Ues and located that ARSK is ubiquitously expressed (Fig. 1). High SMYD3 Inhibitor Storage & Stability expression levels are found in placenta and pancreas, and low expression levels are located in muscle. Other tissues (lung, brain, heart, liver, and kidney) show intermediate expression levels. Due to the fact a distinct signal may be identified in all tissues analyzed, we conclude that ARSK is ubiquitously expressed in most, if not all, human tissues. Expression of Recombinant Arylsulfatase K–The human ARSK-encoding cDNA was obtained by reverse transcription PCR (see “Experimental Procedures”). Its coding sequenceJOURNAL OF BIOLOGICAL CHEMISTRYArylsulfatase K, a Novel Lysosomal SulfataseFIGURE two. Recombinant expression, N-glycosylation, and stability/processing of ARSK in human cells. A, ARSK was stably expressed in HT1080 and HEK293 cells. Cell lysates (C) and medium (M) samples had been analyzed for ARSK expression by Western blotting making use of an anti-RGS-His6 antibody or an anti-ARSK antiserum, as indicated. Untransfected cells served as a manage. The arrow indicates the 68-kDa form of ARSK, as detected in the cell lysates. B, HEK293 cells stably expressing ARSK were lysed, along with the cellular protein was treated with endoglycosidases PNGaseF or EndoH, as indicated. In parallel, ARSK secreted by HEK293 cells and enriched through HisTrap chromatography was subjected to PI3Kδ Inhibitor Synonyms remedy with endoglycosidases. All samples had been analyzed by Western blotting using the anti-RGS-His6 antibody. The black arrow indicates the totally glycosylated 68-kDa form, whereas the white arrows indicate the partially (64-kDa) or completely deglycosylated types (60-kDa). C, HEK293 cells either overexpressing ARSK or not overexpressing ARSK had been metabolically labeled for 1 h with [35S]methionine/cysteine then chased for the indicated occasions. ARSK was immunoisolated from cell extracts employing the anti-ARSK-antibody, separated by SDS-PAGE, and analyzed by autoradiography. ARSK was detected as a 68-kDa protein (black arrow). Also, a 23-kDa fragment (white arrow) appeared during the chase, suggesting processing in the precursor (left panel). A corresponding C-terminal fragment was detected, albeit only weakly, by the anti-RGS-His6 antibody when analyzing ARSK enriched from conditioned medium of producer cells by Western blotting (suitable panel, displaying three elution fractions in the HisTrap column, cf. Fig. 3A).(1608 bp) fully matched GenBankTM accession number AY358596. ARSK was stably expressed in HEK293 cells and HT1080 cells as a C-terminally RGS-His6-tagged variant. These cells have been also stably transfected using the FGE-encoding cDNA mainly because sulfatase activity is determined by posttranslational formylglycine modification. Western blot analyses of untransfected manage and ARSK-expressing HEK293 and HT1080 cells applying a His tag-specific antibody (Fig. 2A, left panel) too as an ARSK-specific antibody (suitable panel) detected a protein with an apparent molecular mass of 68 kDa in transfected cells. The secreted type of ARSK present in conditioned medium from HT1080 cells exhibited a molecular mass of 70 kDa, i.e. slightly larger than the cellular kind (Fig. 2A, lanes 3 and 11). Glycosylation Pattern and Processing–Bioinformatic evaluation predicts seven putative N-glycosylation websites with the consensus sequence NXS/T. To analyze the extent of glycosylation, recombinant ARSK was partially purified from HT1080 or HEK293 cells as well as from conditioned medium by chromatography on nickel-Sepharose and subjected to treatment with the.