Lin H C, Chang J H, Jain S, Gabison E E, Kure T, Kato T, Fukai N, Azar D T
Department of Ophthalmology, Massachusetts Eye and Ear Infirmary and the Schepens Eye Research Institute, Boston, Massachusetts, USA.
Invest Ophthalmol Vis Sci. 2001 Oct;42(11):2517-24.
To localize endostatin and collagen type XVIII in human corneas and to characterize the enzymatic action of matrix metalloproteinases (MMPs) in the cleavage of collagen type XVIII and generation of endostatin in the cornea.
Anti-endostatin and anti-hinge antibodies were generated using peptide fragments corresponding to the endostatin region and the adjacent nonendostatin hinge region of collagen XVIII noncollagenous (NC)1 domain, respectively. Confocal immunostaining was performed to localize collagen XVIII in human corneas. SV40-immortalized corneal epithelial cells were immunoprecipitated and incubated with active MMP-1, -2, -3, -7, or -9, and Western blot analysis was performed to study collagen XVIII cleavage. Incubation with MMP-7 was performed at various concentrations (0, 2, 4, and 6 microg/ml) and time intervals (0, 1, 5, and 12 hours). Purified recombinant NC1 fragment of collagen XVIII was also digested with MMP-7, and the cleavage product was sequenced.
Collagen XVIII was immunolocalized to the human corneal epithelium, epithelial basement membrane, and Descemet membrane. Western blot analysis demonstrated a 180- to 200-kDa band corresponding to collagen XVIII. MMP-7 (but not MMP-1, -2, -3, and -9) cleaved corneal epithelium-derived collagen XVIII to generate a 28-kDa endostatin-spanning fragment in a time- and concentration-dependent fashion. MMP-7 cleaved purified recombinant 34-kDa NC1 fragment of collagen XVIII in the hinge region to generate a 28-kDa fragment.
Collagen XVIII is present in human cornea. MMP-7 cleaves the collagen XVIII NC1 domain to generate a 28-kDa fragment in the cornea.
在人角膜中定位内皮抑素和ⅩⅧ型胶原，并描述基质金属蛋白酶（MMPs）在ⅩⅧ型胶原裂解及角膜内皮抑素生成过程中的酶促作用。
分别使用与ⅩⅧ型胶原非胶原（NC）1结构域的内皮抑素区域及相邻非内皮抑素铰链区域对应的肽段，制备抗内皮抑素抗体和抗铰链抗体。进行共聚焦免疫染色以在人角膜中定位ⅩⅧ型胶原。对SV40永生化角膜上皮细胞进行免疫沉淀，并与活性MMP-1、-2、-3、-7或-9一起孵育，然后进行蛋白质印迹分析以研究ⅩⅧ型胶原的裂解情况。用不同浓度（0、2、4和6μg/ml）和时间间隔（0、1、5和12小时）的MMP-7进行孵育。纯化的重组ⅩⅧ型胶原NC1片段也用MMP-7消化，并对裂解产物进行测序。
ⅩⅧ型胶原免疫定位于人角膜上皮、上皮基底膜和Descemet膜。蛋白质印迹分析显示对应于ⅩⅧ型胶原的180至200 kDa条带。MMP-7（而非MMP-1、-2、-3和-9）以时间和浓度依赖性方式裂解角膜上皮来源的ⅩⅧ型胶原，产生一个28 kDa的跨内皮抑素片段。MMP-7在铰链区域裂解纯化的重组34 kDaⅩⅧ型胶原NC1片段，产生一个28 kDa片段。
ⅩⅧ型胶原存在于人角膜中。MMP-7裂解ⅩⅧ型胶原NC1结构域，在角膜中产生一个28 kDa片段。
