Lectin Binding to Pseudoexfoliative Material and the Ocular Zonules

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1 Lectin Binding to Pseudoexfoliative Material and the Ocular Zonules Barbara W. Sfreeten, Sandra A. Gibson, and Zong-Yi Li Lectin staining of pseudoexfoliative material on the lens capsule, zonules, and iris showed the presence of the /?-D-galactose (1-3) galactosamine sequence, a-d-galactosamine, /J-D-galactose, #-(l-4)-d-glucosamine, and terminal sialic acid. Mono and difucosyl binding sites were also found. The likelihood that the lectins were binding to two different galactosamine-containing moieties was supported by their different disibutions in the photoreceptors. These affinities suggest a complex carbohydrate composition in pseudoexfoliative material, with both O-linked sialomucin-type and N-linked oligosaccharide chains. Invest Ophthalmol Vis Sci 27: , 1986 In the pseudoexfoliative syndrome (PSX) a newly formed fibrillar protein aggregates on most of the tissues surrounding the anterior and posterior chambers, and is often associated with glaucoma. 1 Histochemical staining of this material suggests that it contains a mixture of glycoconjugates, including PAS positive neual polysaccharide and Alcian blue positive mucosubstance. 2 Recently, lectins have been used to characterize sugar residues more specifically in a variety of glycoproteins. In a preliminary survey of ocular tissue affinities for peanut agglutinin, we noted positive binding to pseudoexfoliative material, as commented on by Weinberg and Eagle. 3 For the present study, a panel of lectins was employed to determine whether a pattern of specific binding affinities could be identified in pseudoexfoliative material on the lens capsule, zonules, and iris. Materials and Methods Sixteen surgically exacted lenses and whole eyes with pseudoexfoliative (PSX) disease were studied, and comparison made with similar non-psx surgical tissues and eye bank eyes. Ten of the PSX specimens and conol eyes were fixed in 4% neual buffered formaldehyde and embedded in paraffin. Sections were cut at 6 nm, mounted on slides, rehydrated, and rinsed in From the Departments of Ophthalmology and Pathology, State University of New York, Upstate Medical Center, Syracuse, New York. Supported by NIH Research Grant EY162. Submitted for publication: February 5, Reprint requests: Barbara W. Seeten, MD, Department of Pathology, Upstate Medical Center, 766 Irving Avenue, Syracuse, NY PBS, ph 7.4, for 15 min. Six of the PSX and conol lenses were either fixed for 2-12 hr in 4% formaldehyde and reacted directly with lectins en bloc, or embedded unfixed in OKT medium and cut on a cryostat in 8 nm sections before lectin staining. Fixation was necessary for the en bloc specimens to prevent lens swelling and rupture. Fixation also gave improved histology of the cryo-sections. No significant difference in PSX staining was noted between 4% formaldehyde or acetone brief fixation, or unfixed tissues. All sections and tissues were eated for 5 min with 3% hydrogen peroxide to inactivate endogenous peroxidase, and washed with PBS for 15 min. They were then eated with the following horseradish peroxidase-linked lectins in PBS for 3 min: peanut agglutinin (PNA), soybean agglutinin (SBA), wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Ricinus communis agglutinin I (RCA), Ulex europaeus 1 agglutinin (UEA), asparagus pea (Lotus teagonolobus) agglutinin (ASPA), Limulus polyphemus agglutinin (LPA), and concanavalin A (Con A). PNA was used in a concenation of 1:32. SBA and ASPA were applied in concenations of 1 Mg/ml, and the other lectins at 2 /ug/ml for optimal staining results. Slides were rinsed in PBS several times for 1 hr and reacted with hydrogen peroxide and diaminobenzadine for 2 min in a moist chamber in the dark. After rinsing with PBS three times for ten min each, sections were dehydrated and cover-slipped with permount. Standard inhibitors for each of the lectins were used in.2 or.3 M solutions, added to the lectins before application to the slides. Some sections were eated with neuraminidase (Vibrio cholera 1 IU/ ml) for 3 min to cleave sialic acid. No staining was obtained when lectins were omitted from the incubation solutions as conols. Asparagus pea agglutinin was obtained from Sigma Chemical Company (St. Louis, 1516 Downloaded From: on 2/13/218

2 No. 1 LECTINS, PSEUDOEXFOUATIVE MATERIAL, AND ZONULES / Srreeren er ol Fig. 1. Pseudoexfoliative aggregates on the lens capsule (c) showing binding with peanut agglutinin. A, Bushlike excrescences on the capsule (X288). B, Zonular lamella (arrow) is separating from the capsule. Paler staining zonules (Z) have dense pseudoexfoliative aggregates on them (X288). C> Vertical fibrils within a fibrogranular zone (between the arrows) are stained in the deep lens capsule (X576). D, After neuraminidasc eatment, all pseudoexfoliative material is more deeply stained. Note the density of two adjacent fibrogranular zones (arrows) (X576). B MO) and the rest of the lectins from E. Y. Laboratories (San Mateo, CA). Results Pseudoexfoliative (PSX) material occurs on the anterior and, sometimes, equatorial lens capsule, zonules, and iris, as small, bush-like aggregates. The aggregates on the lens capsule and zonules stained intensely with all of the lectins (Fig. 1A), except for concanavalin A (Con A), which gave the weakest stain (Table 1). Limulus polyphemus (LPA) was inconsistent, binding more songly to the larger aggregates: Ulex europaeus agglutinin (UEA) and asparagus pea agglutinin (ASPA) showed a predilection for staining the tips of the bushlike PSX aggregates more deeply than the cores. PSX aggregates on the pupillary margin of the iris and on the posterior surface of the iris pigment epithelium had a similar lectin profile to those on the capsule, but were generally less intensely staining. The major portion of the lens capsule was unstained, other than for a faint diffuse stain with soybean agglutinin (SBA). However, the thin superficial zonular lamella had a moderately intense reaction with most of the lectins (Fig. IB), Con A having, again, the faintest stain. Downloaded From: on 2/13/218

3 1518 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Ocrober 1986 Vol. 27 Table 1. Lectin binding to pseudoexfoliative material and neighboring tissues PSXon Lens and Zonules FG Zone in Lens Capsule PSXon Iris Lens Capsule Zonular Lamella Zonular Fibers Inner PR Segments:* Outer PNA SBA DBA RCA WGA UEA ASPA LPA Con A * Photoreceptor segments in posterior pole. Of interest was the deep layer of the mid-peripheral lens capsule, which, in PSX disease, has characteristic patches of vertical fibrillar elements and granules called the fibrogranular (FG) zone. 1 These patches share most of the known histochemical staining reactions of PSX aggregates. 2 Their pattern of lectin staining was also similar (Table 1), in that lectins with an affinity for galactosamine and galactose (PNA, SBA, DBA, and RCA) (Table 2) stained the fibrogranular zone as intensely as, or only slightly less than, the typical PSX material (Fig. 1C), as did stains for fucose-containing moieties (UEA and ASPA) (Fig. 2) and glucosamine (WGA). Binding of Con A for mannose and glucose was negative, and LPA for sialic acid was variable to negative. Preeatment of sections with neuraminidase before binding with PNA resulted in increased staining of all PSX material, raising the intensity to 5+ in most areas (Fig. ID). This change indicated the presence of some terminal sialic acid adjacent to the binding sites for PNA. Table 2. Major lectin specificities 4 ' 5 PNA (Peanut SBA (Soybean DBA (Dolichos biflorus RCA (Ricinus communis WGA (Wheat germ UEA I (Ulex europeus ASPA (Asparagus pea or lotus teagonolabus LPA (Limulus polyphemus Con A (Concanavalin A) j8-d-gal (l-3)-d-n-acetylgalactosamine a-d-n-acetylgalactosamine, a-dgalactose a-d-n-acetylgalactosamine (8-D-galactose /? (l-4)-d-n-acetylglucosamine) 2 a-l-fucose Difucosyl compounds N-acetylneuraminic acid a-d-mannose, a-d-glucose Lectin binding to the zonules was less marked than to PSX material in all instances (Table 1). The reaction of the zonules with UEA was consistently the songest among the lectins. With 5/9 lectins, the zonular lamella stained slightly more intensely than the zonules. Lectin binding to the photoreceptors (PR) served as an internal conol (Table 1), since PR-lectin affinities are fairly well documented. Our results were generally similar to those of others studying cone-rich areas in mice and primates. 6 " 8 The intensity and disibution of PR binding with the two a-galactosamine-binding lectins (SBA and DBA) were notably different from that with the galactose ( 1-3) galactosamine-binding lectin (PNA). Unlike the intense stain with PNA, the former showed negative to faintly stained PR inner segments with slightly more binding of outer segments (the reverse of PNA affinities). The conol lenses and eyes had no sign of PSX aggregates, and the aging type of lens capsular inclusions were not detectable with any of the lectins. Comparison between lectin staining of normal eye bank lenses after en bloc or cryosectioning and similar paraffin-embedded lenses showed that paraffin embedding resulted in either one grade less intensity of stain on zonules and zonular lamella, or no change for most lectins (Table 3). The affinities of ULEX and ASPA for the zonules was mildly enhanced after paraffin embedding, possibly due to unmasking of binding sites. Because of limited material, not all PSX lenses could be reacted with lectins en bloc (Fig. 3), but frozen sections were almost identical in the staining of PSX aggregates to that in paraffin sections. Staining of PRs and other tissues in the PSX and non-psx syndrome eyes differed usually by only one grade, except for a songer ASPA reaction in the PR outer segments of the limited number of PSX whole eyes. Inhibition with conol sugars was good for most of the lectins, but poor for UEA. The usual UEA inhibitor, L-fucose, was partially effective in a.3-.4 M con- Downloaded From: on 2/13/218

4 No. 1 LECTIN5. PSEUDOEXFOUATIVE MATERIAL, AND ZONULES / Srreeren er ol Fig. 2. Densely stained pseudoexfoliative aggregates on fragments of zonular lamella and zonules near the lens capsule, after reacting with Ulex europaeus (X576). cenation, and.3 M glucosamine was negative, although found positive by Rhodes 9 in this study of vieous and zonular UEA affinity. D-galactose proved an incomplete inhibitor of PNA on PSX material; D-galactosamine was almost complete. Discussion PSX material on the lens capsule shows evidence of a complex carbohydrate composition by lectin staining. N-acetylgalactosamine was indicated by intense staining with PNA and SBA, and moderate staining with DBA. PNA has a higher affinity for the /?-galacto$e (1-3) galactosamine disaccharide than for /3-D-galactose alone, 4 a characteristic supported by the pattern of sugar inhibition and the lesser staining with RCA. This js-galactose (1-3) galactosamine sequence is an important one because of its specificity, and its song association with the linkage of O-Iinked sugars to their protein core in mucin-type glycoproteins. 1 The increase in staining with PNA after the cleavage of terminal sialic acid adds further weight to the likelihood of a sialomucin component. Evidence of mucin-like substances in PSX material has also been deduced from histochemical stains. 2 While this mucin linkage is found primarily in glycoproteins, it also occurs in the proteoglycan, keratan sulfate II (skeletal type), 11 a component which also could not be ruled out in our histochemical study 2 by critical elecolyte concenation testing. Song binding of PSX material and WGA indicates the presence of j3 (1-4) N acetylglucosamine, another typical glycoprotein component, especially in N-linked glycopeptides. The presence of fucose, possibly in both the mono and difucosyl forms as suggested by staining with UEA and ASPA, would be most consistent with N-linked glycoprotein, although O-linked mucin and keratin sulfate also contain fucose. It is of interest, although presently of unknown significance, that these fucose markers stained the tips of PSX aggregates more intensely than the cores, a feature also shown by oxytalan, PAS, and disulfide staining of PSX material. 2 RCA binding is also evidence for N-linkage. It is clear that there is good evidence for both N- linked and O-linked mucin-type oligosaccharides in PSX material as suggested by our previous histochem- Table 3. Lectin binding to normal lens capsule, zonules, and photoreceptors PR Segments: Lens Capsule Zonular Lamella Zonular Fibers Inner Outer PNA SBA DBA RCA WGA UEA ASPA LPA Con A F* Pf F 2- P. F P f) P 1-1- Frozen lenses en bloc or cryosections. t Paraffin sections. Downloaded From: on 2/13/218

5 152 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / October 1986 Vol. 27 Fi. 3. En bloc staining of normal and pseudoexfoliative syndrome whole lenses with peanut agglutinin. A, Normal eye bank lens showing staining of zonular fibers and lamella (X25). B, Surgically exacted PSX disease lens. Most of the zonules and lamella are missing, but PSX aggregates are seen on the zone capsule in the v-tive (arrows), and preequatorial region (asterisk). A tlap of iris pigment epithelium adheres to-the capsule on the upper left (x25). C, Higher power of PSX aggregates from area of asterisk in B. Note fragments of free zonules thickly coated with PSX material (X235). ical study.' However, lectin binding cannot provide sufficient information to assign the identified carbohydrate moieties to a specific chain, so such inferences must be speculative. The presence of the two hexosamines, glucosamine and galactosamine, is usually taken in itself to indicate a content of both -and N-linked Downloaded From: on 2/13/218 glycoprotein, since the two do not occur together in most glycoproteins, although they do in mucin-type glycoproteins and in the aforementioned keratan sulfate. There was evidence for more than one galactosamine-containing element in PSX material, including

6 No. 1 LECTINS, PSEUDOEXFOUATIVE MATERIAL, AND ZONULES / Srreeren er ol the -galactose-galactosamine component shown by PNA and the a-galactosamine demonsated by SBA and DBA. The photoreceptors contained less of the latter component, which appeared to be slightly more plentiful in the outer disc segments. The zonules and zonular lamella had similar lectin profiles to that of PSX material, although less intensely staining in most instances, adding to the similarities noted between these micronbrillar suctures histochemically. 2 The capsular FG zone contained lectin-binding glycoconjugates of almost identical profile and intensity to the rest of PSX material, confirming its kinship to the abnormal aggregates. Lectin binding on photoreceptors resembled that reported elsewhere. 6 " 8 The songer Con A affinity reported in the mouse 6 may relate more to its presence in rods 7 than to the use of frozen sections in the study. For PSX material and zonules, the increase in Con A stain in frozen sections was within the narrow range shown by most of the other lectins, related in some degree to the thinner paraffin sections. It seems unlikely that PSX material contains a significant amount of N-linked high mannose-containing oligosaccharide for which Con A is a marker,'' even as an exactable glycolipid. The much greater () SBA affinity shown on photoreceptors by Ahmed and Rahi 8 than in our study or that of others 67 is difficult to explain. It might relate to their initial fixation of tissues in absolute methanol, but Uehara et al 7 included 85% alcohol in their fixative. Ahmed and Rahi 8 also reported the zonules and zonular lamella negative for SBA staining, whereas they were mildly positive in the present study. Besides the information provided by this study on specific sugars in PSX material, it also suggests that some of the song PSX-lectin affinities could be used in separation and purification of this material for more direct analysis. Key words: lectins, pseudoexfoliative syndrome, glycoproteins, mucin type O-linkage, proteoglyeans, zonules References 1. Dark AJ and Seeten BW: Pseudoexfoliation syndrome. In Pathobiology of Ocular Disease, Part B, Garner A and Klintworth G, editors. New York, Marcel Dekker, 1982, pp Seeten BW, Dark AJ, and Barnes CW: Pseudoexfoliative material and oxytalan fibers. Exp Eye Res 38:523, Weinberg M and Eagle R: Peanut agglutinin binding in normal and diseased human eyes. ARVO Absacts. Invest Ophthalmol Vis Sci 24 (Suppl):247, Goldstein LJ and Hayes CE: The lectins: carbohydrate-binding proteins of plants and animals. Adv Carbohyd Chem Biochem 35:127, Pereira MEA, Kisailus EC, Gruezo G, and Kabat EA: Immunochemical studies on the combining site of the blood group H specific lectin I (1) from Ulex europaeus seeds. Arch Biochem Biophys 185:18, Blanks JC and Johnson LV: Selective lectin binding of the developing mouse retina. J Comp Neurology 221:31, Uehara F, Sameshima M, Muramatsu T, and Ohba N: Localization of fluorescence-labeled lectin binding sites on photoreceptor cells of the monkey retina. Exp Eye Res 36:113, Ahmed AI and Rahi AH: Physiological and pathological significance of ocular glycoproteins. I. Studies using fluorescein labelled glycine max. Br J Ophthalmol 69:162, Rhodes RH: A comparative study of vieous-body and zonular glycoconjugates that bind to the lectin from Ulex europaeus. Histochemisy 78:349, Kornfeld R and Kornfeld S: Sucture of glycoproteins and their oligosaccharide units. In The Biochemisy of Glycoproteins and Proteoglycans, Lennarz WJ, editor. New York, Plenum Press, 198, pp Hascall VC and Hascall GK: Proteoglycans. In Cell Biology of Exacellular Maix, Hay ED, editor. New York, Plenum Press, 1981, p. 41. Downloaded From: on 2/13/218