2025 年 13 巻 1 号 p. 10-16
Aim: This study aimed to investigate the role of the chondroitin sulfate (ChS) chain in the anti-inflammatory effect of urinary trypsin inhibitor (UTI), identify ChS structures that enhance the anti-inflammatory activity of UTI, and develop a more effective means of treatment for preterm birth.
Methods: ChS chain-remodeled UTIs were prepared by the hydrolysis and/or transglycosylation reaction of testicular hyaluronidase. Uterine cervical fibroblasts were treated with native or ChS chain-remodeled UTIs and incubated with lipopolysaccharide to induce inflammation. To assess anti-inflammatory effects, interleukin-8 (IL-8) concentrations in culture supernatants were measured by enzyme-linked immunosorbent assay.
Results: Compared with controls without UTI, Ch6S-transferred UTI at the nonreducing terminus significantly suppressed IL-8 release. Other UTIs showed limited suppression.
Conclusion: The Ch6S chain at the nonreducing terminus of UTI enhances the anti-inflammatory activity of UTI. This finding could lead to the development of innovative treatments for preterm birth.
Amniotic fluid and neonatal urine contain high amounts of urinary trypsin inhibitor (UTI), a proteoglycan composed of a core protein with 143 amino acid residues and low-sulfated chondroitin 4-sulfate (Ch4S) chains covalently linked at serine 10.1,2,3,4) UTI exerts anti-inflammatory effects by inhibiting the production of inflammatory cytokines interleukin (IL)-8 and IL-6.5) UTI also inhibits the activities of proteases, including elastase, thereby indirectly modulating inflammatory cytokine levels and preventing tissue degradation.
Preterm birth is the main cause of perinatal mortality.6) Cervical ripening, a key process in preterm birth, is primarily driven by the degradation of collagen within the extracellular matrix of the cervix and an increase in glycosaminoglycans (GAGs), particularly hyaluronic acid (HA) production.7) Although UTI has been clinically used as a vaginal agent in the treatment of premature birth, its preventive effect remains unclear.
The protease inhibitory activity of UTI is mediated by the Kunitz domains of the core protein. However, the structural and functional significance of the Ch4S region, which accounts for approximately 30% of the molecular weight of UTI, has not been fully elucidated. Previously, we used hyaluronidase-based glycoengineering techniques to remodel the chondroitin sulfate (ChS) chain of UTI without changing the core protein,8,9,10) and showed that transferring Ch4S to the nonreducing terminus of the original ChS chain of UTI enhanced its anti-inflammatory effects.9) This finding highlighted the importance of the ChS chain linking to its core proteins in protease inhibition.10) However, the relationship between the structure of ChS and its inhibitory effect remains unclear.
We hypothesized that the anti-inflammatory effects of the sulfate groups would be replicated in ChS chain-remodeled UTI. Thus, we aimed to investigate the effect of the chondroitin 6-sulfate (Ch6S) chain on the anti-inflammatory activity of UTI and develop a novel treatment for premature birth that is more effective than native UTI.
Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Nippon Suisan Kaisha, Ltd. (Tokyo, Japan). Fetal bovine serum (FBS), lipopolysaccharide (LPS; a somatic component of Gram-negative rod bacteria, Escherichia coli O55:B5), bovine testicular hyaluronidase (BTH; type 1-S), Ch4S from bovine trachea, and Ch6S from shark cartilage were purchased from Sigma-Aldrich (St. Louis, MO, USA). UTI (Biotech Center, Shanghai Institute of Pharmaceutical Industry, China) was purified by DEAE-Cellufine column chromatography.11) Endotoxin-free HA derived from Streptococcus zooepidemicus was kindly provided by Shiseido Co., Ltd. (Tokyo, Japan). The CNBr-activated Sepharose 4 Fast Flow resin was acquired from GE Healthcare Japan (Tokyo, Japan). Amicon Ultra-15 centrifugal filter units were obtained from Merck Millipore, Ltd. (Tullagreen, Ireland). PAGEL (5%–20% polyacrylamide gradient gel) was purchased from Atto Co. (Tokyo, Japan). All other reagents were of analytical grade and were purchased from commercial suppliers. Endotoxin-free reagents and distilled water were utilized throughout the study.
Remodeling of the ChS chain of UTIA remodeled UTI lacking the original ChS chain, leaving only the hexasaccharide linkage region between the ChS chain and core protein, was prepared by the hydrolysis of UTI with BTH (“linkage-UTI”). The hydrolysis reaction of BTH was initiated by incubating UTI in 0.1 M sodium acetate buffer (pH 4.0) containing 150 mM NaCl for 16 h at 37°C in a BTH-immobilized CNBr-activated sepharose column. Subsequently, reaction products were eluted using 0.1 M sodium acetate buffer (pH 4.0) supplemented with 0.5 M NaCl.
The transglycosylation reaction of BTH was carried out using UTI or linkage-UTI as an acceptor and HA or ChS (Ch4S or Ch6S) as a donor in 0.15 M Tris–HCl (pH 7.0) without NaCl for 16 h (for HA), 24 h (for Ch4S), or 72 h (for Ch6S) at 4°C in a BTH-immobilized column. These reactions yielded various ChS chain-remodeled UTIs with varying donor-derived N-terminal structures. Reaction products were then eluted using 0.1 M Tris–HCl buffer (pH 7.0) without NaCl.
UTI-containing fractions were collected by monitoring the absorbance at 280 nm and then desalted. Following this, samples were concentrated using an Amicon Ultra-15 filter. ChS chain-remodeled UTIs with variations in ChS structure are presented in Figure 1. Concentrations of ChS chain-remodeled UTIs were adjusted by ethanol precipitation. Protein concentrations were determined using the Bradford method.
Linkage-UTI lacks the original ChS chain, leaving only the hexasaccharide linkage region between the ChS chain and core protein. HA-linkage-UTI has hyaluronan (HA) linked to linkage-UTI. Ch4S-linkage-UTI has chondroitin 4-sulfate (Ch4S) linked to linkage-UTI. Ch6S-linkage-UTI has chondroitin 6-sulfate (Ch6S) linked to linkage-UTI. HA-UTI has HA linked to UTI. Ch4S-UTI has Ch4S linked to UTI. Ch6S-UTI has Ch6S linked to UTI. GlcUA, glucuronic acid; GlcNAc, N-acetylglucosamine; Gal, galactose; GalNAc, N-acetylgalactosamine; Xyl, xylose; Ser, serine; 4S, 4-O-sulfate; 6S, 6-O-sulfate. n, m, n, m, N, M, N, and M are the numbers of repeating disaccharide units.
SDS-PAGE was performed using PAGEL as previously described by Laemmli.12) Gels were stained with Coomassie brilliant blue (CBB) R-250 for protein detection and alcian blue (pH 2.5, preferred for HA and ChS staining) for GAG detection.
Immunological detectionFor Western blotting, 1 μg of each UTI was loaded onto PAGEL. After separation, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). Membranes were incubated with primary anti-bikunin antibody (1/1,000 dilution in Tris-Saline pH 7.4, CP6, Millipore) overnight at room temperature. Polyclonal goat anti-rabbit immunoglobulins (1/2,000 dilution in Tris-Saline pH 7.4, P0448, Dako, CA, USA) were used as secondary antibodies for detection, visualized using a ChemiDoc imaging system (Bio-Rad, CA, USA).
For dot blotting, 1 μg (for transferred HA detection) or 0.2 μg (for transferred ChS detection) of each UTI was spotted onto a PVDF membrane (Millipore). The membrane was incubated with the primary biotinylated hyaluronan binding protein (b-HABP; 1/2,500 dilution in Tris-Saline pH 7.4, BC41, Hokudo, Hokkaido, Japan) or monoclonal anti-ChS clone CS-56 (1/400 dilution in Tris-Saline pH 7.4, C8035, Sigma-Aldrich) overnight at room temperature. Horseradish peroxidase (HRP)-conjugated streptavidin (streptavidin/HRP) (1/1,000 dilution in Tris-Saline pH 7.4, P0397, Dako) and polyclonal rabbit anti-mouse immunoglobulins/HRP (1/1,000 dilution in Tris-Saline pH 7.4, P0161, Dako) were used as secondary antibodies. A ChemiDoc imaging system (Bio-Rad) was used for visualization and quantification.
Establishment of primary uterine cervical fibroblast (UCF) cultureCervical tissue samples were collected from nonpregnant women who underwent total hysterectomy for uterine myoma at Hirosaki University Hospital with written informed consent. This study was approved by the Research Ethics Committee of Hirosaki University Graduate School of Medicine (Approval no. 2020–182). UCF culture was performed following the methods previously described by K. Tanaka and S. Tanaka.9,13)
Treatment of USFs with LPS and UTIUCFs (1.7×105 cells/well) were cultured in 24-well plates using 10% FBS-supplemented DMEM. To eliminate the effects of FBS on UTIs, cells that reached subconfluence were cultured in serum-free medium for 16 h. Subsequently, various UTIs (i.e., native UTI, linkage-UTI, HA-linkage-UTI, Ch4S-linkage-UTI, Ch6S-linkage-UTI, HA-UTI, Ch4S-UTI, and Ch6S-UTI) were added to each well at a final concentration of 20 μg/ml. After 1 h, UCFs were further incubated with LPS (1 μg/ml) for 24 h.14) Finally, culture supernatants were collected for enzyme-linked immunosorbent assay (ELISA).
ELISAIL-8 concentrations in culture supernatants were quantified using an ELISA kit (Thermo Fisher Scientific, MA, USA).15) The absorbance at 450 nm was determined using a microplate reader (Tecan Sunrise Reader, Männedorf, Switzerland). IL-8 concentrations were normalized to the number of living cells, as determined using alamarBlue (Thermo Fisher Scientific).
Statistical analysisData are presented as means±standard deviations of one independent experiment performed in triplicate. Statistical analyses were performed using one-way analysis of variance, followed by Tukey’s post hoc test. Data analysis was performed using the Bell Curve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan). P<0.05 was considered statistically significant.
ChS chain-remodeled UTIs were prepared (Figure 1), and UTI hydrolysis and transglycosylation were confirmed by SDS-PAGE, Western blotting, and dot blotting (Figure 2).
SDS-PAGE of UTIs stained with Coomassie brilliant blue R250 (A) and Alcian blue (B). Western blotting of UTIs (C). Dot blotting of UTIs detected using biotinylated hyaluronan binding protein (b-HABP) and anti-ChS antibody (D).
Figures 2A and 2B show the results of SDS-PAGE analyses of native UTI and ChS chain-remodeled UTIs. Native UTI and ChS chain-remodeled UTIs were all stained with CBB and alcian blue. Native UTI was predominantly detected as a broad band (approximately 32.5 k), reflecting the heterogeneity of low-sulfated Ch4S chains.11) A minor population of N-terminal-truncated UTIs lacking GAG chains, corresponding to low-molecular-weight forms of UTI, has been observed at approximately 17 k.4,8,16)
Two distinct bands were detected for Ch4S chain-deficient UTI (“linkage-UTI”), at 21.5 k and 17 k (Figure 2A). These bands were also observed for HA-linkage-UTI, Ch4S-linkage-UTI, and Ch6S-linkage-UTI (Figure 2A).
Alcian blue staining confirmed transglycosylation, with the target donor-derived structures linked to either the UTI linkage region or the ChS chain of native UTI, as the bands of HA-UTI, Ch4S-UTI, Ch6S-UTI, HA-linkage-UTI, Ch4S-linkage-UTI, and Ch6S-linkage-UTI appeared broader in high molecular weight areas (Figure 2B). The bands observed by Western blotting support the results of CBB staining (Figure 2C).
Dot blotting using b-HABP showed a reaction only between HA-UTI and HA-linkage-UTI (Figure 2D); this confirmed the transfer of HA from the donor HA to native UTI or linkage-UTI. All UTIs reacted with the anti-ChS antibody, which reacts with Ch4S and Ch6S.17,18) Specifically, Ch4S-UTI, Ch6S-UTI, Ch4S-linkage-UTI, and Ch6S-linkage-UTI exhibited high reactivity, strongly suggesting that the ChS chain was transglycosylated at the nonreducing terminus.
Figure 3 shows the effect of UTIs on IL-8 accumulation in culture supernatants after LPS stimulation. After 24 h of incubation, LPS induced an approximately 11.9-fold increase in IL-8 release per 104 living cells compared with that without LPS and UTI. The addition of native UTI reduced LPS-induced IL-8 release, although the difference was not significant. Notably, the addition of Ch6S-linkage-UTI and Ch6S-UTI significantly reduced IL-8 release to 35.8% and 34.1%, respectively, compared with the control (i.e., without UTI).
Lipopolysaccharide (LPS) significantly stimulated IL-8 release after 24 h of incubation. The addition of Ch6S-linkage-UTI and Ch6S-UTI significantly reduced LPS-stimulated IL-8 release. Values plotted are mean±standard deviation obtained from one independent experiment performed in triplicate. * Significant compared to LPS-free, P<0.05. ** Significant compared to UTI-free, P<0.05.
This study demonstrated that UTI with Ch6S transferred to its nonreducing terminus exerts a stronger anti-inflammatory effect than native UTI. Unfortunately, due to resource limitations, the results were derived from a single independent experiment performed in triplicate. Nonetheless, we believe that the present study provides useful initial data, and the validity of the conclusions will be greatly improved with additional experiments. Since the position of the sulfate group on GalNAc is different between Ch6S and Ch4S, this may have also resulted in differences in biological activity. This aspect will be considered in future studies.
A previous study reported that Ch6S suppresses inflammatory responses by inhibiting the nuclear translocation of NF-κB,19) suggesting that the position of the sulfate group determines its activity. Conversely, another study reported that free Ch4S exhibited a stronger anti-inflammatory effect than Ch6S in mouse chondrocytes stimulated with LPS, an inflammation inducer.20) While the same inflammation inducer (i.e., LPS) was used in both studies, the latter study used GAG containing no aglycone component as the only test substance; this may explain the differences in results. The free ChS chain suppresses the production of inflammatory cytokines by inhibiting the NF-κB signaling pathway and toll-like receptor activity and is used to treat osteoarthritis. It also has anti-inflammatory effects in many other tissues.21,22)
UTI inhibits cervical maturation, which is induced by IL-8.23) For future clinical applications, although UCFs were used in this study, amnion or decidual cells may yield different experimental results given different developmental mechanisms, necessitating further studies. When comparing UCFs during pregnancy and non-pregnancy, significant differences have been observed in proteome changes and cytokine production.24) Since the concentration of inflammatory cytokines is also expected to increase during pregnancy,25) the effects of UTI on cellular reactions may also differ, warranting further investigation. Based on the present observations, Ch6S chain-remodeled UTI may offer a promising means to treat preterm birth, aid in the development of a new treatment for preterm birth, and help solve major issues in perinatal medicine. The synthesis of ChS chain-remodeled UTI is a proprietary technology developed by our group, with the advantage of producing highly pure remodeled UTIs in large quantities. In the future, we will continue to advance research on the molecular stability of UTI and its administration route, with the aim of future clinical application.
This study was supported by The Seiichi Imai Memorial Foundation.
The authors declare no conflict of interest.
