Both N-terminal and C-terminal regions of steroid sulfatase are important for enzyme activity

Abstract

Steroid sulfatase (STS) is localized in the endoplasmic reticulum and catalyzes desulfation of 3 -hydroxysteroid sulfates. X-linked ichthyosis (XLI) is an inherited skin disorder caused by deficiency of STS enzyme activity. We previously reported a case in which XLI with a one-base change in the STS gene and variation in amino acid Q560P developed. In this study, we performed molecular analysis to determine the importance of terminal regions of STS and the effect of mutant STS on STS enzyme activity. To examine the effect of terminal truncated STS on the enzyme activity, N-and C-terminal truncated STS expression vectors were transfected into COS-1 cells. The activity of truncated STS lacking the N-terminal regions declined, and the activity of C-terminal-truncated STS declined with extension of the truncated C-terminal region. Although the results of pulse-chase experiments showed that a one-base mutant STS (Q560P) and C-terminal-truncated STS (C2 (1-559)) had no effects on protein synthesis and degradation, the mutant STS and C-terminal-truncated STS have dominant negative effect on STS enzyme activity when the STS mutant or truncated STS protein and a wild-type STS protein coexist in cells. Results of coprecipitation of the truncated STS with an STS-FLAG fusion protein showed that STS formed a dimer conformation in cells. In this study, we have shown that both the N-terminal region and C-terminal region are important for STS enzyme activity. The C-terminal mutant has a dominant negative effect on wild-type STS.

Effects of black cohosh on estrogen biosynthesis in normal breast tissue in vitro

“…To quantify STS protein in the breast tissue samples studied, we performed western blots of tissue extracts. A specific band corresponding to the Mr of dimeric STS [51] was detected in all breast tissues investigated (Fig. 2).…”

Novel point mutation in the STS gene in a patient with X-linked recessive ichthyosis

“…Accumulation of fatty alcohol has been shown in cultured fibroblasts and in plasma from SLS patients [1]. Numbers of mutations of FALDH gene have been shown, although only three mutations have been identified in Japanese SLS patients [2][3][4]. We here report a SLS patient who is a homozygote for one of the known mutations.…”

“…Up to 90% of the described mutations of this gene are complete deletions, which is one of the highest percentages of chromosomal deletions among all genetic disorders [2,3]. STS is located on Xp22.3 and encodes a 62kDa polypeptide [4]. In the skin STS is expressed within the epidermis and is thought to have a role in steroid production and lipid metabolism.…”

“…The STS gene has 10 exons spread over 146 kilo base pairs encoding a 62 kDa polypeptide [4]. STS gene deficiencies are heterogeneous and in addition to the more common deletions there are to our knowledge 13 described point mutations (Human Gene Mutations Database, 2010-11-23), 12 in the coding region and one in the non-coding region.…”

Effects of mutations and glycosylations on STS activity: A site-directed mutagenesis study

“…Digestion of STS by endoglycosidase H shows that at least two (Asn47 and Asn259) of the STS N-glycosylation sites are used for glycosylation. It has also been reported that although both the N-terminal region and the C-terminal region of the STS enzyme are important for the STS enzymatic function (Sugawara et al, 2006), the key catalytic residue in sulphatase is a unique C-formylglycine, which is generated from a cysteine precursor. The C-formylglycine functions as a nucleophilic aldehyde hydrate in the initial addition reaction of sulphate ester hydrolysis (von Figura et al, 1998).…”