ABSTRACT
Steroid sulphatase (STS) catalyses the formation of active steroids from inactive steroid sulphates. High levels of intra-tumoural STS mRNA are associated with a poor prognosis in post-menopausal patients with oestrogen receptor positive breast cancer. In this study, analysis of the mutated STS protein showed that N- and C-terminal truncated STS constructs are inactive. Histidine 136, located inside the active site, is crucial for STS activity whereas proline 212, which allows the protein turn into the membrane, is not. Mutations in glycosylation sites asparagine 47 and 259 decreased STS activity while asparagine 333 and 459 mutations did not affect it. However, immunoblot studies revealed that all four N-linked sites are glycosylated to some extent. In addition, a polyclonal antibody raised in rabbits against human STS was developed and characterised. These data increase our knowledge of the STS enzyme structure and may help design new STS inhibitors.
Subject(s)
Mutagenesis, Site-Directed , Point Mutation/genetics , Steryl-Sulfatase/genetics , Steryl-Sulfatase/metabolism , Amino Acid Sequence , Animals , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Glycosylation , Humans , Immune Sera , Molecular Sequence Data , Mutant Proteins/metabolism , Steryl-Sulfatase/chemistry , Steryl-Sulfatase/immunologyABSTRACT
BACKGROUND: Estradiol (E(2)) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent. METHODS: In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17beta-hydroxysteroid dehydrogenases (17beta-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)] in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures. RESULTS: Aromatase and 17beta-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17beta-HSD, EST and STS were readily detectable. Only 17beta-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17beta-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies. CONCLUSIONS: In endometriosis lesions, the balance is tilted in favor of enzymes producing E(2). This is due to a suppression of types 2 and 4 17beta-HSD, and an increased expression of aromatase and type 1 17beta-HSD in ectopic endometrium.
