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D.W. Schultz, K.A. Cox, J.M. Bradley, Y. Chen, A.M. Anderssohn, T.S. Acott; Characterization of Hemicentin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3065.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To characterize the hemicentin protein and transcripts. Previously, a glutamine to arginine mutation was found in a conserved calcium–binding epidermal growth factor (cb–EGF) domain of hemicentin in affected members of a large family with age–related macular degeneration (AMD). To begin investigating the role of this hemicentin mutation in AMD, we evaluated its localization within the retina, protein expression and sizes on gels and alternative splicing of transcripts. Methods: Four peptide antibodies to amino acids, 72–118, 1342–1382, 2654–2697, and 5572–5609 of hemicentin were generated in rabbits. Confocal immunohistochemistry was used to localize hemicentin within cryosectoins of human retina and in retinal pigment epithelial (RPE) and skin fibroblast cell cultures. Western immunoblots and immunoprecipitation were used to determine approximate protein sizes and expression. RT–PCR, DNA sequencing, and BLAST searches were used to identify and map alternative transcripts in human skin fibroblasts and RPE. Results: Western blots of cellular extracts and of immunoprecipitates from cellular extracts with all four antibodies showed a band at around 600 kDa and several smaller apparent breakdown products. Immunohistochemistry of tissue suggests the presence of hemicentin in the RPE, in Bruch's membrane, around choroidal blood vessels and microcapillaries. Immunostaining was also present at lower levels throughout the retina and choroid and more abundant in the nerve fiber layer. In cell cultures, extracellular and cell surface staining was punctate and fibrillar. Perinuclear and cytoplasmic staining was also apparent. Alternative splicing of exon 104 is present in most tissues and alternative transcription start sites are seen at low levels. Conclusions: Hemicentin is expressed in several retinal and other tissues and appears to be predominantly, but not exclusively, an extracellular protein. Alternative transcript forms are expressed in the several tissues evaluated.
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