Mapping the differential distribution of proteoglycan core proteins in the adult human retina, choroid, and sclera

Abstract

Purpose: To examine the presence and distribution of proteoglycan (PG) core proteins in the adult human retina, choroid, and sclera.

Methods: Postmortem human eye tissue was dissected into Bruch's membrane/choroid complex, isolated Bruch's membrane, or neurosensory retina. PGs were extracted and partially purified by anion exchange chromatography. Trypsinized peptides were analyzed by tandem mass spectrometry and PG core proteins identified by database search. The distribution of PGs was examined by immunofluorescence microscopy on human macular tissue sections.

Results: The basement membrane PGs perlecan, agrin, and collagen-XVIII were identified in the human retina, and were present in the internal limiting membrane, blood vessel walls, and Bruch's membrane. The hyalectans versican and aggrecan were also detected. Versican was identified in Bruch's membrane, while aggrecan was distributed throughout the retina, choroid, and sclera. The cartilage link protein HAPLN1 was abundant in the interphotoreceptor matrix and sclera, while HAPLN4 (brain link protein 2) was found throughout the retina and choroid. The small leucine-rich repeat PG (SLRP) family members biglycan, decorin, fibromodulin, lumican, mimecan, opticin, and prolargin were present, with different patterns of distribution in the retina, choroid, and sclera.

Conclusions: A combination of proteomics and immunohistochemistry approaches has provided for the first time a comprehensive analysis of the presence and distribution of PG core proteins throughout the human retina, choroid, and sclera. This complements our knowledge of glycosaminoglycan chain distribution in the human eye, and has important implications for understanding the structure and functional regulation of the eye in health and disease.

Figure 1.

Localization of basement membrane proteoglycans in retina, choroid, and sclera. (A) Human ocular tissue sections (macula) were labeled (green fluorescence) using antibodies against perlecan, agrin and collagen-XVIII. The accompanying images, shown below, are where the tissue sections were not exposed to the primary antibody. Here, and in all other control experiments (see Supplementary Material and Supplementary Figs. S1–S3, http://www.iovs.org/content/53/12/7528/suppl/DC1), the sections exhibited autofluorescence in the RPE only. In all figures, the images shown are representative of three separate donors, the blue color results from DAPI staining of cell nuclei, and the white scale bar indicates 100 μm. In all figures, the following abbreviations are used: ILM, internal limiting membrane; NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner photoreceptor layer; INL, inner nuclear layer; OPL, outer photoreceptor layer; ONL, outer nuclear layer; IPM, interphotoreceptor matrix; Br M, Bruch's membrane. (B) Higher magnification images of the inner retina are shown in the lower panels. The white arrows show labeling at the ILM, and the asterisks illustrate labeling of blood vessel walls.

Figure 2.

Localization of hyalectans in retina, choroid, and sclera. (A) Human ocular tissue sections (macula) were labeled using antibodies against versican, aggrecan, and brevican. In all cases, tissue sections were digested with chondroitin ABC lyase prior to application of antibody (see Table 1). The asterisks show labeling of versican in sclera. In all figures, the white scale bar indicates 100 μm. (B) Higher magnification images of staining for versican in Bruch's membrane. The white arrow shows labeling of versican in choroidal blood vessel walls.

Figure 3.

Localization of SLRPs in retina, choroid, and sclera. Human ocular tissue sections (macula) were labeled using antibodies against prolargin, opticin, fibromodulin, biglycan, decorin, lumican, and mimecan. The white wedge symbol shows the decreasing gradient of labeling for opticin from the internal limiting membrane through the neurosensory retina toward the choroid. The asterisks show the labeling of blood cells (e.g., leukocytes and erythrocytes) in the lumen of choroidal blood vessels and in the neurosensory retina. The plus sign illustrates artifactual separation of the neurosensory retina and RPE from Bruch's membrane and choroid. In all figures, the white scale bar indicates 100 μm.

Figure 4.

Localization of CD44 and link proteins in retina, choroid, and sclera. Human ocular tissue sections (macula) were labeled using antibodies against CD44v3, HAPLN1 and HAPLN4. The white arrow shows very strong labeling of HAPLN1 in the interphotoreceptor matrix, and the asterisks show staining (HAPLN1 and HAPLN4) of leukocytes in the choroid. In all figures, the white scale bar indicates 100 μm.