Mouse Hyal3 encodes a 45- to 56-kDa glycoprotein whose overexpression increases hyaluronidase 1 activity in cultured cells

Abstract

Hyaluronidases are enzymes that mediate the breakdown of hyaluronan (HA), a large polysaccharide abundant in the extracellular matrix of vertebrate tissues. Six genes have been predicted to encode hyaluronidases in humans, but the protein products of only SPAM1, HYAL1, and HYAL2 have been characterized. We have now expressed the mouse Hyal3 gene product, hyaluronidase 3 (Hyal3), in Baby Hamster Kidney (BHK) cells and demonstrated the presence of multiple forms of Hyal3 ranging from approximately 45 to 56 kDa in expression lysates. Complete and partial digestions of the expressed protein with PNGase F showed three N-linked oligosaccharides accounted for all forms of Hyal3 detected in expression lysates. Most of these oligosaccharides were Endo H sensitive, indicating that they were high mannose or hybrid N-linked oligosaccharides. Subcellular fractionation of Hyal3-expressing BHK cells by density gradient centrifugation revealed most Hyal3 in a low-density vesicular population. Low levels of Hyal3 were detected in higher density vesicles, but no colocalization with the late endosomal/lysosomal marker Lamp1 was found by immunofluorescence microscopy. BHK cells stably expressing Hyal3 had increased acid-active hyaluronidase activity, but no such activity was detected when Hyal3 was transfected into Hyaluronidase 1 (Hyal1)-deficient fibroblasts. Overexpression of Hyal3 in BHK cells increased the Hyal1 protein and mRNA levels, suggesting that the increased hyaluronidase activity in these cells was due to Hyal1 rather than Hyal3. The results indicate that Hyal3 overexpressed in cultured cells lacks intrinsic hyaluronidase activity and that Hyal3 may contribute to HA metabolism by augmenting the activity of Hyal1.

Mammalian Hyaluronidase Induces Ovarian Granulosa Cell Apoptosis and Is Involved in Follicular Atresia

“…4A). The two Hyal-1 protein bands observed (50 and 55 kDa) were presumably from a different glycosylation level of this enzyme (17). The in situ hybridization results supported those of the protein expression; the level of Hyal-1 mRNA (localized predominantly in small antral follicles) was higher in the younger mice (Fig.…”

“…6), which contained no enzymatic activity (Fig. 5A) (16,17), strongly suggested that the hyaluronan processing (or the hyaluronidase enzymatic activity) was not relevant to cell apoptosis. Nonetheless, the close association between the hyaluronidase expression and cell apoptosis remained.…”

“…Because Hyal-1 is a highly active hyaluronidase at acidic pH (11,45) and Hyal-3 has been reported to be devoid of enzymatic activity (16,17), proper protein folding was monitored using enzyme assays previously reported for Hyal-1 activity (17,37). First, using HA zymography, a clear band of digested HA can be visualized in the lane where cell lysates of Hyal-1/EGFP transfectants were loaded (Fig.…”

“…However, this notion has not yet been substantiated. Hyal-3 has also recently been reported to be devoid of hyaluronan enzymatic activity (16,17). In mice, Hyal-5 and Hyalp1 have similar properties as PH20/Spam1 in male reproduction (4,7).…”

NADPH Oxidases Regulate CD44 and Hyaluronic Acid Expression in Thrombin-treated Vascular Smooth Muscle Cells and in Atherosclerosis

“…Furthermore, our data indicating that that NADPH oxidase regulates Hyal3 expression in VSMC treated with thrombin is significant because increased HA during pathological conditions is the sum of its synthesis and catabolism. This is also an interesting observation because Hyal3 Ϫ/Ϫ mice do not exhibit any HA accumulation (57) and cellular overexpression of Hyal3 does not impart intrinsic hyaluronidase activity (58). In contrast, up-regulation of Hyal3 was reported in chondrocytes treated with cytokines (59).…”