Rat cystathionine beta-synthase: expression of four alternatively spliced isoforms in transfected cultured cells.
The gene for rat cystathionine beta-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 mM Km 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 (t1/2 = 18 h) was degraded at 1/3 the rate of type I (t1/2 = 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.
Roper
M D
MD
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262.
Kraus
J P
JP
D017398:Alternative Splicing; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003541:Cystathionine beta-Synthase / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D003541:Cystathionine beta-Synthase