Analysis of the thyrotropin-releasing hormone-degrading ectoenzyme by site-directed mutagenesis of cysteine residues. Cys68 is involved in disulfide-linked dimerization

Abstract

Thyrotropin-releasing hormone-degrading ectoenzyme is a member of the M1 family of Zn-dependent aminopeptidases and catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH2). Cloning of the cDNA of this enzyme and biochemical studies revealed that the large extracellular domain of the enzyme with the catalytically active site contains nine cysteine residues that are highly conserved among species. To investigate the functional role of these cysteines in TRH-DE we used a site-directed mutagenesis approach and replaced individually each cysteine by a serine residue. The results revealed that the proteolytically truncated and enzymatically fully active enzyme consists of two identical subunits that are associated noncovalently by protein-protein interactions but not via interchain S-S bridges. The eight cysteines contained within this region are all important for the structure of the individual subunit and the enzymatic activity, which is dramatically reduced in all mutant enzymes. This is even true for the four cysteines that are clustered within the C-terminal domain remote from the Zn-binding consensus sequence HEICH. In contrast, Cys68, which resides within the stalk region seven residues from the end of the hydrophobic membrane-spanning domain, can be replaced by serine without a significant change in the enzymatic activity. Interestingly, this residue is involved in the formation of an interchain disulfide bridge. Covalent dimerization of the subunits, however, does not seem to be essential for efficient biosynthesis, enzymatic activity and trafficking to the cell surface.

Figure 1

Schematic representation of theprimary structure of rat TRH‐DE, rat APN and mouse APA. Cysteine residues and their individual positions are indicated by vertical lines. The open box indicates the N‐terminal intracellular domain and the black box indicates the transmembrane domain. The C‐terminal extracellular domain, with the zinc‐binding site (HEXXH‐motif) is represented by the hatched box. aa, Amino acids.

Figure 2

Western blot analysis of wild‐type andmutated rat TRH‐DEs expressed in BHK‐cells. The top diagram shows the schematic representation of the primary structure of rat TRH‐DE in pcDNA3.1. The complete open‐reading frame was fused in‐frame C‐terminal to the polyhistidine tag. The nine cysteine residues of the wild‐type transcript are indicated as vertical lines on the top of the diagram. The closed box indicates the transmembrane spanning domain and the hatched box indicates the Zn‐binding consensus motif. Following transient transfection of the BHK‐cells with pcDNA3.1, pcDNA3.1/lacZ, wild‐type and mutants, the membrane fractions containing the ectoenzyme were subjected to SDS/PAGE as described under Experimental procedures. The His‐tagged proteins were detected using the SuperSignal HisProbe Western Blotting Kit (Pierce). The positions of the molecular mass markers (in kDa) are indicated.

Figure 3

Enzymatic activity of rat TRH‐DE cysteine‐mutants. Rat TRH‐DE activity in the membrane fractions of transfected BHK cells is expressed as percentage of wild‐type rat TRH‐DE activity. Measurements are the mean ± SD of three independent transfections. The mean value for wild‐type rat TRH‐DE corresponds to 250 nmol of substrate hydrolyzed per min per mg enzyme.

Figure 4

Immunoblot analysis of recombinantTRH‐DEs and truncated ectoenzyme. (A) SDS/PAGE and immunoblotting of the membrane proteins from BHK cells transfected with the FLAG‐tagged wild‐type TRH‐DE cDNA (lanes 2 and 5), the corresponding C68S mutant (lanes 3 and 6) and the pFLAG‐CMV1/BAP cDNA (lanes 1 and 4). The samples were incubated on ice for 30 min in SDS buffer in the presence (+) or absence (–) of dithiothreitol (DTT). After SDS/PAGE, the anti‐FLAG monoclonal antibody M2 was used for immunoblotting. (B) SDS/PAGE and immunoblotting of the purified ectoenzyme from rat brain (trypsin‐fragment) under reducing (lane 7) and nonreducing (lane 8) conditions. After SDS/PAGE separation the samples were analyzed by immunoblotting using an affinity‐purified polyclonal rabbit antibody against rat TRH‐DE.

Figure 5

Heat inactivation of wild‐type and C68S mutant. Membrane preparations of recombinant wild‐type (▪) or the C68S mutant (□) enzyme were preincubated for 40 min and then assayed at the temperatures indicated. [pyroGlu‐³H]TRH was used as substrate.

Figure 6

Subcellular distribution of the truncated GFP‐fusion proteins with either cysteine or serine at position 68, as analyzed by confocal laser fluorescence microscopy. BHK cells were transfected either with the empty pEGFP‐N1 vector (A), the EGFP‐tagged, truncated wild‐type cDNA of TRH‐DE (B) or the corresponding C68S mutant (C) and processed as described in Experimental procedures.

Figure 7

Expression of wild‐type and C68S mutant as truncated GFP‐fusion proteins in BHK‐cells. The large extracellular domain of TRH‐DE with the eight cysteine residues was replaced by EGFP. BHK‐cells were transfected either with the empty pEGFP‐N1 vector (lanes 1 and 4), the EGFP‐tagged, truncated wild‐type cDNA of TRH‐DE (lanes 2 and 5) or the corresponding C68S mutant (lanes 3 and 6). The membrane fractions were subjected to SDS/PAGE (12% gel) in the presence (+) or absence (–) of dithiothreitol and analyzed by immunoblotting using the living colors peptide antibody‐AP conjugate (Clontech). The positions of the molecular mass markers (in kDa) are indicated.