Molecular structure and function analysis of bikunin on down-regulation of tumor necrosis factor-α expression in activated neutrophils
Introduction
Preterm delivery is thought to be mediated by proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and other inflammatory mediators released from activated leukocytes [1]. Lipopolysaccharide (LPS), an inflammatory mediator, has been shown to activate neutrophils directly or indirectly through induction of MAPK-dependent NF-kB activation and expression of proinflammatory cytokine [2]. Because proinflammatory cytokines have been shown to activate neutrophils [3], inhibition of either neutrophil activation or proinflammatory cytokine production by some therapeutic agents might contribute to reduce inflammation-induced preterm delivery.
Bikunin, a well known Kunitz-type protease inhibitor (also known as urinary trypsin inhibitor [UTI] in Japan) purified from human urine, is clinically used for management of preterm delivery [4], [5], [6]. Several clinical studies demonstrated that bikunin has proved effective in patients developing preterm delivery. It is possible that intravaginal administered bikunin will reduce preterm delivery by inhibiting neutrophil activation. We have demonstrated previously that bikunin plays a critical role in anti-inflammation: it prevents the release of proinflammatory cytokines, activation of MAPK, phosphorylation of IkB-α, degradation of IkB-α, and nuclear translocation of NF-kB in macrophages stimulated with LPS [7], [8]. Such inhibitory activities of bikunin on cell activation may contribute to reduce LPS-induced preterm delivery in in vivo experiments [9], [10], [11], [12], [13]. In the previous study, this possibility was examined in vitro [14]. However, the molecular networks underlying this event in bikunin-mediated anti-inflammation remain poorly understood.
Bikunin is produced as a light chain of inter-α-inhibitor (IαI) by liver cells [15]. The protein is heavily glycosylated at two sites, Ser10 and Asn45. It is a 40-kDa glycoprotein comprised of several structurally and functionally distinct domains [16]. This glycoprotein is composed of three domains: an N-terminal-domain with O-glycoside side chain (Ala1-Asp21), a middle-domain with N-glycoside side chain (Kunitz domain I [kI], Thr23-Trp77), and a C-terminal-domain (Kunitz domain II [kII], anti-tryptic activity, Ser78-Ala143) [16]. The anti-catalytic domain is covered by the C-terminal kII-domain. Bikunin has several conserved residues in the N-terminal first Kunitz domain (kI) and the C-terminal second Kunitz domain (kII), including six cysteines. Both domains are predicted to have a similar structure, presumably maintained by disulfide bonds between the conserved cysteines [16].
It was shown that bikunin inhibited LPS-mediated increases in intracellular calcium (Ca2+) [17]. LPS recognition requires its binding protein (LBP), CD14, and MD-2, which is directly involved in ligand binding and subsequent receptor activation [18]. Then, LPS activates neutrophil signal transduction in a receptor-dependent manner, which is related to LPS-mediated increases in Ca2+. On the other hand, Ca2+-sensitive ionophore ionomycin directly causes changes in cytosolic Ca2+ level, and also activates MAP kinases in a receptor-independent manner [19]. It is unclear whether bikunin-mediated suppression of MAP kinase activation is a receptor-dependent.
In the present study, we performed a detailed molecular analysis of bikunin-mediated anti-inflammation using a truncated form of bikunin, to investigate whether the effects of bikunin were mediated by its Kunitz structure or glycoside side chain. We finally examined the suppressive effect of bikunin on LPS- or ionomycin-induced changes in both p38 and p42/44 MAP kinase activation.
Section snippets
Materials
Bikunin was purified to homogeneity from human urine. A highly purified preparation of bikunin was kindly supplied by Mochida Pharmaceutical (Tokyo, Japan). The kII (HI-8) was purified from bikunin as previously described [16]. Metalloendopeptidase (EC 3.4.2.4), chondroitinase ABC lyase (EC 4.2.2.4), neuraminidase (EC 3.2.1.18), endo-β-N-acetylglucosaminidase F2 (Endo F2; EC 3.2.1.92), and hyaluronidase were purchased from Seikagaku Kogyo (Tokyo, Japan). Trypsin (EC 3.4.21.4) was purchased from
Results
The recently published structure of bikunin shown in Fig. 1 was used as the basis for preparing several truncated forms of the intact bikunin to examine the structural requirements for the regulatory function of this glycoprotein. There are three recognizable sub-domains within bikunin. We delineated structural motifs regulating bikunin’s function.
Discussion
Bikunin is a member of Kunitz-type protease inhibitor family. The domain of bikunin can be divided into three regions with prominent structural features. This work is a follow up of earlier publications of our group, which demonstrated the capacity of bikunin to prevent proinflammatory cytokine release, MAPK activation and NF-kB activation in LPS-stimulated cells [7], [8]. The protein truncation procedure was also used in preceding studies, which revealed the importance of O-glycoside site for
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.
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