Heparin-binding properties of selenium-containing thioredoxin reductase from HeLa cells and human lung adenocarcinoma cells

Abstract

Mammalian selenocysteine-containing thioredoxin reductase (TR) isolated from HeLa cells and from human lung adenocarcinoma cells was separated into two major enzyme species by heparin-agarose affinity chromatography. The low-affinity enzyme forms that were not retained on heparin agarose showed strong crossreactivity in immunoblot assays with anti-rat liver TR polyclonal antibodies, whereas the high-affinity enzyme forms that were retained by the heparin column were not detected. Both low and high heparin-affinity enzyme forms contained FAD, were indistinguishable on SDS/PAGE analysis, and exhibited similar catalytic activities in the NADPH-dependent DTNB [5,5'-dithiobis(2-nitrobenzoate)] assay. The C-terminal amino acid sequences of 75Se-labeled tryptic peptides from lung adenocarcinoma low- and high heparin-affinity enzyme forms were identical to the predicted C-terminal sequence of human placental TR. These two determined peptide sequences were -Ser-Gly-Ala-Ser-Ile-Leu-Gln-Ala-Gly-Cys-Secys-(Gly). Occurrence of the Se-carboxymethyl derivative of radioactive selenocysteine in the position corresponding to TGA in the gene confirmed that UGA is translated as selenocysteine. The presence of cysteine followed by a reactive selenocysteine residue in this C-terminal region of the protein may explain some of the unusual properties of the mammalian TRs.

Figure 1

Separation of two forms of TR from transformed cell lines on a heparin-5PW Progel-TSK HPLC column. The enzyme preparations applied to the column had been purified by chromatography on DEAE Sepharose followed by 2′,5′-ADP agarose-affinity chromatography.

Figure 2

SDS/PAGE analysis of low heparin and high heparin-affinity enzyme forms on a 12% polyacrylamide gel. Protein bands stained with Coomassie brilliant blue (Upper) and ⁷⁵Se-labeled protein bands detected by PhosphorImager (Lower). Lane 1, prestained standard proteins (Novex) in 250, 98, 64, 50, 36, 30, 16, and 6 kDa; lane 2, 1 μg of high heparin-affinity HeLa cell enzyme (2,100 cpm); lane 3, 1 μg of low heparin-affinity human lung adenocarcinoma cell enzyme (1,200 cpm); lane 4, mixture of 1 μg of each (3,300 cpm).

Figure 3

Immunoblot analysis of low heparin-affinity enzyme forms. TR isolated from human lung adenocarcinoma cells and from HeLa cells was chromatographed on heparin agarose and 1 μg samples of enzyme fractions were subjected to electrophoresis and transferred to a poly(vinylidene difluoride) membrane followed by immunoblotting with a polyclonal anti-rat liver TR antibody (1:1,000 dilution). Goat anti-rabbit IgG (H+L), 5-bromo-4-chloro-3-indolyl phosphate, and nitroblue tetrazolium were used for visualization of reactive protein bands. Transferred standards: 98 kDa at 3.2 cm and 64 kDa at 3.9 cm (lane 1), low heparin-affinity enzyme from lung cells (lane 2) and from HeLa cells (lane 3). The high heparin-affinity enzyme forms were not detected.

Figure 4

Automated Edman degradation amino acid sequences of tryptic selenopeptides derived from carboxymethylated heparin high-affinity and heparin low-affinity forms of TR isolated from human lung adenocarcinoma cells. The amino acid sequence of each ⁷⁵Se-labeled tryptic peptide was determined using Hewlett–Packard Protein Sequencer G100A. Eluates from each cycle were collected and analyzed for ⁷⁵Se using a gamma counter. The amino acids identified in each cycle were identical for both peptides and are indicated in the line below the figure. The profile of radioactivity from the ⁷⁵Se-labeled tryptic peptide isolated from heparin high-affinity TR is shown in the bar graph of the figure. The peak of radioactivity collected from the heparin low-affinity ⁷⁵Se-labeled peptide, likewise, was centered in cycle 11 with the following profile: cycle 10, 245 cpm; cycle 11, 660 cpm; cycle 12, 317 cpm; cycle 13, 117 cpm. In this peptide, the amino acid residue at cycle 11 was identified as dehydroalanine. In both peptides, S-carboxymethylcysteine was identified in cycle 10. The yield of the expected C-terminal glycine residue from both peptides was too low to be detected. The gene sequence of the C-terminal region of human placental TR (11) is: