Enhanced Translation of Rat β-Glucuronidase cDNA Is Conferred by 155-bp Segment of Internal Coding Sequence

When cDNAs for human and rodent β-glucuronidases were expressed in COS-7 cells using several different promoters, rodent β-glucuronidases were produced three times more than human β-glucuronidase, although their transcriptional levels were similar. Similar observations were also recorded in LMTK−cells using SV40 early or chicken β-actin promoters. In hopes of enhancing yields of recombinant human β-glucuronidase for enzyme replacement therapy, we sought to determine the region within the linear sequences responsible for the higher levels of expression of the rodent cDNAs. To do so, we made various rat–human chimeric cDNAs utilizing conserved restriction enzyme sites. The levels of products expressed from these chimeric cDNAs in COS cells were assessed by activity assay and by metabolic labeling of the proteins followed by immunoprecipitation and SDS–PAGE. From the results of these expression studies, we identified a 155-bpClaI (643)–AflII (797) fragment in the rat open reading frame responsible for the increased rate of translation of the rat β-glucuronidase (RBG) cDNA. Replacement of the homologousClaI (683)–AflII (838) fragment in human β-glucuronidase (HBG) with this 155-bp fragment from RBG increased the translation level of the resulting chimeric HRaH. Conversely, substitution of the 155-bp human fragment for that of rat in RBG cDNA reduced the total synthesis of the resulting chimeric RHaR. Placement of the 155-bp segment between the initiation ATG and the promoter has only negative effects on the expression of either cDNA. A more stable secondary structure of the human cDNA in this region might explain a reduced rate of translation. However, secondary structure analysis of mRNAs from the 155-bp fragment of rat and human cDNAs predicted that, while both can form stem–loops, the rat fragment (ΔG0= −42.1 kcal/mol) is actually more stable than the human fragment (ΔG0= −32.1 kcal/mol). In fact, the free energy of stability of the first 50 bp within thisClaI–AflII fragment from rat (−10.3 kcal/mol) indicates that the secondary structure is considerably more stable than the corresponding 50 bp from human (−2.1 kcal/mol). This segment of the rat sequence also contains atar-like sequence in a stem–loop. Althoughtar-like sequences can enhance rates of translation, altering this sequence by mutagenesis had no effect on the rate of synthesis of rat β-glucuronidase. Thus, although the region conferring enhanced rate of synthesis from rat cDNA has been identified, the mechanism by which it does so is not yet clear.