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Subcellular Trafficking and Activity of Hyal‐1 and Its Processed Forms in Murine Macrophages
Author(s) -
Puissant Emeline,
Gilis Florentine,
Dogné Sophie,
Flamion Bruno,
Jadot Michel,
Boonen Marielle
Publication year - 2014
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/tra.12162
Subject(s) - endosome , biology , mannose , endocytosis , mannose 6 phosphate , lysosome , cell fractionation , biochemistry , mannose 6 phosphate receptor , microbiology and biotechnology , intracellular , extracellular , mannose receptor , enzyme , receptor , macrophage , growth factor , in vitro
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 Percoll TM 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.