PMID- 10617644
OWN - NLM
STAT- MEDLINE
DCOM- 20000131
LR  - 20190508
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 275
IP  - 1
DP  - 2000 Jan 7
TI  - In vitro synthesis of hyaluronan by a single protein derived from mouse HAS1 gene
      and characterization of amino acid residues essential for the activity.
PG  - 497-506
AB  - 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.
FAU - Yoshida, M
AU  - Yoshida M
AD  - Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, 
      Aichi 480-1195, Japan.
FAU - Itano, N
AU  - Itano N
FAU - Yamada, Y
AU  - Yamada Y
FAU - Kimata, K
AU  - Kimata K
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Membrane Proteins)
RN  - 0 (Oligosaccharides)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Xenopus Proteins)
RN  - 1398-61-4 (Chitin)
RN  - 17479-04-8 (UDP-glucosamine)
RN  - 528-04-1 (Uridine Diphosphate N-Acetylglucosamine)
RN  - 9004-61-9 (Hyaluronic Acid)
RN  - EC 2.- (Transferases)
RN  - EC 2.4.- (Glycosyltransferases)
RN  - EC 2.4.1.17 (Glucuronosyltransferase)
RN  - EC 2.4.1.212 (HAS1 protein, Xenopus)
RN  - EC 2.4.1.212 (Hyaluronan Synthases)
RN  - N08U5BOQ1K (Glucosamine)
SB  - IM
MH  - Amino Acid Motifs
MH  - Amino Acid Sequence
MH  - Animals
MH  - Cell-Free System
MH  - Chitin/metabolism
MH  - Glucosamine/analogs & derivatives/metabolism
MH  - Glucuronosyltransferase/genetics/isolation & purification/*metabolism
MH  - *Glycosyltransferases
MH  - Hyaluronan Synthases
MH  - Hyaluronic Acid/*biosynthesis
MH  - *Membrane Proteins
MH  - Membranes/metabolism
MH  - Mice
MH  - Molecular Sequence Data
MH  - Oligosaccharides/metabolism
MH  - Recombinant Proteins/isolation & purification/metabolism
MH  - *Transferases
MH  - Uridine Diphosphate N-Acetylglucosamine/metabolism
MH  - *Xenopus Proteins
EDAT- 2000/01/05 00:00
MHDA- 2000/01/05 00:01
CRDT- 2000/01/05 00:00
PHST- 2000/01/05 00:00 [pubmed]
PHST- 2000/01/05 00:01 [medline]
PHST- 2000/01/05 00:00 [entrez]
AID - 10.1074/jbc.275.1.497 [doi]
PST - ppublish
SO  - J Biol Chem. 2000 Jan 7;275(1):497-506. doi: 10.1074/jbc.275.1.497.