title: Molecular structure and function analysis of bikunin on down-regulation of tumor necrosis factor-alpha expression in activated neutrophils.
abstract: OBJECTIVE
  We performed a detailed molecular analysis of bikunin-mediated anti-inflammation (suppressive effect of cytokine release, MAP kinase activation, and nuclear translocation of NF-kB) using a truncated form of bikunin.
  
  
  METHODS
  We obtained bikunin derivatives that contained O-glycoside-linked N-terminal glycopeptide (Bik-m1), N-glycoside-linked C-terminal tandem Kunitz domains (Bik-m2), bikunin lacking O-glycoside (Bik-c), asialo bikunin (Bik-a), bikunin lacking N-glycoside (Bik-n), and purified C-terminal Kunitz domain II (kII) of bikunin (HI-8). Enzyme-linked immunosorbent assay and Western blot were carried out to measure secreted TNF-alpha and MAP kinase activation.
  
  
  RESULTS
  We examined the TNF-alpha secretion in control and lipopolysaccharide (LPS)-treated neutrophils and did not see any changes of its protein levels in the cells pretreated with Bik-m1, Bik-m2, Bik-c, or HI-8. In all of the derivatives tested, only the derivatives that lacked N-glycoside side chain showed a significant suppression of TNF-alpha secretion by LPS. Only a small (21 amino acids) deletion of the N-terminal portion of bikunin (which corresponds to Bik-m2) abolished its suppressing activity of TNF-alpha secretion, thus suggesting that the N-terminal 21 amino acids play a critical role in anti-inflammation. Bik-m1 alone failed to show anti-inflammatory response. Bikunin failed to inhibit ionomycin-induced phosphorylation of MAP kinases.
  
  
  CONCLUSIONS
  These data allow us to conclude that the cytokine expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain. Our results also suggest a possible role of bikunin for receptor-dependent MAP kinase activation.
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Kanayama
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Yamada
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Onogi
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shigetomi
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Sachiyo
authors.initials: S
authors.lastname: Ueda
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Yoriko
authors.initials: Y
authors.lastname: Tsuji
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Haruta
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Ryuji
authors.initials: R
authors.lastname: Kawaguchi
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Shozo
authors.initials: S
authors.lastname: Yoshida
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Sakata
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Sado
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kitanaka
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Oi
authors.affiliation: NetForce Co., Ltd., Nakamura, Nagoya, Aichi, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Yagyu
authors.affiliation: Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan. Electronic address: hirokoba@naramed-u.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kobayashi
mesh_terms: D000510:Alpha-Globulins / Q000737:chemistry* / Q000502:physiology*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000502:physiology*; D006801:Humans; D018836:Inflammation Mediators / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D015394:Molecular Structure; D018375:Neutrophil Activation / Q000502:physiology*; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000510:Alpha-Globulins; D018836:Inflammation Mediators; D014409:Tumor Necrosis Factor-alpha; C016001:alpha-1-microglobulin; D020928:Mitogen-Activated Protein Kinases