARS activities in B, C, and D give the means of three independent experiments. Bars indicate standard deviations. The error bars in B were omitted because the differences between samples were obvious.

Figure 4

Arylsulfatase activity of media. A: Arylsulfatase A (ARSA) products were analyzed with western blot. ARPE-19 cells were transfected with 5 μg ARSA-FLAG and 1 μg SUMF1-FLAG, and cultured in serum-containing media for 2 h and then in serum-free media for 22 h. Loaded was 12.5% of the total sample. B: The ARS activity of ARSA-FLAG was measured using 0.4% of the total medium. C: Arylsulfatase I (ARSI) products were analyzed with western blot. Transfected were 8 μg ARSI-FLAG, 8 μg ARSI-C93S-FLAG, and 1 μg SUMF1-FLAG into ARPE-19 cells. D: The trichloroacetic acid (TCA)-induced precipitate of the medium was analyzed with western blot. E: The ARS activity of ARSI-FLAG was measured using 20% of the total medium. F: The pH dependence of ARS activities was shown. The ARS activities in B, E, and F give the means of three independent experiments. Bars indicate standard deviations.