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Mammalian cells contain a second nucleocytoplasmic hexosaminidase.

ABSTRACT: Some thirty years ago, work on mammalian tissues suggested the presence of two cytosolic hexosaminidases in mammalian cells; one of these has been more recently characterized in a recombinant form and has an important role in cellular function due to its ability to cleave beta-N-acetylglucosamine residues from a variety of nuclear and cytoplasmic proteins. However, the molecular nature of the second cytosolic hexosaminidase, named hexosaminidase D, has remained obscure. In the present study, we molecularly characterize for the first time the human and murine recombinant forms of enzymes, encoded by HEXDC genes, which appear to correspond to hexosaminidase D in terms of substrate specificity, pH dependency and temperature stability. Furthermore, a Myc-tagged form of this novel hexosaminidase displays a nucleocytoplasmic localization. Transcripts of the corresponding gene are expressed in a number of murine tissues. On the basis of its sequence, this enzyme represents, along with the lysosomal hexosaminidase subunits encoded by the HEXA and HEXB genes, the third class 20 glycosidase to be identified from mammalian sources.

SUBMITTER: Gutternigg M

PROVIDER: S-EPMC2850170 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Mammalian cells contain a second nucleocytoplasmic hexosaminidase.

Gutternigg Martin M Rendić Dubravko D Voglauer Regina R Iskratsch Thomas T Wilson Iain B H IB

The Biochemical journal 20090401 1

Some thirty years ago, work on mammalian tissues suggested the presence of two cytosolic hexosaminidases in mammalian cells; one of these has been more recently characterized in a recombinant form and has an important role in cellular function due to its ability to cleave beta-N-acetylglucosamine residues from a variety of nuclear and cytoplasmic proteins. However, the molecular nature of the second cytosolic hexosaminidase, named hexosaminidase D, has remained obscure. In the present study, we ...[more]

PMID: 19040401

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Project description:When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903phikan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 1-->4 glycosidic bond of beta-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble beta-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.

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Mammalian cells contain a second nucleocytoplasmic hexosaminidase.

Publications

Mammalian cells contain a second nucleocytoplasmic hexosaminidase.

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