Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing.
OBJECTIVE
To purify and characterize the glycoprotein lumican, isolated from human amniotic membrane (AM), and to examine its efficacy in treating corneal epithelium debridement.


METHODS
An affinity-purified, anti-human lumican antibody-conjugated protein A Sepharose column was used to purify soluble lumican protein from human AM. The purified AM lumican was characterized by two-dimensional and SDS gel electrophoresis, plus Western blot analysis with anti-lumican antibody. The effects of lumican on corneal epithelial wound healing were examined in an organ culture mouse eye model.


RESULTS
Lumican was found to be abundantly present in the stroma of human AM. It was extracted from the AM by isotonic, 1 M NaCl, and 4 M guanidine HCl solutions, suggesting that it is present in both the soluble and matrix-bound states. In two-dimensional gel electrophoresis, the 50-kDa human amniotic lumican purified by antibody-conjugated affinity chromatography migrated in a smear between pH 3.0 and 6.0. After endo-beta-galactosidase digestion, it existed as a single core protein at pH 6.0, suggesting that native human amniotic lumican is a glycoprotein with short sugar moiety. Addition of purified human AM lumican to cultured medium promoted re-epithelialization and enhanced cell proliferation of wild-type mouse corneal epithelial cells in an organ culture. In lumican-knockout (lum(-/-)) mice, the effect of human lumican on promoting corneal epithelial wound healing was even more dramatic than in wild-type mice.


CONCLUSIONS
The diversified functions of lumican include modulation of epithelial cells in wound healing and serving as an extracellular matrix component. Administration of lumican may be beneficial for treating epithelial defects in the cornea and other tissues.
Yeh
Lung-Kun
LK
Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida 33136, USA.
Chen
Wei-Li
WL
Li
Wei
W
Espana
Edgar M
EM
Ouyang
Jie
J
Kawakita
Tetsuya
T
Kao
Winston W-Y
WW
Tseng
Scheffer C G
SC
Liu
Chia-Yang
CY
D000650:Amnion / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification* / Q000494:pharmacology*; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D019573:Epithelium, Corneal / Q000187:drug effects*; D006801:Humans; D007124:Immunoenzyme Techniques; D007632:Keratan Sulfate / Q000302:isolation & purification* / Q000494:pharmacology*; D000071234:Lumican; D051379:Mice; D008810:Mice, Inbred C57BL; D012995:Solubility; D014945:Wound Healing / Q000187:drug effects*
D011508:Chondroitin Sulfate Proteoglycans; C000604306:LUM protein, human; C000604307:Lum protein, mouse; D000071234:Lumican; D007632:Keratan Sulfate