PRIMARY STRUCTURE AND EXPRESSION OF A HUMAN CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE

Human CTP:phosphocholine cytidylyltransferase (CT) cDNAs were isolated by PCR amplification of a human erythroleukemic K562 cell library. In itially two degenerate oligonucleotide primers derived from the sequen ce of the rat liver CT cDNA were used to amplify a centrally located 2 30 bp fragment. Subsequently overlapping 5' and 3' fragments were ampl ified, each using one human CT primer and one vector-specific primer. Two cDNAs encoding the entire translated domain were also amplified. T he human CT (HCT) has close homology at the nucleotide and amino acid level with other mammalian CTs (from rat liver, mouse testis or mouse B6SutA hemopoietic cells and Chinese hamster ovary). The region which deviates most from the rat liver CT sequence is near the C-terminus, w here 7 changes are clustered within 34 residues (345-359), of the puta tive phosphorylation domain. The region of the proposed catalytic doma in (residues 75-235) is 100% identical with the rat liver sequence. Si gnificant homology was observed between the proposed catalytic domain of CT and the Sacchnromyces cereuisiae MUQ1 gene product, and between the proposed amphipathic alpha-helical membrane binding domains of CT and soybean oleosin, a phospholipid-binding protein. There are several shared characteristics of these amphipathic helices. An approx. 42000 Da protein was over-expressed in COS cells using a pAX142 expression vector containing one of the full-length HCT cDNA clones. The specific activity of the HCT in COS cell homogenates was the same as that of a nalogously expressed rat liver CT. The activity of HCT was lipid depen dent. The soluble form was activated 3 to 4-fold by anionic phospholip ids and by oleic acid or diacylglycerol-containing PC vesicles.