Versican and fibrillin-1 form a major hyaluronan-binding complex in the ciliary body.
OBJECTIVE
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.
Ohno-Jinno
Akiko
A
Department of Ophthalmology, Aichi Medical University, Nagakute, Aichi, Japan.
Isogai
Zenzo
Z
Yoneda
Masahiko
M
Kasai
Kenji
K
Miyaishi
Osamu
O
Inoue
Yoko
Y
Kataoka
Takuya
T
Zhao
Jing-Song
JS
Li
Huili
H
Takeyama
Masayuki
M
Keene
Douglas R
DR
Sakai
Lynn Y
LY
Kimata
Koji
K
Iwaki
Masayoshi
M
Zako
Masahiro
M
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002924:Ciliary Body / Q000378:metabolism* / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D000071837:Fibrillins; D006820:Hyaluronic Acid / Q000378:metabolism*; D007150:Immunohistochemistry; D020894:Microfibrils / Q000378:metabolism / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D053675:Versicans / Q000235:genetics / Q000378:metabolism*; D014822:Vitreous Body / Q000378:metabolism
D000071837:Fibrillins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D053675:Versicans; D006820:Hyaluronic Acid