Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain

Abstract

Human 3 -hydroxysteroid dehydrogenase/steroid 5 -4 -isomerase (3 -HSD/isomerase) is a bifunctional, single enzyme protein that is membranebound in the endoplasmic reticulum (microsomes) and mitochondria of cells in the placenta (type I) and in the adrenals and gonads (type II). Two membrane-binding domains (residues 72-89 and 283-310) have been predicted by analyses of hydrophobicity in the type I and II isoenzymes (90% regional homology). These putative membrane domains were deleted in the cDNA by PCR-based mutagenesis, and the two mutant enzymes were expressed by baculovirus in insect Sf9 cells. Differential centrifugation of the Sf9 cell homogenate containing the 283-310 deletion mutant revealed that 94% of the 3 -HSD and isomerase activities were in the cell cytosol, 6% of the activities were in the microsomes, and no activity was in the mitochondria. This is the opposite of the subcellular distribution of the wild-type enzyme with 94% of the activities in the microsomes and mitochondria and only 6% activity in the cytosol. The organelle distribution of the 72-89 deletion mutant lies between these two extremes with 72% of the enzyme activity in the cytosol and 28% in the microsomes/ mitochondria. The integrity of the subcellular organelle preparations was confirmed by electron microscopy. Western immunoblots confirmed the presence of the 283-310 deletion mutant enzyme and the absence of the wild-type enzyme in the insect cell cytosol. The unpurified, cytosolic 383-310 deletion mutant exhibited 3 -HSD (22 nmol/min per mg) and isomerase (33 nmol/min per mg) specific activities that were comparable with those of the membrane-bound, wild-type enzyme. The isomerase reaction of the cytosolic 283-311 deletion mutant requires activation by NADH just like the isomerase of the microsomal or mitochondrial wild-type enzyme. In contrast, the 72-89 deletion mutant had low 3 -HSD and isomerase specific activities that were only 12% of the wild-type levels. This innovative study identifies the 283-310 region as the critical membrane domain of 3 -HSD/isomerase that can be deleted without compromising enzyme function. The shorter 72-89 region is also a membrane domain, but deletion of this NH 2 -terminal region markedly diminishes the enzyme activities. Purification of the active, cytosolic 283-310 deletion mutant will produce a valuable tool for crystallographic studies that may ultimately determine the tertiary/ quaternary structure of this key steroidogenic enzyme.

Human 3β-hydroxysteroid dehydrogenase types 1 and 2: Gene sequence variation and functional genomics

“…None of the HSD3B1 nonsynonymous cSNPs observed during our gene resequencing studies were located in any of these regions. In addition, two putative membrane-binding domains have been identified in these enzymes that are located between residues 72 and 89 in the N-terminus of the proteins and between resides 283 and 310 in the C-terminal region [11,37]. Two of the nonsynonymous cSNPs in HSD3B1 (Ile79Val and Phe286Leu), and one in HSD3B2 (Asp74Asn), were located in these putative membrane-binding domain regions.…”

“…Since the HSD3B1 Val79 and Leu286 and the HSD3B2 Asn74 alterations in amino acid sequence were located within putative membrane-binding domains [11,37], confocal microscopy was performed to determine the possible effects of these polymorphisms on the subcellular localization of these isoforms. Specifically, with calnexin as an endoplasmic reticulum marker, immunofluorescent studies were performed using COS-1 cells transiently transfected with constructs encoding HSD3B1 WT, Val79, Leu286 and a combination of the codon 79 and 286 variants, as well as HSD3B2 WT and Asn74 constructs.…”

The engineered, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase: purification, characterization and crystallization

“…The codons were deleted for amino acids 283-310 in the cDNA encoding human type I 3 -HSD/ isomerase as previously described (Thomas et al 1999). This truncated protein is referred to as the 283-310 deletion-mutant or the cytosolic enzyme in this report.…”

“…One hydrophobic segment lies in the NH 2 -terminal region (residues 72-89), and the other segment is in the COOH-terminal region (residues 283-310) of the human type I and type II 3 -HSD/isomerase (Thomas et al 1999). Homologous hydrophobic domains have been identified in 15 deduced proteins from the 3 -HSD gene family in several species (Simard et al 1996).…”

“…Whereas the deletion of the NH 2 -terminal hydrophobic region (residues 72-89) produced a partially soluble protein with significantly reduced enzyme activities, the deletion of the COOHterminal hydrophobic domain (residues 283-310) produced a fully active cytosolic form of the enzyme that possessed 3 -HSD and isomerase activities comparable with those of the wild-type enzyme (Thomas et al 1999). The presence of the detergents needed to solubilize the wild-type enzyme from microsomes and mitochondria (Thomas et al 1989, Zhou et al 1996 may have prevented the crystallization and structural analysis of this important steroidogenic enzyme.…”