Department of Biochemistry, Hoshi University School of Pharmacy, 2-4-41, Ebara, Shinagawa-ku 142-8501, Tokyo, Japan.
Department of Microbiology, Hoshi University School of Pharmacy, 2-4-41, Ebara, Shinagawa-ku 142-8501, Tokyo, Japan.
Int J Mol Sci. 2024 Jun 6;25(11):6281. doi: 10.3390/ijms25116281.
Mast cells take up extracellular latent heparanase and store it in secretory granules. The present study examined whether the enzymatic activity of heparanase regulates its uptake efficiency. Recombinant mouse heparanase mimicking both the latent and mature forms (L-Hpse and M-Hpse, respectively) was internalized into mastocytoma MST cells, peritoneal cell-derived mast cells, and bone marrow-derived mast cells. The internalized amount of L-Hpse was significantly higher than that of M-Hpse. In MST cells, L-Hpse was continuously internalized for up to 8 h, while the uptake of M-Hpse was saturated after 2 h of incubation. L-Hpse and M-Hpse are similarly bound to the MST cell surface. The expression level of cell surface heparan sulfate was reduced in MST cells incubated with M-Hpse. The internalized amount of M-Hpse into mast cells was significantly increased in the presence of heparastatin (SF4), a small molecule heparanase inhibitor that does not affect the binding of heparanase to immobilized heparin. Enzymatically quiescent M-Hpse was prepared with a point mutation at Glu335. The internalized amount of mutated M-Hpse was significantly higher than that of wild-type M-Hpse but similar to that of wild-type and mutated L-Hpse. These results suggest that the enzymatic activity of heparanase negatively regulates the mast cell-mediated uptake of heparanase, possibly via the downregulation of cell surface heparan sulfate expression.
肥大细胞摄取细胞外潜伏的肝素酶并将其储存在分泌颗粒中。本研究探讨了肝素酶的酶活性是否调节其摄取效率。模拟潜伏和成熟形式的重组小鼠肝素酶（L-Hpse 和 M-Hpse）被内化到肥大细胞瘤 MST 细胞、腹腔细胞衍生的肥大细胞和骨髓衍生的肥大细胞中。内化的 L-Hpse 量明显高于 M-Hpse。在 MST 细胞中，L-Hpse 可连续内化长达 8 小时，而 M-Hpse 的摄取在孵育 2 小时后达到饱和。L-Hpse 和 M-Hpse 与 MST 细胞表面相似结合。在用肝素酶抑制剂 SF4（一种不影响肝素酶与固定化肝素结合的小分子肝素酶抑制剂）孵育的 MST 细胞中，细胞表面硫酸乙酰肝素的表达水平降低。在肝素酶抑制剂 SF4 的存在下，M-Hpse 进入肥大细胞的内化量显著增加，该抑制剂不影响肝素酶与固定化肝素的结合。用点突变在 Glu335 制备了酶失活的 M-Hpse。突变型 M-Hpse 的内化量明显高于野生型 M-Hpse，但与野生型和突变型 L-Hpse 相似。这些结果表明，肝素酶的酶活性负调节肝素酶介导的肥大细胞摄取，可能通过下调细胞表面硫酸乙酰肝素的表达。
