Subcellular trafficking and activity of hyal-1 and its processed forms in murine macrophages

Research output: Contribution to journalArticle

Abstract

The hyaluronidase Hyal-1 is an acid hydrolase that degrades hyaluronic acid (HA), a component of the extracellular matrix. It is often designated as a lysosomal protein. Yet few data are available on its intracellular localization and trafficking. We demonstrate here that in RAW264.7 murine macrophages, Hyal-1 is synthesized as a glycosylated precursor that is only weakly mannose 6-phosphorylated. Nevertheless, this precursor traffics to endosomes, via a mannose 6-phosphate-independent secretion/recapture mechanism that involves the mannose receptor. Once in endosomes, it is processed into a lower molecular mass form that is transported to lysosomes, where its activity could be detected using native gel zymography. Indeed, this activity co-distributed with lysosomal hydrolases in the densest fraction of a self-forming PercollTM density gradient. Moreover, it shifted toward the lower density region, in parallel with those hydrolases, when a decrease of lysosomal density was induced by the endocytosis of sucrose. Interestingly, the activity of the processed form of Hyal-1 was largely underestimated when assayed by zymography after SDS-PAGE and subsequent renaturation of the proteins, by contrast to the full-length protein that could efficiently degrade HA in those conditions. These results suggest that noncovalent associations support the lysosomal activity of Hyal-1.

Original languageEnglish
Pages (from-to)500-15
Number of pages16
JournalTraffic (Copenhagen, Denmark)
Volume15
Issue number5
Early online date6 Feb 2014
DOIs
Publication statusPublished - 2014

Fingerprint

Macrophages
Hydrolases
Endosomes
Hyaluronic Acid
Protein Renaturation
Hyaluronoglucosaminidase
Molecular mass
Mannose
Endocytosis
Lysosomes
Extracellular Matrix
Sucrose
Polyacrylamide Gel Electrophoresis
Proteins
Gels
Acids

Keywords

  • Animals
  • Endocytosis
  • Endosomes
  • Glycosylation
  • Hyaluronoglucosaminidase
  • Hydrolases
  • Lectins, C-Type
  • Lysosomes
  • Macrophages
  • Mannose-Binding Lectins
  • Mannosephosphates
  • Mice
  • Protein Transport
  • Receptors, Cell Surface
  • Sucrose

Cite this

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title = "Subcellular trafficking and activity of hyal-1 and its processed forms in murine macrophages",
abstract = "The hyaluronidase Hyal-1 is an acid hydrolase that degrades hyaluronic acid (HA), a component of the extracellular matrix. It is often designated as a lysosomal protein. Yet few data are available on its intracellular localization and trafficking. We demonstrate here that in RAW264.7 murine macrophages, Hyal-1 is synthesized as a glycosylated precursor that is only weakly mannose 6-phosphorylated. Nevertheless, this precursor traffics to endosomes, via a mannose 6-phosphate-independent secretion/recapture mechanism that involves the mannose receptor. Once in endosomes, it is processed into a lower molecular mass form that is transported to lysosomes, where its activity could be detected using native gel zymography. Indeed, this activity co-distributed with lysosomal hydrolases in the densest fraction of a self-forming PercollTM density gradient. Moreover, it shifted toward the lower density region, in parallel with those hydrolases, when a decrease of lysosomal density was induced by the endocytosis of sucrose. Interestingly, the activity of the processed form of Hyal-1 was largely underestimated when assayed by zymography after SDS-PAGE and subsequent renaturation of the proteins, by contrast to the full-length protein that could efficiently degrade HA in those conditions. These results suggest that noncovalent associations support the lysosomal activity of Hyal-1.",
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author = "Emeline Puissant and Florentine Gilis and Sophie Dogn{\'e} and Bruno Flamion and Michel Jadot and Marielle Boonen",
note = "{\circledC} 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.",
year = "2014",
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TY - JOUR

T1 - Subcellular trafficking and activity of hyal-1 and its processed forms in murine macrophages

AU - Puissant, Emeline

AU - Gilis, Florentine

AU - Dogné, Sophie

AU - Flamion, Bruno

AU - Jadot, Michel

AU - Boonen, Marielle

N1 - © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

PY - 2014

Y1 - 2014

N2 - The hyaluronidase Hyal-1 is an acid hydrolase that degrades hyaluronic acid (HA), a component of the extracellular matrix. It is often designated as a lysosomal protein. Yet few data are available on its intracellular localization and trafficking. We demonstrate here that in RAW264.7 murine macrophages, Hyal-1 is synthesized as a glycosylated precursor that is only weakly mannose 6-phosphorylated. Nevertheless, this precursor traffics to endosomes, via a mannose 6-phosphate-independent secretion/recapture mechanism that involves the mannose receptor. Once in endosomes, it is processed into a lower molecular mass form that is transported to lysosomes, where its activity could be detected using native gel zymography. Indeed, this activity co-distributed with lysosomal hydrolases in the densest fraction of a self-forming PercollTM density gradient. Moreover, it shifted toward the lower density region, in parallel with those hydrolases, when a decrease of lysosomal density was induced by the endocytosis of sucrose. Interestingly, the activity of the processed form of Hyal-1 was largely underestimated when assayed by zymography after SDS-PAGE and subsequent renaturation of the proteins, by contrast to the full-length protein that could efficiently degrade HA in those conditions. These results suggest that noncovalent associations support the lysosomal activity of Hyal-1.

AB - The hyaluronidase Hyal-1 is an acid hydrolase that degrades hyaluronic acid (HA), a component of the extracellular matrix. It is often designated as a lysosomal protein. Yet few data are available on its intracellular localization and trafficking. We demonstrate here that in RAW264.7 murine macrophages, Hyal-1 is synthesized as a glycosylated precursor that is only weakly mannose 6-phosphorylated. Nevertheless, this precursor traffics to endosomes, via a mannose 6-phosphate-independent secretion/recapture mechanism that involves the mannose receptor. Once in endosomes, it is processed into a lower molecular mass form that is transported to lysosomes, where its activity could be detected using native gel zymography. Indeed, this activity co-distributed with lysosomal hydrolases in the densest fraction of a self-forming PercollTM density gradient. Moreover, it shifted toward the lower density region, in parallel with those hydrolases, when a decrease of lysosomal density was induced by the endocytosis of sucrose. Interestingly, the activity of the processed form of Hyal-1 was largely underestimated when assayed by zymography after SDS-PAGE and subsequent renaturation of the proteins, by contrast to the full-length protein that could efficiently degrade HA in those conditions. These results suggest that noncovalent associations support the lysosomal activity of Hyal-1.

KW - Animals

KW - Endocytosis

KW - Endosomes

KW - Glycosylation

KW - Hyaluronoglucosaminidase

KW - Hydrolases

KW - Lectins, C-Type

KW - Lysosomes

KW - Macrophages

KW - Mannose-Binding Lectins

KW - Mannosephosphates

KW - Mice

KW - Protein Transport

KW - Receptors, Cell Surface

KW - Sucrose

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U2 - 10.1111/tra.12162

DO - 10.1111/tra.12162

M3 - Article

C2 - 24502338

VL - 15

SP - 500

EP - 515

JO - Traffic (Copenhagen, Denmark)

JF - Traffic (Copenhagen, Denmark)

SN - 1398-9219

IS - 5

ER -