The cytidine deaminase catalyzes irreversibly deamination of cytidine or deoxycytidine to uridine or deoxyuridine respectively. The active form of HCD is formed by a homotetramer and each subunit has a molecular weight of 16.2 kDa, which in turn it comes from the translation of a mRNA with 438 nt of open reading frame. Genomic sequence of HCD is formed by 31 kb with 4 exons and several alternative splicing signals. In this report, we describe the cloning and characterization of a small form of HCD, denominated HSCD (human small cytidine deaminase), and it is likely to be a product of alternative splicing of HCD.
For cloning of HSCD, total RNA from HL-60 cells cultivated for 3 days with 1-a, 25-dihydroxycholecalciferol were isolated and primers designed based on the HCD sequence were used to RT-PCR. Two bands of DNA were identified by agarose gel electrophoresis: a band of 735 bp of HCD and another of 600 bp, which was denominated HSCD. Alignment of HSCD DNA sequence with HCD suggests that the HSCD should be the product of an alternative splicing, because all sequence of HSCD match with the HCD in two parts, except for a deletion of 170 bp. Based on the HCD genome organization, the exons 1 and 4 should be joined and all sequences of introns and exons 2 and 3 should be deleted by splicing. This alternative splicing shifted the translation of reading frame from the point of splicing and formed a protein with 29 % of similarity from the C terminal of HCD. The estimated molecular weight based on the sequence is 9.8 kDa, and this value was confirmed by Western blot and mass spectroscopy after expressing the gene fused with GST in pGEX vector. The deletion and shift of reading frame should lead to lose of CD activity according to the another study, and this was confirmed by enzyme assay in vitro and also with NIH3T3 cells expressing HSCD challenged against cytosine arabinoside, which is toxic to cells without CD activity. Transcription of HSCD was found in several cell types, but its biological role has to be determined.
Supported by FAPESP