Molecular structure and function analysis of bikunin on down-regulation of tumor necrosis factor-alpha expression in activated neutrophils.
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.
Kanayama
Seiji
S
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Yamada
Yoshihiko
Y
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Onogi
Akira
A
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Shigetomi
Hiroshi
H
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Ueda
Sachiyo
S
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Tsuji
Yoriko
Y
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Haruta
Shoji
S
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Kawaguchi
Ryuji
R
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Yoshida
Shozo
S
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Sakata
Mariko
M
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Sado
Toshiyuki
T
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Kitanaka
Takashi
T
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Oi
Hidekazu
H
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan.
Yagyu
Tatsuo
T
NetForce Co., Ltd., Nakamura, Nagoya, Aichi, Japan.
Kobayashi
Hiroshi
H
Department of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Nara, Kashihara 634-8522, Japan. Electronic address: hirokoba@naramed-u.ac.jp.
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
D000510:Alpha-Globulins; D018836:Inflammation Mediators; D014409:Tumor Necrosis Factor-alpha; C016001:alpha-1-microglobulin; D020928:Mitogen-Activated Protein Kinases