LOCALIZATION AND CHARACTERIZATION OF STANNIN - RELATIONSHIP TO CELLULAR-SENSITIVITY TO ORGANOTIN COMPOUNDS

The cDNA encoding the protein stannin was isolated previously via subt ractive hybridization, using differential expression after trimethylti n (TMT) intoxication, as a basis for isolating mRNA which may be expre ssed in TMT-sensitive cells. Initial characterization revealed a novel gene product which was differentially expressed in several tissues se nsitive to TMT. In the current study, biochemical and molecular techni ques were used to quantitate stannin expression at the cellular and su bcellular Levels. Northern blot analysis showed that the stannin 3.0 k b mRNA transcript was present, in decreasing amounts, in: spleen, hipp ocampus, neocortex, cerebellum, striatum, midbrain, kidney and lung. L iver, heart, skeletal muscle and testis showed no detectable expressio n of stannin mRNA. Immunoblot analysis using antipeptide antisera rais ed against stannin indicated a high level of expression in spleen, fol lowed by brain and kidney. Stannin mRNA was present during early brain development and consolidated by post-natal day (PND) 20 to patterns a nd levels seen in adults. In situ hybridization studies showed widespr ead neuronal expression of stannin mRNA at PND 1, which shifted to a r estricted pattern of expression in specific regions by PND 20. Stannin was partially purified from rodent brain and spleen using cation exch ange, sizing and hydrophobic interaction chromatography. It behaved as a monomer throughout purification. Stannin was also expressed in a ba culovirus system, using a series of constructs containing the entire c DNA, 1.0 kb of DNA flanking the open reading frame, and a 400 bp open reading frame minimal construct. While all constructs expressed stanni n, the best expression was seen with the entire cDNA. Based on current findings, we suggest that stannin expression is necessary but not suf ficient for TMT toxicity. (C) 1997 Elsevier Science Ltd. All rights re served.