BIOCHEMICAL-PROPERTIES OF RECOMBINANT HUMAN BETA-GLUCURONIDASE SYNTHESIZED IN BABY HAMSTER-KIDNEY CELLS

The cDNA sequence encoding human beta-glucuronidase [Oshima, Kyle, Mil ler, Hoffmann, Powell, Grubb, Sly, Troplak, Guise and Gravel (1987) Pr oc. Natl. Acad. Sci. U.S.A. 84, 685-689] was expressed in baby hamster kidney (BHK) cells. After purification from the culture supernatant i n one step by use of immunoaffinity chromatography, the biochemical pr operties 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 degrees C, this protein has c haracteristics very similar to those described for beta-glucuronidase from human placenta [Brot, Bell and Sly (1978) Biochemistry 17, 385-39 1. However, the recombinant product has several structural properties not previously reported for beta-glucuronidase isolated from natural s ources. First, recombinant beta-glucuronidase is synthesized as a tetr amer consisting of two disulphide-linked dimers. As can be inferred fr om the cDNA sequence, the enzyme possesses five cysteine residues afte r cleavage of the signal peptide. By introducing a C-terminal truncati on, we eliminated the last cysteine at position 644. In the mutant, co valent 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 a nd (ii) it is still able to form an enzymically active tetramer. A sec ond feature that has not previously been observed for beta-glucuronida se from any origin is the existence of two enzymically active species for recombinant beta-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 con ditions revealed that the dimers consist of subunits that have been pr oteolytically processed at their C-terminus losing 3-4 kDa in peptide mass. Controlled proteolysis demonstrates that the enzyme's overall pr otein backbone as well as its activity are resistant to a number of pr oteases. Only the C-terminal portion is susceptible to protease action , and the disulphide-linked form is readily converted into non-disulph ide-bonded subunits. Pulse-chase analysis shows that human beta-glucur onidase 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 beta-glucuronidase , the intracellular form behaves as a dimer on gel filtration, indicat ing that the processing event in the cell leads to a different oligome ric structure of the enzyme.