Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Abstract

The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD) to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3 (rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1 Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in K m (2 µM) for rT3 as compared with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125 I]T3 affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and 32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity. Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal sequence and the membrane-spanning domain (amino acid residue 2-35), did not result in the production of a soluble D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further structure-function analysis.

New insights into the structure and mechanism of iodothyronine deiodinases

“…The biophysical and structural characterization of deiodinases is severely aggravated by the fact that they are integral transmembrane proteins and selenoenzymes. The mechanism of Sec incorporation differs between eukaryotes and bacteria, hampering the expression of fully active, recombinant mammalian deiodinase protein in the widely used, efficient prokaryotic expression systems (Kuiper et al 2005b). Expression of a Cys-mutant Dio1 in yeast yielded an active but heterogenous protein that did not allow structural studies (Kuiper et al 2005b).…”

“…The mechanism of Sec incorporation differs between eukaryotes and bacteria, hampering the expression of fully active, recombinant mammalian deiodinase protein in the widely used, efficient prokaryotic expression systems (Kuiper et al 2005b). Expression of a Cys-mutant Dio1 in yeast yielded an active but heterogenous protein that did not allow structural studies (Kuiper et al 2005b). In the absence of experimental structural information, modeling of human deiodinases suggested that a Dio-specific insertion in the Trx-like sequence forms a glycoside hydrolase like insertion in the basic Trx fold (Callebaut et al 2003).…”

Iodotyrosine Deiodinase Is the First Mammalian Member of the NADH Oxidase/Flavin Reductase Superfamily

“…For ID, excision of its predicted transmembrane anchor still did not yield soluble protein (44). For IYD, limited proteolysis by trypsin did generate a soluble and active protein, presumably by hydrolysis of a single terminal peptide anchor.…”

“…Enzyme Solubilization, Isolation, and Identification-Purification and detailed mechanistic study of both IYD and ID have been hindered by their association with cell membranes (36,44). For ID, excision of its predicted transmembrane anchor still did not yield soluble protein (44).…”

Expression of a soluble form of iodotyrosine deiodinase for active site characterization by engineering the native membrane protein from Mus musculus

“…Cell culture remains the most common source of ID 22,23 despite recent success in heterologous expression of ID in yeast. 24 Attempts to generate a soluble form of ID have not yet been successful. Deletion of its N-terminal membrane anchor yielded only inactive protein.…”