In Vitro Synthesis of Hyaluronan by a Single Protein Derived from Mouse HAS1 Gene and Characterization of Amino Acid Residues Essential for the Activity
HAS1 was expressed as a FLAG-tagged HAS1 fusion protein in COS-1 cells. This recombinant protein was extracted with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) from the membrane fraction and purified by anti-FLAG affinity chromatography and subsequent SDS-polyacrylamide gel...
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Veröffentlicht in: | The Journal of biological chemistry 2000-01, Vol.275 (1), p.497-506 |
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Zusammenfassung: | HAS1 was expressed as a FLAG-tagged HAS1 fusion protein in COS-1 cells. This recombinant protein was extracted with CHAPS
(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) from the membrane fraction and purified by anti-FLAG affinity
chromatography and subsequent SDS-polyacrylamide gel electrophoresis. A protein solubilized from the one single band on the
gel was able to synthesize hyaluronan when incubated with UDP-GlcNAc and UDP-GlcA as donor substrates without any further
additions. The detergent-solubilized and purified HAS1 protein, however, exhibited quite different kinetic properties from
the membrane-bound protein. When assayed under the reconstitutive conditions where the reaction mixture was layered onto the
buffer containing high concentration of CHAPS, the activity was enhanced and the kinetic properties became similar to those
of the membrane-bound protein. In addition, a HAS1 gene product by an in vitro transcription/translation system also showed HAS1 activity under the reconstitutive conditions. To our surprise, when incubated
with UDP-GlcNAc alone, the protein was found to synthesize chito-oligosaccharide. Taking advantage of these enzyme reaction
properties, active sites on the protein involved in for hyaluronan and chito-oligosaccharide synthesis were characterized.
Site-directed mutagenesis induced in the cytoplasmic central loop domain of the protein revealed that several amino acid residues
conserved among those domains of various proteins of a HAS family were essential for both hyaluronan and chito-oligosaccharide
syntheses but one of them was not for chito-oligosaccharide synthesis. The substitutions that caused partial or severe loss
of the activity gave no significant changes of the K
m values of the mutated proteins, suggesting that no conformational or other indirect changes were involved in the effect.
Taken together, the results suggest that the HAS1 protein alone is able to synthesize hyaluronan and different amino acid
residues on the cytoplasmic central loop domain are involved in transferring GlcNAc and GlcA residues, respectively. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.275.1.497 |