title: CD44-dependent intracellular and extracellular catabolism of hyaluronic acid by hyaluronidase-1 and -2.
abstract: Hyaluronic acid (HA) is a high molecular weight glycosaminoglycan involved in a wide variety of cellular functions. However, its turnover in living cells remains largely unknown. In this study, CD44, a receptor for HA, and hyaluronidase-1, -2, and -3 (Hyal-1, -2 and -3) were stably expressed in HEK 293 cells and the mechanism of HA catabolism was systematically investigated using fluorescein-labeled HA. CD44 was essential for HA degradation by both endogenous and exogenously expressed hyaluronidases. Hyal-1 was not able to cleave HA in living cells in the absence of CD44. Intracellular HA degradation was predominantly mediated by Hyal-1 after incorporation of HA by CD44. Although Hyal-1 was active only in intracellular space in vivo, a certain amount of the enzyme was secreted to extracellular space. This extracellular Hyal-1 was found to be incorporated by cells and such uptake of Hyal-1 was, in part, involved in the intracellular degradation of HA. Hyal-2 was involved in the extracellular degradation of HA. Hyal-2 activity was also dependent on the expression of CD44 in both living cells and enzyme assays. Immunofluorescent microscopy demonstrated that both Hyal-2 and CD44 are present on the cell surface. Without CD44 expression, Hyal-2 existed in a granular pattern, and did not show hyaluronidase activity, suggesting that localization change could contribute to Hyal-2 function. A convenient and quantitative enzyme assay was established for the measurement of Hyal-2 activity. Hyal-2 activity was detected in the membrane fraction of cells co-expressing Hyal-2 and CD44. The pH optimum for Hyal-2 was 6.0-7.0. The membrane fraction of cells expressing Hyal-2 alone did not show hyaluronidase activity. Hyal-3 did not show any hyaluronidase activity in our experimental conditions. Based on these findings, Hyal-1 and -2 contribute to intracellular and extracellular catabolism of HA, respectively, in a CD44-dependent manner, and their HA degradation occurs independently from one another.
authors.affiliation: Lead Discovery Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan. hosami_harada@merck.com
authors.forename: Hosami
authors.initials: H
authors.lastname: Harada
authors.forename: Masaaki
authors.initials: M
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D006821:Hyaluronoglucosaminidase; D006863:Hydrogen-Ion Concentration; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid; D051548:Histone Acetyltransferases; C115866:OGA protein, human; D006821:Hyaluronoglucosaminidase; D001619:beta-N-Acetylhexosaminidases