The Expression and Localization of LOC387715 and HTRA1

Abstract

Purpose:: Age-related macular degeneration (AMD) is the leading cause of blindness in the industrialized world. Recent studies have linked polymophisms within the predicted gene, LOC387715 and the promoter region of high temperature requirement factor A (HTRA1) with the development of AMD. These two susceptibility variants are in complete LD, hence it is not possible to genetically distinguish between them. This study examines the localization and expression patterns of both LOC387715 and HTRA1 in order to elucidate the potential role they may play in the pathogenesis of AMD.

Methods:: RT-PCR was used to detect the presence of LOC387715 and HTRA1 transcripts in isolated human neural retinal and human RPE mRNA . Protein was extracted from human placental tissue, human and rhesus monkey neural retinal tissue, human and rhesus monkey RPE tissue, and isolated human primary RPE cells using RIPA buffer. Standard polyacrylamide gel electrophoresis and western blot analyses were used to determine the level of expression and the approximate protein size. Immunocytochemical and immunohistochemical techniques were used to localize both LOC387715 and HTRA1 in human primary RPE cells and posterior eye sections.

Results:: Individual transcripts of both LOC387715 and HTRA1 were identified in human neural retinal tissue and human RPE tissue. Although we evaluated the possibility that they were cotranscribed as a single mRNA, we could find no evidence to support this. Western blot analyses of LOC387715 revealed major bands present at approximately 50kDa in human and rhesus monkey RPE and human placental tissue, and at 70 kDa in both human and rhesus monkey RPE, as well as in human retina. In contrast, the major forms of HTRA1 were approximately 50 and 70 kDa in all eye tissues examined. Both LOC387715 and HTRA1 appear to bind to different locations on the same filamentous structures in primary human RPE cells, perhaps on intermediate filaments or microtubules. Neither protein colocalized with actin. LOC387715 protein was localized most highly in the rods and cones of the neural retina with lower levels of staining found in the RPE and subsequent layers of the neural retina.

Conclusions:: Both LOC387715 and HTRA1 mRNA are individually expressed in neural retinal and RPE tissue of humans. Antibodies against LOC387715 and HTRA1 detected products in both human and rhesus monkey ocular tissues. Although LOC387715 and HTRA1 do not colocalize with actin, both proteins do appear to bind to different locations on the same filamentous structures.

Keywords: age-related macular degeneration • proteins encoded by disease genes • immunohistochemistry