Abstract
Purpose To analyse high-molecular-weight matrix glycoproteins in trabecular meshwork, cornea and sclera using SDS/PAGE and immuno- and lectin blotting.
Method Extracts of normal trabecular meshwork (TM), cornea and sclera were analysed under reducing conditions on SDS/ PAGE. Western blots were stained for total protein, and major high-molecular-weight components were identified by immunoblotting with antibodies to fibronectin (FN) and type VI collagen. Lectin blotting with PSA, MPA and DSA identified some of the glycoprotein glycans.
Results FN antibody bound to the 240 kDa band in TM, cornea and sclera. Type VI collagen antibody bound more strongly to one band and less so to two other bands at ~200 kDa in normal TM and to a ladder of bands in cornea and sclera. PSA and DSA bound at 240, 200 and 140 kDa in TM, cornea and sclera. MPA bound at 240, 200 and 140 kDa in TM and at 240, 200 and ~120 kDA in cornea and sclera.
Conclusions FN is a component of the band at 240 kDA in TM, cornea and sclera. Normal TM was found to contain relatively more of one of the isoforms of the α3 (VI) chain whilst cornea and sclera contained all the α3 (VI) isoforms. Complex N-linked bi/tri-antennary glycans were localised in FN and the α1, α2 and α3 (VI) chains in TM, cornea and sclera. O-linked glycans (identified by MPA binding) were located in FN and α3 (VI) chains of TM, cornea and sclera.
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This work was funded by the Guide Dogs for the Blind Association and Manchester Royal Eye Hospital Research Endowments
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Chapman, S., Ayad, S., O'Donoghue, E. et al. Glycoproteins of trabecular meshwork, cornea and sclera. Eye 12, 440–448 (1998). https://doi.org/10.1038/eye.1998.102
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DOI: https://doi.org/10.1038/eye.1998.102