Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties

Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recen tly been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes i n COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluro nan coat. The pericellular coats formed by HAS1 transfectants were signific antly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic stud ies of these enzymes in the membrane fractions isolated from HAS transfecta nts demonstrated that HAS proteins are distinct from each other in enzyme s tability, elongation rate of HA, and apparent K-m values for the two substr ates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyalur onan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to similar to 2 x 10(6) Da. Furthermore, comparisons of hyalu ronan secreted into the culture media by stable HAS transfectants showed th at HAS1 and HAS2 generated hyaluronan with broad size distributions (molecu lar masses of 2 x 10(5) to similar to 2 x 106 Ha), whereas HAS2 generated h yaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enz ymatic characteristics may provide the cells with flexibility in the contro l of hyaluronan biosynthesis and functions.