A novel testicular Mu class GST subfamily

Abstract

Glutathione S-transferases (GSTs) are a family of proteins that catalyze the conjugation of reduced glutathione to a variety of electrophiles, and also function as lipophilic binding proteins. Although one Pi class, five Mu class and two Alpha class human GST genes have been identified, many of their corresponding gene products were not identified from human tissue sources. Purified GSTs from a variety of human tissues were used for western blotting, reversed-phase HPLC and electrospray ionization mass spectrometry to identify GST subunits and their tissue specific distribution patterns. GST subunit patterns were found to be tissue-dependent and characteristic of a given normal adult tissue. A unique hGSTM3 subunit, containing 4 amino acid N-terminal and 3 amino acid C-terminal extensions relative to other Mu-class GSTs was expressed in testis and brain and shown to be developmentally regulated in the testis; it was undetectable in fetal testis and highly expressed in adult testis.;To fully investigate the developmental changes in the expression of the hGSTM3 subunit, an animal model was needed. A rat homologue also found in testis and brain, rGSTMS, was identified and its primary structure determined. A mouse subunit homologue, mGSTMS, was also identified from mouse testis. These three subunits represent a new subfamily of Mu class GSTs; they all contain extended N- and C-termini, have greater homologies within the subfamily than to other known Mu class GSTs and share unique enzymatic and tissue distribution characteristics.;The expression of rGSTMS was studied in rat testis and brain at several ages as a model of hGSTM3 expression. rGSTM3 was undetectable in fetal rat testis and expressed at about 10% of adult levels in testes from 1, 2 and 3 week old rats. Brain expression of rGSTMS in 2 and 3 week old rats was comparable to the levels in adult rats. These results were consistent with observed patterns of expression of hGSTM3 in human fetal, infant and adult tissue. Rats were hypophysectomized to study hormonal regulation of rGSTMS. At five weeks post-hypophysectomy, the rGSTMS subunit was barely detectable in testis. In contrast, there were only minor differences in the GST composition from matched rat brain. GSTs were purified from human sperm to test the hypothesis that this subunit family is expressed in spermatozoa. Resolution of sperm GST subunits showed hGSTM3 to be the predominant GST subunit in these cells. These results account for the observed developmental pattern of rat and human testicular GSTs.