Cloning and biochemical characterisation of two novel PDE4A cAMP phosphodiesterases

A large multi-gene family encodes many different phosphodiesterase (PDE) enzymes with alternative mRNA splicing generating additional complexity. This thesis details the discovery and cloning of two new PDE4A cyclic AMP specific phosphodiesterases. These two enzymes have been named hRDl (HSPDE4A1) and PDE4A10. hRDl is a PDE4A short-form and PDE4A10 is a PDE4A long-form. Previous to this study only a single active PDE4A short-form from rat RDl (RNPDE4A1A) had been cloned and two active PDE4A long-forms, namely PDE4A5 (mammalian and rat) and PDE4A8 (rat). Reverse transcriptase PCR analysis carried out in a previous study had indicated that RDl homologues were conserved across species and sequencing of the HSPDE4A gene revealed the existence of the human homologue. The sequencing study located the 5' exon encoding the unique N-terminal region of the human homologue of the rodent isoform RDl and also the splice junction used to produce this sliort-form. The work carried out in this thesis details how this information was used to clone the human short-forms hRDl as both the sequence encoding the unique N-terminal and the position of the splice junction had both been revealed. The open reading frame encoding hRDl (HSPDE4A1A, GenBank U97583) was engineered for expression in the plasmid pcDNA3 (Invitrogen) and this was transiently expressed in COS-1 cells. The mature hRDl protein was detected as an 83kDa species, the majority of which was associated with the high-speed membrane fraction. The kinetic properties of this enzyme were also investigated and hRDl displayed a Km for cAMP of around 3 muM, an IC50 value for inhibition by the PDE4-selective inhibitor rolipram of around 0.3 muM and the thermostability of the enzyme was found to be considerably more thermostable than rat RDl. The membrane binding capability of the enzyme was also investigated. This was done by generating human RDl as a mature 80kDa species in an in vitro transcription-translation system which displayed the ability to bind to COS-1 cell membranes. The data from these experiments showed that the rat and human homologues displayed very similar properties, particularly their intracellular location. Km for cAMP and sensitivity to rolipram inhibition. They displayed marked differences in their thermostability which may be a result of conformational differences between the proteins in regions outside of their active sites. The human PDE4A long-form PDE4A10 was cloned initially by Graham Rena in this laboratory. The sequence for the unique N-terminus of this isoform was based on a rat cDNA containing sequence which indicated a novel N-terminus. Existence of the human homologue was confirmed by reverse transcriptase analysis which detected PDE4A10 in human cell lines. Further sequencing of human cosmid clones containing HSPDE4A sequence confirmed the sequence and also revealed the position of the PDE4A10 splice junction. This thesis details how PDE4A10 was engineered for expression studies. The construct which was originally engineered apparently contained three potential in-frame initiating methionines (ATG). This study defined the native ATG of PDE4A10 and a construct was engineered in the plasmid pSV-SPORT for expression in COS-1 cells. The mature PDE4A10 protein was detected as a species of around 125kDa on traditional 8% cross- linked SDS-polyacrylamide gels. This was very similar to the other PDE long-form PDE4A4B (PDE46) and the difference in molecular weight between these two proteins could only be resolved on a pre-cast Tris-Acetate 3-8% polyacrylamide gel system (Novex). Using this system the calculated molecular weight of PDE4A10 was now around 120kDa compared to 125kDa for PDE4A4B calculated using the same system. The majority of PDE4A10 expressed in COS-1 cells was detected in the cytosolic fraction and the majority of PDE4A10 activity was also found in the cytosolic fraction. Kinetic studies revealed that the enzyme displayed a Km for cAMP of around 4 muM and an IC50 value for inhibition by the PDE4-selective inhibitor rolipram of around 0.3-1 muM in the membrane fractions and around 0.02 muM in the cytosolic fraction