Molecular Cloning and Characterization of a cDNA Encoding the Third Putative Mammalian Hyaluronan Synthase

Abstract

We report the isolation of a cDNA encoding the third putative hyaluronan synthase, HAS3. Partial cDNAs and genomic fragments of mouse Has3 were obtained using a degenerate polymerase chain reaction approach. Partial clones facilitated the isolation of genomic and cDNA clones representing the mouse Has3 open reading frame. The open reading frame of 554 amino acids predicted a protein of 63.3 kDa with multiple transmembrane domains and several consensus HA binding motifs. Sequence comparisons indicated that mouse Has3 is most closely related to Has2 (71% amino acid identity) and also related to Has1 (57% identity), Xenopus laevis DG42 (56% identity), and Streptococcus pyogenes HasA (28% identity). Isolation of a genomic fragment of human HAS3 indicated high conservation between mouse and human sequences, similar to those observed for HAS1 and HAS2. Expression of the mouse Has3 open reading frame in transfected COS-1 cells led to high levels of hyaluronan synthesis, as determined through a classical particle exclusion assay, and by in vitro HA synthase assays. These results suggest that there are three putative mammalian hyaluronan synthases encoded by three separate but related genes which comprise a mammalian hyaluronan synthase (HAS) gene family. Hyaluronan (HA)1 is a linear unbranched glycosaminoglycan (GAG) composed of repeating disaccharide units of D-glucuronic acid(␤133)N-acetylglucosamine(␤134). HA is a major constituent of the extracellular matrix of most tissues and organs, especially during embryonic development, where it has been proposed to play important roles in cell migration, proliferation, and the development of tissue architecture (1-3). In addition, HA has been implicated in tumorigenesis, and defects in HA metabolism are a hallmark of several important diseases including rheumatoid arthritis, Grave's ophthalmopathy, cirrhosis of the liver, and accelerated aging in Werner's syndrome (1-4). Unlike other GAGs, which are synthesized within the Golgi network and attached to protein, HA is synthesized at the inner face of the plasma membrane and is subsequently extruded to the outside of the cell (5, 6). Recently, we and others (7-11) have identified two mammalian genes, HAS1 and HAS2, encoding putative plasma membrane HA synthases related to the Streptococcus pyogenes HA synthase, HasA. Expression of either HAS1 or HAS2 by cells led to high levels of HA biosynthesis, consistent with both proteins playing critical roles in HA biosynthesis, possibly as the HA synthases themselves.While attempting to isolate fragments of the human HAS2 gene using a degenerate PCR approach, we isolated fragments of an additional related gene in the mouse and human. We now report the molecular cloning and characterization of a cDNA encoding the third putative mammalian hyaluronan synthase, HAS3. EXPERIMENTAL PROCEDUREScDNA and Genomic Cloning and Expression-Previously described degenerate oligonucleotide primer pools (10), DEG 1 and DEG 5, were utilized in an attempt to amplify fragments of HAS genes from human and mous...

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