title: Expression and cellular localization of human hyaluronidase-2 in articular chondrocytes and cultured cell lines.
abstract: OBJECTIVE
  There is debate whether hyaluronan (HA) can be enzymatically degraded within the extracellular matrix of cartilage and other tissues or whether its catabolism occurs strictly within the lysosomal compartment of chondrocytes and other cell types. Previous studies have suggested that one of the lysosomal hyaluronidases (hyaluronidase-2) can be expressed as a functionally-active glycosyl phosphatidylinositol-linked protein at the surface of mammalian cells. If this form of hyaluronidase expression occurs in chondrocytes, this could represent a possible mechanism for extracellular HA cleavage. Thus, which hyaluronidases are expressed and where was the objective of this study.
  
  
  METHODS
  mRNA for hyaluronidases was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and enzymatic activity by HA zymograms. Recombinant forms of hyaluronidase-2 were generated and expressed in model cell lines. A peptide-specific polyclonal antiserum was prepared to localize endogenous human hyaluronidase-2 in human articular chondrocytes.
  
  
  RESULTS
  Hyaluronidase-2 is the principal mRNA transcript expressed by primary human articular chondrocytes as well as various model cell lines. Recombinant hyaluronidase-2, containing N-terminal or C-terminal epitope tags, was strictly localized intracellularly and not released by treatment with a phosphatidylinositol-specific phospholipase. Endogenous hyaluronidase-2 expressed by human chondrocytes as well as HeLa cells could only be detected following detergent permeabilization of the plasma membranes.
  
  
  CONCLUSIONS
  These data suggest that on chondrocytes and other cell types examined, hyaluronidase-2 is not present or functional at the external plasma membrane. Thus, local turnover of HA is dependent on receptor-mediated endocytosis and delivery to low pH intracellular organelles for its complete degradation.
authors.affiliation: Department of Biochemistry, Rush Medical College, Rush University Medical Center, 1735 West Harrison Avenue, Chicago, IL 60612, USA.
authors.forename: G
authors.initials: G
authors.lastname: Chow
authors.forename: C B
authors.initials: CB
authors.lastname: Knudson
authors.forename: W
authors.initials: W
authors.lastname: Knudson
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods; D019556:COS Cells; D002358:Cartilage, Articular / Q000201:enzymology*; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D019902:Chondrocytes / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D058851:GPI-Linked Proteins; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000032:analysis* / Q000235:genetics; D007106:Immune Sera / Q000302:isolation & purification; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods
chemical_list: D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D007106:Immune Sera; D012333:RNA, Messenger; D011994:Recombinant Proteins; C473096:Hyal2 protein, human; D006821:Hyaluronoglucosaminidase