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 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.

Hyaluronidase-1 Is Mainly Functional in the Upper Granular Layer, Close to the Epidermal Barrier

“…In macrophages and liver extracts, the processed forms of HYAL1 are localized in lysosomes (Boonen et al, 2014;Puissant et al, 2014). In accordance, our data confirm co-localization between HYAL1 and lysosomal proteins cathepsins B and D inside granular keratinocytes (Supplementary Figure S2).…”

Hyaluronidase Hyal1 Increases Tumor Cell Proliferation and Motility through Accelerated Vesicle Trafficking

“…Sucrose gradient fractionation confirmed that chloroquine treatment induced a shift in the content of Hyal1WT-tdT (fractions 12-17 in Fig. 4G) to higher density fractions (fractions [13][14][15][16][17][18][19] as well as a small portion localized to the least dense fractions 4 and 6. Moreover, the lower molecular mass band decreased in intensity, consistent with reduced cleavage of Hyal1 as occurs in acidified vesicles.…”

“…3G and 4, G and H). The Hyal1 band consistent with the intact secreted mass of Hyal1-tdT is ϳ100 kDa and can be observed to co-fractionate in all three cell lines with EEA1 (fractions 4 -6), cathepsin D (two bands; fractions 13-17), and calnexin (fractions [13][14][15][16][17][18][19], which correspond to fractions of early endosomes, lysosomes, and microsomes (ER), respectively. However, close comparison of the fractions reveals a greater portion of uncleaved enzyme species in the Hyal1E131Q-tdT cell line that specifically extends throughout the denser fractions (fractions 14 -19) of the sucrose gradient in contrast to the wild-type and Y202F mutant, which are more similar to the profile of cathepsin D fractions (fractions [13][14][15][16][17].…”

“…In these cells, Hyal1 is secreted via the ER-Golgi pathway where only a small portion of it is mannose 6-phosphorylated for lysosomal targeting (18). Rather, the majority of the protein is released to the extracellular space and is thought to traffic to lysosomes via endocytosis following capture by the cell surface mannose receptor.…”

Hyaluronidase 1 Deficiency Preserves Endothelial Function and Glycocalyx Integrity in Early Streptozotocin-Induced Diabetes

“…HYAL1 is the only hyaluronidase present in human and mouse plasma (3). In all cell types, its enzymatic activity occurs at pH levels ,4.0, which requires the enzyme to undergo endocytosis (4).…”