Biochemical properties of recombinant human β-glucuronidase synthesized in baby hamster kidney cells

Abstract

The cDNA sequence encoding human β-glucuronidase was expressed in baby hamster kidney (BHK) cells. After purification from the culture supernatant in one step by use of immunoaffinity chromatography, the biochemical properties of the enzyme were examined. With a pH optimum of 4.0, a K(m) of 1.3 mM and thermal stability up to 68°C, this protein has characteristics very similar to those described for β-glucuronidase from human placenta. However, the recombinant product has several structural properties not previously reported for β-glucuronidase isolated from natural sources. First, recombinant β-glucuronidase is synthesized as a tetramer consisting of two disulphide-linked dimers. As can be inferred from the cDNA sequence, the enzyme possesses five cysteine residues after cleavage of the signal peptide. By introducing a C-terminal truncation, we eliminated the last cysteine at position 644. In the mutant, covalent linkage between two monomers is no longer observed, indicating that Cys-644 is involved in intermolecular disulphide-bond formation. The functional role of the disulphide bond remains elusive, as it was shown that (i) intracellular transport of the mutant is not impaired and (ii) it is still able to form an enzymically active tetramer. A second feature that has not previously been observed for β-glucuronidase from any origin is the existence of two enzymically active species for recombinant β-glucuronidase, when examined by gel filtration on a TSK 3000 column. With apparent molecular masses of 380 kDa and 190 kDa we propose that they represent tetramers and dimers respectively. Partial N-terminal sequencing and electrophoresis under denaturing conditions revealed that the dimers consist of subunits that have been proteolytically processed at their C-terminus losing 3-4 kDa in peptide mass. Controlled proteolysis demonstrates that the enzyme's overall protein backbone as well as its activity are resistant to a number of proteases. Only the C-terminal portion is susceptible to protease action, and the disulphide-linked form is readily converted into non-disulphide-bonded subunits. Pulse-chase analysis shows that human β-glucuronidase remaining intracellular in BHK cells after synthesis undergoes a similar proteolytic processing event, i.e. a reduction in mass of 3-4 kDa. Like purified and proteolytically processed β-glucuronidase, the intracellular form behaves as a dimer on gel filtration, indicating that the processing event in the cell leads to a different oligomeric structure of the enzyme.