SDS-PAGE on a 15 gel. The gel was dried and analyzed by
SDS-PAGE on a 15 gel. The gel was dried and analyzed by phosphorimaging.Results Endogenous Expression of Arylsulfatase K in Human Tissues– To verify endogenous expression of human ARSK, we initial analyzed its mRNA amounts. We looked for tissue-specific expression by RT-PCR of normalized cDNA samples from various human tissues and identified that ARSK is ubiquitously expressed (Fig. one). High expression levels are found in placenta and pancreas, and lower expression amounts are located in muscle. Other tissues (lung, brain, heart, liver, and kidney) demonstrate intermediate expression amounts. Due to the fact a certain signal could possibly be found 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 2. 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 Adenosine A2B receptor (A2BR) Antagonist Synonyms medium (M) samples have been analyzed for ARSK expression by Western blotting working with an anti-RGS-His6 antibody or an anti-ARSK antiserum, as indicated. Untransfected cells served being a handle. The arrow indicates the 68-kDa type of ARSK, as detected in the cell lysates. B, HEK293 cells stably expressing ARSK have been lysed, along with the cellular protein was taken care of with endoglycosidases PNGaseF or EndoH, as indicated. In parallel, ARSK secreted by HEK293 cells and enriched via HisTrap chromatography was subjected to therapy with endoglycosidases. All samples have been analyzed by Western blotting applying the anti-RGS-His6 antibody. The black arrow indicates the totally glycosylated 68-kDa kind, whereas the white arrows indicate the partially (64-kDa) or totally deglycosylated forms (60-kDa). C, HEK293 cells both overexpressing ARSK or not overexpressing ARSK were metabolically labeled for 1 h with [35S]methionine/cysteine then chased for your indicated instances. ARSK was immunoisolated from cell extracts employing the anti-ARSK-antibody, separated by SDS-PAGE, and analyzed by autoradiography. ARSK was detected like a 68-kDa protein (black arrow). In addition, a 23-kDa fragment (white arrow) appeared for the duration of the chase, suggesting processing with the precursor (left panel). A corresponding C-terminal fragment was detected, albeit only weakly, through the anti-RGS-His6 antibody when analyzing ARSK enriched from conditioned medium of producer cells by Western blotting (right panel, showing 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 were also stably transfected with all the FGE-encoding cDNA for the reason that sulfatase action depends on posttranslational formylglycine modification. Western blot SGK1 Storage & Stability analyses of untransfected control and ARSK-expressing HEK293 and HT1080 cells working with a His tag-specific antibody (Fig. 2A, left panel) too as an ARSK-specific antibody (correct panel) detected a protein with an apparent molecular mass of 68 kDa in transfected cells. The secreted kind of ARSK present in conditioned medium from HT1080 cells exhibited a molecular mass of 70 kDa, i.e. slightly greater than the cellular kind (Fig. 2A, lanes 3 and 11). Glycosylation Pattern and Proces.