Versican and Fibrillin-1 Form a Major Hyaluronan-Binding Complex in the Ciliary Body
Akiko Ohno-Jinno 1,
Zenzo Isogai 2,
Masahiko Yoneda 3,
Kenji Kasai 4,
Osamu Miyaishi 5,
Yoko Inoue 1,
Takuya Kataoka 1,
Jing-Song Zhao 1,
Huili Li 6,
Masayuki Takeyama 6,
Douglas R. Keene 7,
Lynn Y. Sakai 8,
Koji Kimata 9,
Masayoshi Iwaki 1,
and
Masahiro Zako 10*
1 Ophthalmology, Aichi Medical University, Aichi, Japan
2 Dermatology, National Center for Geriatrics and Gerontology, Aichi, Japan
3 Biochemistry, Aichi Prefectural College of Nursing and Health, Aichi, Japan
4 Department of Pathology, Aichi Medical University, Aichi, Japan
5 Pathology, Chubu-Rosai Hospital, Aichi, Japan
6 Ophthalmology, Aichi Medical University, Aichi-gun, Aichi-Ken, Japan
7 Shriners Hospital for Children, Portland, Oregon, United States
8 Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon, United States
9 Aichi Medical University, Institute for Molecular Science of Medicine, Aichi, Japan
10 Ophthalmology, Aichi Medical University, Nagakute, Aichi-gun, Aichi-ken, 480-1195, Japan
* To whom correspondence should be addressed. E-mail: zako{at}aichi-med-u.ac.jp.
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Abstract |
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PURPOSE. This study was conducted to examine whether versican, which is known to bind fibrillin-1, interacts with fibrillin-1 in the ciliary body and vitreous body, and whether the versican in this complex binds to hyaluronan, by utilizing biochemistry, molecular biology, immunohistochemistry, and electron microscopy techniques.
METHODS. The new polyclonal antibodies against amino- and carboxy-termini of versican were raised and characterized. The mRNA expression levels of versican and fibrillin-1 were analyzed by RT-PCR and real-time PCR, while their protein levels were evaluated by Western blotting and immunohistochemistry. Isolation of versican bound to fibrillin-1-containing microfibrils from ciliary bodies was performed by extraction studies. Slot blot analyses and rotary shadowing electron microscopy were applied to identify versican associated with fibrillin-1-containing microfibrils after gel filtration chromatography and density gradient centrifugation.
RESULTS. The newly prepared polyclonal antibodies recognized amino- and carboxy-termini of chicken versican. Versican, principally V0 and V1, was found to be securely bound to fibrillin-1-containing microfibrils, forming a major hyaluronan-binding structure in the ciliary non-pigmented epithelium. In addition, Western blotting revealed two cleaved complexes, the carboxy-terminal end of versican bound to fibrillin microfibrils and the amino-terminal end of versican bound to hyaluronan in the vitreous body.
CONCLUSIONS. Fibrillin-1, versican, and hyaluronan form a unique complex in the ciliary non-pigmented epithelium, and two cleavage products of this complex were shown to exist in the vitreous body. This newly clarified fibrillin-versican-hyaluronan (FiVerHy) complex and its cleavage products may be indispensable for the physiological properties important to the ciliary body and vitreous body.
Key Words:
extracellular matrix, ciliary epithelium, vitreous humor, fibrillin-1, versican, hyaluronan
