Molecular cloning of a novel human PAPS synthetase which is differentiallyexpressed in metastatic and nonmetastatic colon carcinoma cells

Subtractive hybridisation was used to select for genes which are differenti ally expressed between a highly metastatic human colon carcinoma cell line, KM12SM, and the isogenetic non-metastatic cell line, KM12C. This led to th e isolation of cDNA clones for a novel human adenosine 5'-phosyhosulphate k inase/ATP sulphurylase (PAPS synthetase). Northern hybridisation revealed a single 4.2 kb mRNA species which showed an approximately 20-fold higher le vel of expression in the non-metastatic cell line than in the metastatic ce ll line. The overlapping cDNA clones together covered 3,774 bp including th e entire coding region of 1,842 bp encoding a protein of 614 amino acids (c alculated molecular mass of 69,496 Da). The protein contains consensus sequ ences for APS kinase and ATP sulphurylase, in its amino- and carboxy-termin al regions, respectively, as well as other sequences that are highly conser ved amongst ATP sulphurylases and APS kinases. Interestingly, consensus seq uences for GTPase activity were also identified, indicating that enzyme act ivity may be regulated by an intrinsic GTPase mechanism. Overall the new pr otein is 78% homologous with a previously described human PAPS synthetase ( PAPSS1) indicating that we have identified the second member of a gene fami ly which we have provisionally named PAPSS2. The gene locus for PAPSS2 was identified on chromosome 10 at 10q23.1-q23.2. This locus has synteny with t he mouse brachymorphic gene recently identified as a PAPS synthetase (SK2). PAPSS2 appears to be the human homologue of this gene and thus PAPSS2 is l ikely to be important in human skeletogenesis. (C) 1999 Elsevier Science Lt d. All rights reserved.