Synthesis of hyaluronate in differentiated teratocarcinoma cells. Characterization of the synthase

Prehm, Peter

doi:10.1042/bj2110181

Cited by 123 publications

(79 citation statements)

References 18 publications

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“…In addition, the pattern observed in digests of total mouse genomic DNA was identical to that observed in equivalent digests of recently isolated mouse Has2 genomic clones. 2 Low stringency wash conditions failed to identify any further hybridizing fragments including those fragments corresponding to the related mouse HAS (17) gene (data not shown). This suggests that the level of sequence identity (55%) between mouse Has2 and mouse HAS, and possibly other Has-related genes, is not sufficient to permit detection through Southern hybridization.…”

Section: Resultsmentioning

confidence: 99%

“…We have recently isolated clones for a second human Has gene, which shares greater than 95% amino acid identity to mouse Has2 and thus is predicted to represent the human HAS2 gene. 2 This suggests that there are at least two related Has genes in both mouse and humans.…”

Section: Resultsmentioning

confidence: 99%

“…Cells were analyzed 36 h after lipofection (transient transfection). The COS-1 cell line and the mouse 3T6 (Swiss embryonic fibroblast) cell line (22) were routinely maintained at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine, in a humidified chamber at 5% CO 2 .…”

Section: Northern and Southern Analysis-mouse Multiple Tissue Northermentioning

confidence: 99%

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Molecular Cloning and Characterization of a Putative Mouse Hyaluronan Synthase

Spicer

Augustine

McDonald

1996

Journal of Biological Chemistry

156

104

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We report the isolation of a novel mouse gene which encodes a putative hyaluronan synthase. The cDNA was identified using degenerate reverse transcriptase-polymerase chain reaction. Degenerate primers were designed based upon an alignment of the amino acid sequences of Streptococcus pyogenes HasA, Xenopus laevis DG42, and Rhizobium meliloti NodC. A mouse embryo cDNA library was screened with the resultant polymerase chain reaction product, and multiple cDNA clones spanning 3 kilobase pairs (kb) were isolated. The open reading frame predicted a 63-kDa protein with several transmembrane sequences, multiple consensus phosphorylation sites, and four putative hyaluronan binding motifs. The amino acid sequence displayed 55% identity to mouse HAS, 56% identity to Xenopus DG42, and 21% identity to Streptococcus HasA. Northern analysis identified transcripts of 4.8 kb and 3.2 kb, which were expressed highly in the developing mouse embryo and at lower levels in adult mouse heart, brain, spleen, lung, and skeletal muscle. Transfection experiments demonstrated that mouse Has2 could direct hyaluronan coat biosynthesis in transfected COS cells, as evidenced by a classical particle exclusion assay. These results suggest that mammalian HA synthase activity is regulated by at least two related genes. Accordingly, we propose the name Has2 for this gene.Hyaluronan (HA 1 ) is a linear unbranched polymer made up of repeating disaccharide units of D-glucuronic acid(␤133)Nacetylglucosamine(␤134). More than 60 years after the isolation of hyaluronan from the vitreous humor (1), its synthetic pathway remains incompletely characterized. HA is synthesized as a free, linear polymer at the inner face of the plasma membrane of eukaryotic cells and is subsequently extruded to the outside of the cell (2-7). HA biosynthesis in mammalian cells may be regulated in part through signaling cascades (8, 9).Certain bacteria can synthesize an HA polymer that is identical to the polymer synthesized by mammalian cells (10 -12). Indeed, investigation of HA biosynthesis in the Group A Streptococcus, Streptococcus pyogenes, has recently led to the identification and cloning of several genes that encode enzymes critical for HA biosynthesis in this bacterium (11). However, until very recently, no genes have been identified that encode enzymes with similar activities in mammalian cells.Degenerate reverse transcriptase-PCR has been a useful tool in the identification and cloning of many genes and gene families (13-15). This approach relies upon conserved sequences deduced from alignments of related gene or protein sequences. The hasA gene of S. pyogenes encodes hyaluronan synthase in this bacterium (11). Sequence analysis predicts that this protein is a membrane protein with a large intracellular loop encoding the active site of the enzyme (11). Similarly, in mammalian cells, the HA synthase has been localized to the plasma membrane, with the active site on the inner face of the membrane (4, 5). Data base searches have identified the Rhizobium sp. nodulation fa...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Northern and Southern Analysis-mouse Multiple Tissue Northermentioning

confidence: 99%

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Molecular Cloning and Characterization of a Putative Mouse Hyaluronan Synthase

Spicer

Augustine

McDonald

1996

Journal of Biological Chemistry

156

104

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“…It is, however, found in locations which are not connective, mainly in the nerve system. Hyaluronan is synthesized at the cell membrane [2], and readily forms a pericellular matrix in culture. With rare exceptions, its distribution is essentially extracellular.…”

Section: Distribution In Naturementioning

confidence: 99%

Hyaluronan: fundamental principles and applications in cancer

Delpech

Girard

Bertrand

et al. 1997

Journal of Internal Medicine

146

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“…The two glycosyltransferase activities in HAS add glucuronic acid and N-acetylglucosamine into their alternating positions in the chain, using UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) as substrates (18). Sufficient supply of both UDP-GlcUA and UDP-GlcNAc is important for hyaluronan synthesis.…”

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confidence: 99%