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Versican and Fibrillin-1 Form a Major Hyaluronan-Binding Complex in the Ciliary Body

¹From the Departments of Ophthalmology and ⁴Pathology and the ⁸Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi, Japan; the ²Department of Dermatology, National Center for Geriatrics and Gerontology, Aichi, Japan; the ³Aichi Prefectural College of Nursing and Health, Nagoya, Aichi, Japan; the ⁵Department of Pathology, Chubu-Rosai Hospital, Nagoya, Aichi, Japan; and the ⁶Shriners Hospital for Children and the ⁷Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon.

PURPOSE. In this study, biochemistry, molecular biology, immunohistochemistry, and electron microscopy techniques were used to examine whether versican, which is known to bind fibrillin-1, interacts with fibrillin-1 in the ciliary body and vitreous, and whether the versican in this complex binds to hyaluronan.

METHODS. The new polyclonal antibodies against the amino and carboxyl 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, and their protein levels were evaluated by Western blot analysis 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 carboxyl 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 nonpigmented epithelium. In addition, Western blot analysis revealed two cleaved complexes, the carboxyl-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 nonpigmented 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.