Expression of the TIGR gene in the iris, ciliary body, and trabecular meshwork of the human eye.
Mutations in the the glaucoma gene GCL1A, also known as trabecular meshwork glucocorticoid response (TIGR) or myocilin (Myoc), have been shown to be associated with juvenile-onset primary open-angle glaucoma. Very little is known about the pattern of expression of the TIGR gene in human ocular tissues. In-situ hybridization experiments demonstrated the localization of TIGR mRNA in cells throughout the iris, ciliary muscle, and the filtering portion of the trabecular meshwork of normal eye donors. The expression of TIGR protein was investigated by Western blot using an epitope-directed antibody to the carboxy terminus region of TIGR. This antibody was able to distinguish a recombinant TIGR fusion protein from a truncated TIGR form containing the naturally occurring Gln(368)-->stop mutation. In tissue extracts from the iris, ciliary body, and trabecular meshwork, the antibody recognized a major protein band of 57-kDa molecular mass. Deglycosylation treatment with PNGase F, NANase II, and O-glycosidase indicated that the 57-kDa protein in these tissues was unglycosylated. In agreement with this observation, in coupled in-vitro transcription/translation systems, the 57-kDa TIGR protein was unaffected by the presence of the processing and glycosylation activities of canine pancreatic microsomal membranes. These findings support the view that the expression of TIGR mRNA in cells of the iris, ciliary body, and trabecular meshwork correlates with that of TIGR protein, and that the 57-kDa TIGR protein was unglycosylated. These results, which are in contrast with earlier reports, raise the possibility that the TIGR protein might be processed into distinct forms in a tissue-specific manner.
Huang
W
W
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Jaroszewski
J
J
Ortego
J
J
Escribano
J
J
Coca-Prados
M
M
D000328:Adult; D000368:Aged; D015153:Blotting, Western; D002924:Ciliary Body / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D007498:Iris / Q000378:metabolism*; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014129:Trabecular Meshwork / Q000378:metabolism*; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D017931:DNA Primers; D005136:Eye Proteins; D006023:Glycoproteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000099179:Myocilin