Genomic identification and biochemical characterization of a second spermidine/spermine N1-acetyltransferase
Genomic identification and biochemical characterization of a second spermidine/spermine N1-acetyltransferase
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DOI：
10.1042/bj20030734
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发表时间：
2003-08-01
影响因子：
4.1
通讯作者：
Porter, CW
中科院分区：
文献类型：
--
作者：
Chen, Y;Vujcic, S;Porter, CW
在多胺逆向转化途径中，精胺和亚精胺首先被亚精胺/精胺N - 1 - 乙酰转移酶（SSAT - 1）乙酰化，然后被多胺氧化酶氧化，分别生成亚精胺和腐胺。在此，我们应用同源性搜索方法来鉴定属于第二种SSAT（SSAT - 2）的新序列，其染色体定位在17p13.1，这与位于Xp22的SSAT - 1不同。从小细胞肺癌中获得的人SSAT - 2 cDNA经推导可编码一个含170个氨基酸的蛋白质，它与SSAT - 1具有46%的序列同一性和64%的序列相似性。当瞬时转染到HEK - 293细胞中时，SSAT - 1使亚精胺和精胺库减少约30%，同时显著增加腐胺、N - 1 - 乙酰亚精胺、N - 1 - 乙酰精胺和N - 1,2 - 二乙酰精胺库。相比之下，转染的SSAT - 2对细胞内多胺或乙酰化多胺库没有影响。当对转染细胞的酶提取物进行酶活性测定时，SSAT - 1和SSAT - 2都显示出比载体转染细胞高得多的乙酰化活性。数据表明，在完整细胞中，SSAT - 2可能被分隔开来，或者在细胞内多胺浓度较低时效率低下。通过在酶测定中替换候选底物，我们确定SSAT - 1对底物的偏好为降亚精胺 = 亚精胺＞精胺＞N - 1 - 乙酰精胺＞腐胺，而SSAT - 2的偏好为降亚精胺＞亚精胺 = 精胺＞N - 1 - 乙酰精胺 = 腐胺。通过数字Northern（一种基于网络的工具，可根据EST序列虚拟显示查询基因的表达谱）对细胞系中的mRNA水平以及来自各种组织的EST（表达序列标签）进行分析表明，SSAT - 1往往比SSAT - 2表达更广泛且水平更高。虽然在多种细胞系中多胺类似物可诱导SSAT - 1 mRNA的表达，但不能诱导SSAT - 2。一种有活性但可能被隔离的SSAT - 2酶的存在表明，在某些条件下，它可能被招募参与基础的或受干扰的多胺代谢。
In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N-1-acetyltransferase (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 approximate to 30 %, while, at the same time, significantly increasing putrescine, N-1-acetylspermidine, N-1-acetylspermine and N-12 -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 vectortransfected 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 much greater than spermine > N-1-acetylspermine > putrescine, whereas SSAT-2 shows the preference norspermidine >spermidine = spermine much greater than N-1-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.