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C.–L. Kuo, W. Li, H. He, Y. Matsumoto, S.C. G. Tseng; NC1 Domains And Intact 1 And 2 Chains of Type IV Collagen Are Masked in Corneal Stroma and in Cornea Epithelial Basement Membrane Respectively . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2715.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies believed that α1 and α2 chains of type IV collagen are not expressed or speculated that they are masked in the corneal epithelial basement membrane (BM). We underwent studies to resolve this dispute.
Human corneolimbal cross–sections were immunostained with α1, α2, α3, or α5 chain–specific type IV collagen antibodies. Sections were pretreated with different protocols including DTT (with or without subsequent alkylation by iodoacetamide (IAA)), glycine/urea, proteinase K, trypsin, and Antigen Unmasking Solution (Vector). Human central cornea, peripheral cornea, limbus, and sclera were extracted with RIPA buffer, and remaining insoluble residues were further digested with collagenase. Both water–soluble and collagenase–digested protein extracts were subjected to Western blot analysis using NC1 domain–specific α1, α2 (IV) chain antibodies.
Without any unmasking treatment, immunofluorescence staining showed that central corneal BM had α3 and α5 (IV) chains, whereas limbal BM contained α1, α2, and α5 (IV) chains and conjunctival BM contained α1 and α2 (IV) chains. Nevertheless, when sections were pre–treated with 200 mM DTT followed by IAA, α1 and α2 (IV) chains were readily detected in corneal BM, while none of the other retrieval methods could unmask such antigenicity. Western blot analyses showed NC1 domains of α1 and α2 (IV) chains, but not the intact IV chain nor triple helical topomers [(α1)2α2] after DTT reduction were detected in water–soluble protein extracts from the central cornea, the peripheral cornea, and the limbus, but barely detected in the sclera. NC1 domains of α1 and α2 (IV) chains were also present in collagenase–digested water–insoluble extracts of central cornea, peripheral cornea, and limbus but not that of sclera.
Under the normal circumstance, α1 and α2 chains of type IV collagen are present but masked in the corneal epithelial BM. Because its antigenicity can be unmasked by DTT treatment, it suggests that intra–chain disulfide bonds play a role in masking mechanism. In the corneal stroma, surprisingly, NC1 domains of α1 and α2 chains of type IV collagen, known to be anti–angiogenic, are also present and can only be unmasked by DTT treatment. Further investigation of the masking mechanism may help unravel how the corneal stroma is maintained avascular.
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