Nobuaki Higashi

Heparanase 1 (HPSE1) is the only mammalian endoglycosidase that cleaves heparan sulfate (HS), a glycosaminoglycan (GAG), and is frequently upregulated in cancers, thereby promoting tumor progression. Despite extensive efforts to develop inhibitors of its HS-degrading activity, its non-enzymatic functions limit therapeutic efficacy and pose a major challenge for therapeutic development. Thus, inhibiting HPSE1 expression is critical for controlling its enzymatic and non-enzymatic functions; however, no FDA-approved inhibitors are currently available. Here, we identify auranofin (AUF), an oral gold-containing drug used to treat rheumatoid arthritis, as a potent inhibitor of HPSE1 promoter activity. High-throughput screening revealed that an atypical protein kinase C (aPKC)–NF-κB signaling axis is a key regulator of HPSE1 expression. Notably, AUF treatment reduced HPSE1 expression and significantly suppressed the invasive capacity of MDA-MB-231 cells in a Transwell migration assay. We then investigated the role of HPSE1 in the invasive activity of MDA-MB-231 cells, which produce higher levels of hyaluronan (HA) and HS than non-invasive cells. Neither HS degradation, HA supplementation in Matrigel during Transwell migration, nor HPSE1 overexpression alone was sufficient to drive invasion, suggesting that invasive capacity depends on mesenchymal features and coordinated induction of HPSE1 and GAGs rather than HS degradation. Collectively, our findings demonstrate that AUF-mediated inhibition of aPKC suppresses HPSE1 expression, thereby inhibiting both its enzymatic and non-enzymatic functions and limiting cancer progression, metastasis, and angiogenesis. These results highlight the therapeutic potential of AUF for targeting HPSE1-driven tumor progression and support its repurposing for cancer treatment.

A chimeric protein of heparanase and Ig-Fc was designed as a novel tool to expand the detection of structurally heterogeneous heparan sulfate (HS) and related glycosaminoglycans. The whole mouse heparanase gene was combined with the gene segment encoding the mouse IgG1 hinge-Fc domain. A point mutation E335A was inserted to disable putative HS degradation activity. Chimeric proteins consisted of the latent form of the enzyme devoid of HS degradation activity. The chimeric proteins bound to heparin, N-desulfated heparin, and O-sulfated N-acetylheparosan. Their binding spectrum to glycosaminoglycans differed from that of anti-HS mAb 10E4. The chimeric proteins bound to Kato III and A549 cell lines. The binding was reduced by knocking down EXT1 gene expression. One of the chimeric proteins stained the epidermal cells in the hyperplastic spinous layer of inflamed atopic dermatitis skin and inflammatory cells in the dermis, which were not stained with mAb 10E4. The protein stained a polarized structure on the surface of monocytic U937 and THP1 cells. Similar polarized structures were observed with anti-syndecan-1 antibody staining. The chimeric protein and anti-syndecan-1 antibody precipitated similar sets of proteins that included syndecan-1 from the lysates of U937 cells. These novel chimeric proteins are useful to detect HS abundant in O-sulfation in histochemical, cytochemical, and biochemical studies.

Glycosaminoglycans (GAGs), including hyaluronic acid (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), heparan sulfate (HS)/heparin (HP), and keratan sulfate (KS), play pivotal roles in living organisms. Generally, GAGs are analyzed after enzymatic digestion into unsaturated or saturated disaccharides. Due to high structural similarity between disaccharides, however, separation during analysis is challenging. Additionally, little is known about the structures of GAGs and their functional relationships. Elucidating the function of GAGs requires highly sensitive quantitative analytical methods. We developed a method for the simultaneous analysis of 18 types of disaccharides derived from HA (1 type), CS/DS (7 types), HS/HP (8 types), and KS (2 types) potentially detectable in analyses of human urine. The simple method involves HPLC separation with fluorescence detection following derivatization of GAG-derived disaccharides using 4-aminobenzoic acid ethyl ester (ABEE) as a pre-labeling agent and 2-picoline borane as a reductant. The ABEE derivatization reaction can be performed under aqueous conditions, and excess derivatization reagents can be easily, rapidly, and safely removed. This method enables highly sensitive simultaneous analysis of the 18 abovementioned types of GAG-derived disaccharides using HPLC with fluorescence detection in small amounts of urine (1 mL) in a single run. The versatile method described here could be applied to the analysis of GAGs in other biological samples. Graphical Abstract

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.

A cyclin-dependent kinase (CDK) inhibitor, p57^Kip2, is an important molecule involved in bone development; p57^Kip2-deficient (p57-/-) mice display neonatal lethality resulting from abnormal bone formation and cleft palate. The modulator 1α,25-dihydroxyvitamin D₃ (l,25-(OH)₂VD₃) has shown the potential to suppress the proliferation and induce the differentiation of normal and tumor cells. The current study assessed the role of p57^Kip2 in the 1,25-(OH)₂VD₃-regulated differentiation of osteoblasts because p57^Kip2 is associated with the vitamin D receptor (VDR). Additionally, 1,25-(OH)₂VD₃ treatment increased p57^KIP2 expression and induced the colocalization of p57^KIP2 with VDR in the osteoblast nucleus. Primary p57-/- osteoblasts exhibited higher proliferation rates with Cdk activation than p57+/+ cells. A lower level of nodule mineralization was observed in p57-/- osteoblasts than in p57+/+ cells. In p57+/+ osteoblasts, 1,25-(OH)₂VD₃ upregulated the p57^Kip2 and opn mRNA expression levels, while the opn expression levels were significantly decreased in p57-/- cells. The osteoclastogenesis assay performed using bone marrow cocultured with 1,25-(OH)₂VD₃-treated osteoblasts revealed a decreased efficiency of 1,25-(OH)₂VD₃-stimulated osteoclastogenesis in p57-/- cells. Based on these results, p57^Kip2 might function as a mediator of 1,25-(OH)₂VD₃ signaling, thereby enabling sufficient VDR activation for osteoblast maturation.

We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (−1)- and (−2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.

マスト細胞はその巨大な細胞内分泌顆粒によって特徴づけられるが、この顆粒内にはグリコサミノグリカン、すなわちヘパリンや高硫酸化コンドロイチンが大量に蓄積されている。顆粒複合体は一種のタンパク質–高分子電解質複合体であり、グリコサミノグリカン、プロテアーゼ、生理活性アミンや他の様々なメディエータから構成されている。近年の知見から、マスト細胞から放出された後の顆粒複合体の運命が多様であることが示唆されている。すなわち複合体の構成物は、血流に乗る、マスト細胞近傍の結合組織に留まる、上皮・間質の細胞や免疫細胞に取り込まれて獲得免疫の引き金や組織ホメオスタシスの維持に関わる、リンパ流に乗って所属リンパ節に運ばれる、などである。これらの運命の選択は顆粒複合体内の糖鎖により、特にヘパラナーゼの切断によって加工されたヘパリンにより大きく影響される。本総説では、マスト細胞の顆粒複合体の構成に関する最近の進歩に焦点を当て、特に顆粒内ヘパリンのプロセシング、顆粒複合体が放出された後の運命と役割、免疫調節物質としての役割の可能性、またこの複合体を模倣した人工的な送達システムの構築の試みについて述べる。さらにこれらの機能における糖鎖の関与についても議論する。

Mast cells are characterized by large, intracellular secretory granules that accumulate large amounts of glycosaminoglycans, heparin and highly sulfated chondroitin depending on the mast cell type. The granular complex, a kind of protein-polyelectrolyte complex, is composed of these glycosaminoglycans, proteases, biogenic amines, and other mediator molecules. Recent findings suggest that, after the granular complex is released, it may undergo various fates, i.e., components of the complex are either transferred into the blood flow; or they may be retained in the connective tissues near the mast cells; or ingested by epithelial, interstitial or immune cells to trigger the adaptive immune responses or to maintain tissue homeostasis; or they may be transferred to the draining lymph nodes through lymph flow. The choice of fate is greatly influenced by carbohydrates in the granular complex, especially by processed heparin via heparanase-mediated cleavage. The present review focuses on recent developments on the formation of granular complex, especially on the processing of the granular heparin, the fates and roles of the complex after being released, its potential role as an immunomodulator, and a recent attempt to mimic the complex as an artificial delivery system. The involvement of carbohydrates in these functions is also discussed.

Purpose: Pin1 is a peptidyl prolyl cis/trans-isomerase (PPIase) that regulates phosphorylated protein function by cis-trans-isomerization. Pin1 catalytic activity has been associated with the pathogenesis of cancer, asthma, neurodegenerative diseases, nonalcoholic steatohepatitis, and viral infections. Recently, imidazole dipeptide molecules from natural products have attracted interest as functional food components. Here, we evaluated the effects of natural imidazole dipeptides on the PPIase activity of Pin1 to identify novel Pin1 inhibitors. Methods: PPIase catalytic activity of Pin1 was measured using chymotrypsin-coupled isomer specific protein degradation. The test substance was preincubated with recombinant Pin1 to form their complex. Results: Our results showed that among the imidazole dipeptide molecules, L-balenine, and not L-anserine and L-carnosine, reduced the PPIase activity of Pin1. Both β-alanine and 1-methyl-L-histidine obtained by hydrolysis of L-balenine did not affect Pin1 PPIase activity. These results suggested that the structure of L-balenine allows it to occupy the active site of the Pin1 enzyme. Conclusion: Among imidazole dipeptides from natural products, L-balenine inhibited the PPIase catalytic activity of Pin1.