EXPRESSION CLONING AND MOLECULAR CHARACTERIZATION OF HAS PROTEIN, A EUKARYOTIC HYALURONAN SYNTHASE

We developed a mammalian transient expression system to isolate cDNA c lones that determine hyaluronan expression. HAS(-), a mouse mammary ca rcinoma mutant cell line, which is defective in hyaluronan synthase ac tivity, was first established and used as a recipient for the expressi on cloning, One cloned cDNA that overcame the deficiency was isolated, The cDNA termed HAS contains an open reading frame of 1749 base pairs encoding a new protein of 583 amino acids, Homology analysis of the a mino acid sequence suggests that HAS protein is related to streptococc al hyaluronan synthase and also to Xenopus laevis DG42 protein that wa s found to be homologous to bacterial hyaluronan synthase. Expression of HAS cDNA in HAS(-) cells complemented not only their mutant phenoty pes such as deficient hyaluronan-matrix deposition but also hyaluronan synthase activity itself, Therefore, HAS cDNA is responsible for the activity of the hyaluronan synthase, a key enzyme of hyaluronan synthe sis in eukaryotic cells.