The Sialome of the Retina, Alteration in Age-Related Macular Degeneration Pathology, and Potential Impacts on Complement Factor H

Abstract

Purpose: Little is known about sialic acids of the human retina, despite their integral role in self /non-self-discrimination by complement factor H (FH), the alternative complement pathway inhibitor.

Methods: A custom sialoglycan microarray was used to characterize the sialic acid-binding specificity of native FH or recombinant molecules where IgG Fc was fused to FH domains 16 to 20 (which contains a sialic acid-binding site), domains 6 and 7 (which contains a glycosaminoglycan-binding site), or the FH-related proteins (FHRs) 1 and 3. We analyzed macular and peripheral retinal tissue from postmortem ocular globes for the amount, type, and presentation (glycosidic linkage type) of sialic acid in individuals with age-related macular degeneration (AMD) and age-matched controls using fluorescent lectins and antibodies to detect sialic acid and endogenous FH. Released sialic acids from neural retina, retinal pigmented epithelium (RPE) cells, and the Bruch's membrane (BrM) were labeled with 1,2-diamino-4,5-methylenedioxybenzene-2HCl (DMB), separated and quantified by high-performance liquid chromatography (HPLC).

Results: Both native FH and the recombinant FH domains 16 to 20 recognized Neu5Ac and Neu5Gc that is α2-3-linked to the underlying galactose. 4-O-Actylation of sialic acid and sulfation of GlcNAc did not inhibit binding. Different linkage types of sialic acid were localized at different layers of the retina. The greatest density of α2-3-sialic acid, which is the preferred ligand of FH, did not colocalize with endogenous FH. The level of sialic acids at the BrM/choroid interface of the macula and peripheral retina of individuals with AMD were significantly reduced.

Conclusions: The sialome of the human retina is altered in AMD. This may affect FH binding and, consequently, alternative complement pathway regulation.

Figure 1.

Human complement factor H (CFH) recognition of sialic acid. (A) Recombinant CFH domains 16 to 20 fused to Fc (rCFH16-20; 0.5 µg/mL) the composition of the sialogycans recognized are depicted. (B) Native CFH purified from human serum (10 µg/mL). (C) An overlay to compare the recognition of rCFH 16 to 20 and native CFH. Relative fluorescent units (RFUs). Error bars represent standard deviation of four replicate spots. For a list of all glycan compositions included on the array, see Supplementary Table S2.

Figure 2.

The distribution of different sialic acid linkages throughout the retina of individuals with and without age-related macular degeneration (AMD). (A) Hematoxylin and eosin stain (H&E). (B) SGRP3^Hsa recognition of α2-3-linked sialic acid and SNA α2-6-linked sialic acid. (C) Monoclonal antibody 735 (mAb735) binds α2-8 polysialic acid. (D) Endogenous FH detected with goat polyclonal anti-human FH detecting endogenous FH. The yellow scale bar is 100 µm. The pink arrowhead indicated drusen. Comparison of individual variation can be observed in Supplementary Figure S1.

Figure 3.

Identification and quantification of sialic acid in the human retina with and without age-related macular degeneration (AMD). (A) Representative DMB-HPLC profile. Retention time (RT) of peaks detected in the retinal samples (lower panel, black) are compared to RT of well- characterized distinct types of sialic acids present in bovine submaxillary mucins (BSM; upper panel, blue). Additionally, the released sialic acids are base treated (lower panel, red) to remove any potential O-acetyl groups, confining the modification of the sialic acid. (B) The quantity of Neu5Ac in each sample from both the peripheral retina (PR) and macula (Mac) was determined by comparisons to standards of known concentration. An unpaired t-test with Welch correction and no assumption of variance of each group, error bars represent the standard error of the mean.