RAT CYSTATHIONINE BETA-SYNTHASE - EXPRESSION OF 4 ALTERNATIVELY SPLICED ISOFORMS IN TRANSFECTED CULTURED-CELLS
RAT CYSTATHIONINE BETA-SYNTHASE - EXPRESSION OF 4 ALTERNATIVELY SPLICED ISOFORMS IN TRANSFECTED CULTURED-CELLS
复制标题
DOI：
10.1016/0003-9861(92)90443-z
复制
发表时间：
1992-11-01
影响因子：
3.9
通讯作者：
KRAUS, JP
中科院分区：
文献类型：
--
作者：
ROPER, MD;KRAUS, JP
大鼠胱硫醚β-合酶基因由 17 个外显子组成。它的转录物被选择性剪接，形成四种不同的 mRNA 种类。 III 型由外显子 1 至 12、14、15 和 17 组成； I型还包含外显子16。IV型的开放阅读框跨越外显子1→13； type II, 3→ 13。我们将相应的 cDNA 克隆到适当的表达载体中，并将构建体插入大肠杆菌（I、III 和 IV）和中国仓鼠 (CH) 细胞（I 至 IV）中；所有序列均被转录和翻译。仅在转染的 CH 细胞和转化的大肠杆菌的裂解物中观察到 I 型和 III 型的催化活性。尽管结构不同（外显子 16），但 I 和 III 的催化和动力学性质相同。两种同工型均表现出 6 m m K m 的同型半胱氨酸常数，AdoMet 将其降低约八倍；这阐明了 AdoMet 调节合酶活性的机制。转染的培养细胞对四种亚型进行了差异性降解。 III型(t 1 2= 18 h)的降解速度是I型(t 1 2= 6 h)的1 3 倍；因此，外显子 16 编码的 14 个氨基酸残基似乎增强了 CBS 的降解。 II 型和 IV 型的半衰期明显较短（约 1 小时）。将大鼠肝脏与转染的 CH 细胞裂解物进行蛋白质印迹比较，结果显示肝细胞表达两种亚型。 III 型占主导地位，正如其半衰期更长和 mRNA 更丰富所预测的那样。对不同组织的 cDNA 进行 PCR 分析表明，III 型 mRNA 在肝脏、肾脏和心脏中优先；在大脑中发现了等量的 I 和 III。
The gene for rat cystathionine β-synthase consists of 17 exons. Its transcripts are alternatively spliced, forming four distinct mRNA species. Type III consists of exons 1 through 12, 14, 15, and 17; type I also contains exon 16. The open reading frame of type IV spans exons 1→ 13; type II, 3→ 13. We cloned the corresponding cDNAs into appropriate expression vectors and inserted the constructs into Escherichia coli (I, III, and IV) and Chinese hamster (CH) cells (I through IV); all sequences were transcribed and translated. Catalytic activity was observed only for types I and III in lysates of transfected CH cells and transformed E. coli. The catalytic and kinetic properties of I and III were identical despite their structural difference (exon 16). Both isoforms exhibited 6 m m K m constants for homocysteine which were reduced approximately eightfold by AdoMet; this elucidates the mechanism by which AdoMet regulates synthase activity. The four isoforms were differentially degraded by transfected cultured cells. Type III (t 1 2= 18 h) was degraded at 1 3 the rate of type I (t 1 2= 6 h); thus the 14 amino acid residues encoded by exon 16 appear to enhance degradation of CBS. The half-lives of both types II and IV were markedly shorter (ca. 1 h). Western blots comparing rat liver to lysates from transfected CH cells revealed that hepatocytes express both isoforms. Type III was predominant, as predicted by its longer half-life and more abundant mRNA. PCR analysis of cDNA from various tissues revealed that type III mRNA was preferred in liver, kidney, and heart; equal amounts of I and III were found in brain.