In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N1-acetyl-transferase (SSAT-1) and then oxidized by polyamine oxidase to produce spermidine and putrescine respectively. Herein we apply homology-search methods to identify novel sequences belonging to a second SSAT, SSAT-2, with a chromosomal location at 17p13.1, which is distinct from SSAT-1 at Xp22. Human SSAT-2 cDNA derived from small-cell lung carcinoma was deduced to encode a 170-amino-acid protein having 46% sequence identity and 64% sequence similarity with SSAT-1. When transiently transfected into HEK-293 cells, SSAT-1 decreased spermidine and spermine pools by ≈30%, while, at the same time, significantly increasing putrescine, N1-acetylspermidine, N1-acetylspermine and N1,N12-diacetylspermine pools. By contrast, transfected SSAT-2 had no effect on intracellular polyamine or acetylated polyamine pools. When enzyme activity was assayed on enzyme extracts from transfected cells, both SSAT-1 and SSAT-2 demonstrated much higher acetylating activity than vector-transfected cells. The data suggest that, in intact cells, SSAT-2 may be compartmentalized or it may be inefficient at low intracellular polyamine concentrations. By substituting candidate substrates in the enzyme assay, we determined that SSAT-1 shows the substrate preference norspermidine=spermidine≫spermine>N1-acetylspermine>putrescine, whereas SSAT-2 shows the preference norspermidine>spermidine=spermine≫N1-acetylspermine=putrescine. Analysis of mRNA levels in cell lines and ESTs (expressed sequence tags) from various tissues by digiNorthern (a web-based tool for virtually displaying expression profiles of query genes based on EST sequences) indicated that SSAT-1 tends to be more widely and highly expressed than SSAT-2. While SSAT-1 mRNA was inducible by polyamine analogues in a variety of cell lines, SSAT-2 was not. The existence of an active, but possibly sequestered, SSAT-2 enzyme suggests that, under certain conditions, it may be recruited into basal or perturbed polyamine metabolism.
Abbreviations used: N1-AcSpd, N1-acetylspermidine; N1-AcSpm, N1-acetylspermine; DASpm, N1,N12-diacetylspermine; DESpm, N1,N12-diethylspermine; DEHSpm, N1,N14-diethylhomospermine; DENSpm, N1,N11-diethylnorspermine; EST, expressed sequence tag; GNAT, generic N-acetyltransferase; norSpd, norspermidine; ODC, ornithine decarboxylase; PAO, polyamine oxidase; Put, putrescine; SMO, spermine oxidase; Spd, spermidine; Spm, spermine; SSAT, spermidine/spermine N1-acetyltransferase.
Shared first authorship.