Identification of a novel chondroitin hydrolase in Caenorhabditis elegans.
Hyaluronidases have been postulated to be the enzyme acting at the initial step of chondroitin sulfate (CS) catabolism in vivo. Since chondroitin (Chn) but not hyaluronic acid (HA) has been detected in Caenorhabditis elegans, the nematode is a good model for elucidating the mechanism of the degradation of CS/Chn in vivo. Here we cloned the homolog of human hyaluronidase in C. elegans, T22C8.2. The Chn-degrading activity in vitro was first demonstrated when it was expressed in COS-7 cells. The enzyme cleaved preferentially Chn. CS-A and CS-C were also depolymerized but to lesser extents, and HA was hardly degraded. In order of preference, the substrates ranked Chn >> CS-A > CS-C >> HA. The products of the degradation of Chn by the enzyme were characterized by anion-exchange high performance liquid chromatography and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The structure of the major component in the digest was determined as GlcUAbeta1-3GalNAcbeta1-4GlcUAbeta1-3GalNAc, where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively, indicating that this enzyme is a Chn hydrolase, an endo-beta-galactosaminidase specific for Chn. Investigation of the effects of pH on the activity revealed the optimum pH of Chn hydrolase to be 6.0. Since Chn in C. elegans has been demonstrated to play critical roles in cell division, Chn hydrolase possibly regulates the function of Chn in vivo. This is the first demonstration of a Chn hydrolase in an animal.
Kaneiwa
Tomoyuki
T
Laboratory of Proteoglycan Signaling and Therapeutics, Hokkaido University Graduate School of Life Science, Nishi-11-choume, Kita 21-jo, Kita-ku, Sapporo, Japan.
Yamada
Shuhei
S
Mizumoto
Shuji
S
Montaño
Adriana M
AM
Mitani
Shohei
S
Sugahara
Kazuyuki
K
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology*; D002809:Chondroitin Sulfates / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006596:Hexosaminidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000502:physiology
D029742:Caenorhabditis elegans Proteins; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D006596:Hexosaminidases; C028551:endo-beta-glucosaminidase