Is Hyal-1 an authentic lysosomal enzyme ?

  • Christine Gasingirwa

Student thesis: Doc typesDocteur en Sciences

Résumé

Introduction: Hyaluronan is a glucosaminoglycan (GAG) found in the extracellular matrix (ECM) of many tissues where it influences many biological processes such as cell migration and proliferation. Its levels under normal cellular conditions are held in check by the balance between its synthesis by HA synthases and its catabolism by a family of depolymerising enzymes, Hyaluronidases, six of which are already known in mammals (Csoka et al., 2001). The most predominant and active of these are Hyal-1 and Hyal-2. They degrade the polymer to progressively smaller and different sizes of fragments which exert a wide and occasionally opposing spectrum of biological activities (Stern, 2008). Hyaluronidase-1 is the most ubiquitously distributed hyaluronidase, by virtue of its mRNA expression, in all mammalian tissues with exception of adult brain. The highest specific activity of the protein is found in serum, from which it derives the name, “plasma Hyal-1” (Frost et al., 1997), but it is tightly regulated by association with its inhibitors (Stern, 2005). At the same time, Hyal-1 is the most abundant hyaluronidase in somatic tissues particularly those with the highest turnover rate for HA, its substrate, namely the liver, kidney and the spleen (Csoka et al., 2001; Laurent and Fraser, 1992). Apart from its acidic optimal pH, no other evidence has ever been experimentally established in support of the lysosomal localization that is attributed to the protein. We investigated the subcellular localization of Hyal-1 by classical centrifugation methods, based on comparing the behavior of an intracellular compound under different centrifugation systems with that of known reference marker enzymes under the same conditions. Methods: Experiments were carried out on perfused rodent liver and kidney as well as on human hepatoma cells, HepG2. Detection of the protein was by immunodetection (Western blotting) and zymography activity. These assays were carried out on perfused tissue fractions obtained after differential centrifugation in isoosmotic sucrose solution, according to the scheme of de Duve et al. (1955). Likewise detection was performed on sucrose and Nycodenz density gradients prepared from M, L and P fractions after isopycnic centrifugation. Reference enzymes for different organelles were assayed parallel to these detection methods so as to establish the relative distribution profile of Hyal-1, on the principle that the intensity of signal in the Hyal-1 blot or zymogram would be comparable to the relative specific activity of the marker enzymes in the same fractions. Results: the distribution profile of Hyal-1in blot differed from that of â-galactosidase (lysosomes) in both HepG2 cells and rodent liver. However, it was realized, on testing the antibody used -1D10, with preparation from Hyal-1 -/- mouse liver, that the antibody was not specific. It recognized, in addition to Hyal-1, a contaminant molecule whose behavior could be reminiscent to peroxisomal nature. A non-lysosomal localization of the enzyme was suggested but a specific, non-ambiguous detection technique was sought. Zymography was the choice, supported by total negative results on preparations from both liver and kidney of Hyal-1 -/- mice, thus validating the specificity of the technique for Hyal-1 detection. Differential centrifugation indicated that Hyal-1 was recovered in L and P fractions while it was almost absent in M, fraction in which the bulk of lysosomes are recovered. Isopycnic centrifugation of L and P fractions in sucrose gradients when the animals had received a prior injection of Triton WR 1339, non ionic detergent known to modify lysosome density, revealed that the induced density shift of lysosomes did not affect the distribution of Hyal-1. Furthermore, Hyal-1 remained associated with sedimentable structures when the same fractions were subjected to hypotonic shock contrary to lysosomal enzymes release in the supernatant. Similar results were obtained from both mouse and rat liver and so were results from differential centrifugation of rat kidney. Having been suggested that Hyal-1 in kidney and liver could be originating from serum Hyal-1, the endocytosis of Hyal-1 was followed by injecting the protein in WT and Hyal-1 -/- mice and comparing its distribution with that of endogenous Hyal-1 enzyme. The injection of rh-Hyal-1(recombinant human Hyal-1) labelled with Iodine 125 in WT mice, unlabelled rh-Hyal-1 injected in Hyal-1 -/- mice and serum of a WT mouse in Hyal-1 -/- mice were carried out. The intracellular journey of endocytosed Hyal-1 was followed by analyzing the distribution of the protein in fractions isolated from homogenates prepared from perfused livers of animals injected at increasing times after injection and fractionated by different methods. The distribution of endocytosed Hyal-1 was monitored by determining the radioactivity or by zymography which were compared with that of marker enzymes, â-galactosidase (lysosomes) and alkaline phosphodiesterase (plasma membrane). Results showed that, Hyal-1 is endocytosed and rapidly transported inside liver cells with a maximum uptake being attained a few minutes after injection then followed by a relatively rapid inactivation. Distribution patterns after both differential and isopycnic centrifugation indicate that probably the protein is endocytosed by sinusoidal cells and that on reaching lysosomes, the protein is rapidly inactivated in the presence of lysosomal hydrolases. The same observation was made with in vitro incubation of endogenous and rh-Hyal-1 with purified lysosomes. Conclusion: Our results agree with the proposal that tissue Hyal-1 originates from endocytosed serum enzyme. The intracellular distribution of the enzyme, on the other hand, would be determined by the steady state situation in which the constant concentration of the Hyal-1 in serum and the relatively rapid inactivation of the protein on reaching terminal lysosomes, site of the bulk of lysosomal hydrolases. Conceivably, with the concentration of the endocytosed Hyal-1 remaining high enough in the endosomal compartments, partial digestion of the endocytosed HA would probably be realized in the prelysosomal compartments, prior to inactivation of Hyal-1 on being transferred to terminal lysosomes.
la date de réponse5 déc. 2009
langue originaleAnglais
L'institution diplômante
  • Universite de Namur
SuperviseurMichel Jadot (Promoteur), Bruno Flamion (Copromoteur), Nathalie Caron (Jury), Yves Poumay (Jury), Jean Bosco Gahutu (Jury), Mustapha Cherkaoui Malki (Jury) & P Robberrecht (Jury)

mots-clés

  • Progesterone
  • Cultured cells
  • Density shift
  • Lysosome

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