ORIGINAL PAPER

Differential Binding to Glycotopes Among the Layers of ThreeMammalian Retinal Neurons by Man-Containing N-linkedGlycan, Ta (Galb1–3GalNAca1-), Tn (GalNAca1-Ser/Thr)and Ib/IIb (Galb1–3/4GlcNAcb-) Reactive Lectins

Wei-Chi Wu Æ Chi-Chun Lai Æ Jia-Hau Liu ÆTanuja Singh Æ Lien-Ming Li Æ Willy J. Peumans ÆEls J. M. Van Damme Æ Albert M. Wu

Accepted: 22 February 2006 / Published online: 23 May 2006

� Springer Science+Business Media, Inc. 2006

Abstract Carbohydrate structures between retinal neu-

rons and retinal pigment epithelium (RPE) play an

important role in maintaining the integrity of retinal

adhesion to underlying RPE, and in retinal detachment

pathogenesis. Since relevant knowledge is still in the

primary stage, glycotopes on the adult retina of mongrel

canines (dog), micropigs and Sprague-Dawley rats were

examined by lectino-histochemistry, using a panel of 16

different lectins. Paraffin sections of eyes were stained

with biotinylated lectins, and visualized by streptavidin-

peroxidase and diaminobenzidine staining. Mapping the

affinity profiles, it is concluded that: (i) all sections of

the retina reacted well with Morniga M, suggesting that

N-linked glycans are present in all layers of the retina;

(ii) no detectable human blood group ABH active gly-

cotopes were found among retinal layers; (iii) outer and

inner segments contained glycoconjugates rich in ligands

reacting with Ta (Galb1–3GalNAca1-Ser/Thr) and Tn

(GalNAca1-Ser/Thr) specific lectins; (iv) cone cells of

retina specifically bound peanut agglutinin (PNA), which

recognizes Ta residues and could be used as a specific

marker for these photoreceptors; (v) the retinas of rat,

dog and pig, had a similar binding profile but with dif-

ferent intensity; (vi) each retinal layer had its own

binding characteristic. This information may provide

useful background knowledge for normal retinal physi-

ology and miscellaneous retinal diseases, including reti-

nal detachment (RD) and age-related macular

degeneration (ARMD).

Keywords Carbohydrate Æ Disaccharide structural

units Æ Lectins Æ Retina

Introduction

The retina is a light-sensitive membrane lining the

internal surface of the eye that transduces light intensity

and color into electrical signals. The neurons of the

retina are divided into three layers: (i) the most external

(the outer layer) is the photoreceptor cell layer, which

includes the outer (OS) and inner (IS) segments and the

layer of photoreceptor cell bodies (outer nuclear layer,

ONL); (ii) the layer of intermediate neurons (inner

nuclear layer, INL); and (iii) the layer of ganglion cells

(GCL). The axons of ganglion cells converge to form the

nerve fiber layer (NFL) and exit the eye as the optic

nerve. The synapses are confined to two synaptic, or

plexiform layers—the outer (OPL) and inner (IPL)

plexiform layers. The photoreceptor layer of the retina is

intimately apposed to the retinal pigment epithelium

(RPE). The outer limiting membrane (OLM) is formed

by junctional complexes between cell membranes of the

W.-C. Wu Æ C.-C. Lai Æ J.-H. Liu Æ L.-M. Li

Department of Ophthalmology, Chang Gung Memorial Hospital,

Tao-yuan, Taiwan

T. Singh Æ A. M. Wu (&)

Glyco-Immunochemistry Research Laboratory, Institute of

Molecular and Cellular Biology, Chang-Gung University, 333,

Tao-yuan, Kwei-san, Taiwan

e-mail: amwu@mail.cgu.edu.tw

W. J. Peumans Æ E. J. M. Van Damme

Department of Molecular Biotechnology, Faculty of Bioscience

Engineering, Ghent University, Coupure Links 653, 9000 Gent,

Belgium

Neurochem Res (2006) 31:619–628

DOI 10.1007/s11064-006-9060-8

123

major glial cells, the Muller cells, and the inner seg-

ment of photoreceptor cells. The inner limiting

membrane (ILM) consists of a basement membrane,

which is actually a surface modification of the vitreous

body, and the expanded vitreal processes of Muller cells

[1, 2].

The retinal interphotoreceptor matrix (IPM), which

lies between the apical surfaces of the neural retina and

the RPE, surrounds the ellipsoid portion of the inner and

outer segments of photoreceptor cells. The IPM is

important in mediating the interaction between the pig-

ment epithelium and the neural retina. Carbohydrate

structures in the IPM may play an important role in the

maintenance of the integrity of retinal adhesion to its

underlying RPE structure [3]. However, related infor-

mation is still limited, especially the functional role and

distribution of glycotopes in the retina. We examined the

expression of glycotopes of the mature retina of mongrel

canine (dog), micropig and Sprague-Dawley rat by

lectino-histochemistry, using a panel of 16 different

lectins with mannose-containing N-linked, Ta (Galb1–

3GalNAca1-Ser/Thr)/Tn (GalNAca1-Ser/Thr), I/II

(Galb1–3/4GlcNAc), E (Gala1–4Gal), B (Gala1–3Gal),

A/F (GalNAca1–3Gal/GalNAc) and H (LFuca1–2Gal)

carbohydrate specificities [4, 5]. In order to exclude the

possibility that differences in binding pattern affinity

were due to non-specific interactions, a series of poly-

valent glycotopes occurring in natural glycoproteins were

used to inhibit the interactions of lectin-retinal glycans.

Materials and methods

Animals

Twenty Sprague-Dawley rats (weight range: 150–200 g),

ten mongrel canines (age: 3-year-old, weight: 20 kg) and

ten Yucatan micropigs (age range, 4–6 months old;

weight range, 9–14 kg) were used in this study. The

animals were handled in accordance with the ARVO

statement for the Use of Animal in Ophthalmic and

Vision Research. The rats, dogs, and pigs were

anesthetized with intramuscular injections of an equal

volume mixture of 2-(2,6-xylidino)-5,6-dihydro-4H-1,3-

thiazine-hydrochloride, methylparaben (Rompun; Bayer

AG, Leverkusen, Germany) and 50 mg/ml Ketamine

hydrochloride (Ketalar; Parke-Davis, Morris Plains, NJ)

of 1.5 ml/kg, 0.5 ml/kg, and 1 ml/kg, respectively. The

eyeballs were excised from the anesthetized animals.

After removal of the eyeballs, the animals were sacri-

ficed with intravenous injection of sodium chloride

(Taiwan Biotech, Tao-yuan, Taiwan).

Lectins

Morniga M was purified from Morus nigra bark by a

combination of affinity chromatography and ion exchange

chromatography as previously described [6]. Artocarpus

integrifolia agglutinin (Jacalin; L-7775), Ricinus communis

agglutinin 1 (RCA1 or RCA120; L-7886), Ricin (RCA60;

L-8508), Vicia villosa B4 (VVL-B4; L-7638), Agaricus

bisporus agglutinin (ABA; L-5640), Arachis hypogea

(PNA), wheat germ agglutinin (WGA; L-1005), Griffonia

simplicifolia lectin-I isolectin A4 (GSI-A4; L-1509),

Anguilla anguilla agglutinin (AAA) from fresh water eel,

were purchased from Sigma (St. Louis, MO, USA). Ama-

ranthus caudatus lectin (ACL; B-1255), Erythrina crista-

galli lectin (ECL; B-1145) and Griffonia simplicifolia

lectin-I isolectin B4 (GSI-B4; B-1205) were purchased

from Vector Laboratories (Burlingame, CA, USA). Macl-

ura pomifera lectin (MPL) was prepared as previously

described [7]. Abrus precatorious agglutinin (APA) and

abrin-a were kindly given by Drs. L.P. Chow and J.Y. Lin,

Institute of Biochemistry, College of Medicine, National

Taiwan University, Taipei, Taiwan. Biotinylation of lectins

were performed as described previously [8].

Immunohistochemistry

Immediately after dissection, the eyeballs were fixed

overnight at 4�C with 4% paraformaldehyde in phosphate-

buffered saline (PBS), then dehydrated and embedded in

paraffin. The retinal sections (5 lm thick) were placed on

slides that had been coated with 1% gelatin and 0.1%

chromium potassium sulfate (Sigma) in distilled water to

promote adhesion of the sections to the glass surface.

Paraffin sections of retinas were soaked in xylene to re-

move paraffin, washed with PBS and incubated in 3%

H2O2 for 10 min to block endogenous peroxidase activity.

Samples were then blocked with 1% goat serum and 1%

bovine serum albumin for 30 min after washing in PBS.

Samples were then incubated with a panel of biotinylated

lectins with a concentration of 1 lg/ml for 60 min in a

dark, humidified chamber. Control sections were prepared

by either incubating in buffer solution without lectins or

preincubating the lectins with their corresponding specific

inhibiting sugars at a concentration of 10 lg/ml. The bio-

tinylated lectins and their inhibitory sugars used in this

study are listed in Table 1. After washing with PBS,

samples were incubated with 2 lg/ml peroxidase-labeled

streptavidin (Sigma) for 1 h. The lectin binding sites were

then visualized by adding diaminobenzidine as a chromo-

gen. Hematoxylin and eosin-stained sections served as

controls to assess the histologic quality of the specimens.

The slides were dehydrated, cover slipped and examined

for the location of brown reaction product. Photos were

620 Neurochem Res (2006) 31:619–628

123

taken and printed under the same conditions so that direct

comparisons of binding intensity could be made.

The intensity of lectin binding was quantitatively graded

as: ), negative; +, weak; ++, moderate; and +++, strong.

Results

Sections stained with hematoxylin and eosin (Figs. 1–3)

revealed well-preserved retina in the retinal specimen. In

most of the eyes the retina had become detached during

processing. With some variations, the lectin-binding profile

of all specimens was similar even in different species of

mammalian retinal neurons. Table 2 summarizes the

results of lectin-carbohydrate binding in the three mam-

malian retinal neurons.

Interaction of retinal glycans with a lectin exhibiting

an exclusive specificity towards mannose-containing

N-linked glycans—Morus nigra lectin (Morniga M)

(Figs. 4–6)

In rat retina, Morniga M bound strongly to GCL, moder-

ately to RPE, photoreceptor OS and IS, ONL, OPL, INL,

IPL, NFL, and blood vessel walls. Morniga M bound,

weakly to ILM. In the retina of dog and pig, the stain

showed strong binding to the OS of photoreceptor cell

layer, ganglion cells, and blood vessel walls, and moderate

binding to RPE, photoreceptor IS, ONL, OPL, INL, IPL,

NFL, and ILM. There was no obvious difference in the

binding pattern of Morniga M among the retina of rat, dog,

and pig, implying the presence of N-linked glycans in all

layers of the retina.

Interaction of retinal glycans with GalNAca1-Ser/Thr

(Tn) specific lectin—Vicia villosa isolectin B4

(VVL-B4) (Figs. 7–9)

In the rat retina, VVL-B4 bound moderately to the regions

of photoreceptor OS and IS. Photoreceptor OS were in-

tensely labeled in the retinas of dog and pig. Photoreceptor

IS were moderately stained in the retina of dog, and heavily

stained in pig retina. There was no binding at other retinal

layers.

Interaction of retinal glycans with Galb1–3GalNAca1-

(Ta) and Tn specific lectins

Artocarpus integrifolia agglutinin (Jacalin) (Figs. 10–12)

The OS of the photoreceptor layer were moderately stained

by Jacalin in the retinas of rat and dog, and strongly stained

in the pig retina. The IS were weakly stained by Jacalin in

the three mammalian retinas. There was weak Jacalin

Table 1 Summary of the lectins used in this study [4, 5, 9, 10]

No. Lectins Sources Carbohydrate specificity

(expressed by mammalian

disaccharides)

Polyvalent glycotopes as inhibitors

1 Morniga M Morus nigra mII oligomannosyl residues >>Tri-Man oligomer > Penta-Man

oligomer ‡ Mana1–2,3,or 6 Man

Asialo bovine a1-acid gp (mII)

2 VVL-B4 Vicia villosa isolectin-B4 Tn mainly Asialo OSM (Tn)

3 Jacalin Artocarpus integrifolia T > Tn >>> I(II) Asialo PSM (Tn, Ta, Ah, H) and Asialo human

a1-acid gp (mII)

4 MPL Maclura pomifera T > Tn Asialo PSM (Tn, Ta, Ah, H)

5 ABA Agaricus bisporus T, Tn > I/II > L Asialo PSM (Tn, Ta, Ah, H) and Asialo human

a1-acid gp (mII)

6 ACL Amaranthus caudatus T, Tn > I/II Asialo PSM (Tn, Ta, Ah, H)

7 PNA Arachis hypogea T >>> I/II Asialo fetuin (T, II)

8 Ricin Ricinus communis toxin T > I/II > E > B > Tn Asialo fetuin (T, II)

9 APA Abrus precatorius T > I/II > E > B > Tn Asialo fetuin (T, II)

10 WGA Triticum vulgaris I/II Asialo human a1-acid gp (mII)

11 ECL Erythrina cristagalli mI/II, A and mII clusters,

F > A > mII >> IIAsialo human a1-acid gp (mII)

12 RCA1 Ricinus communis II > I > B > T >> Tn Asialo human a1-acid gp (mII)

13 Abrin-a Abrus precatorius toxin-a E > I/II, L Asialo Bird nest gp (II, E, Ta, Fa)

14 GSI-A4 Griffonia simplicifolia-A4 F > A > and Tn clusters > E > B > I > T >> L Cyst Beach phenol insoluble (B)

15 GSI-B4 Griffonia simplicifolia-B4 B > E Cyst Beach phenol insoluble (B)

16 AAA Anguilla anguilla Ah, Bh, H Hog gastric mucin #4 (Ah, H)

Carbohydrate specificity of lectins as expressed by mammalian disaccharide structural units – Fp, Forssman pentasaccharide (GalNAca1–

3GalNAcb1–3Gala1–4Galb1–4Glc); F, GalNAca1–3GalNAc; A, GalNAca1–3Gal; Ah, GalNAca1–3(LFuca1–2)Gal; Tn, GalNAca1-Ser/Thr;

B, Gala1–3Gal; E, Gala1–4Gal; I/II, Galb1–3/4GlcNAcb1-; L, Galb1–4Glc; T, Galb1–3GalNAc; Ta, Galb1–3GalNAca1-; mII, multivalent II

Neurochem Res (2006) 31:619–628 621

123

Table 2 Lectin binding profiles in the retinae of rat, dog, and pig

No. Lectina Specificityb RPEc OSd ISe ONLf OPLg INLh IPLi GCLj NFLk ILMl BVm

1 Morniga M MIIRat ++n ++ ++ ++ ++ ++ ++ +++o ++ +p ++

Dog ++ +++ ++ ++ ++ ++ ++ +++ ++ ++ +++

Pig ++ +++ ++ ++ ++ ++ ++ +++ ++ ++ +++

2 VVL-B4 TnRat )q ++ ++ ) ) ) ) ) ) ) )Dog ) +++ ++ ) ) ) ) ) ) ) )Pig ) +++ +++ ) ) ) ) ) ) ) )

3 Jacalin T/Tn, IIRat ) ++ + ) ) ) ) ) ) ) )Dog ) ++ + ) ) ) ) ) ) ) )Pig ) +++ + ) ) ) ) ) ) + )

4 MPL T/TnRat ) + + ) ) ) ) ) ) ) )Dog ) +++ +++ ) ) ) ) ) ) ) )Pig ) +++ +++ ) ) ) ) ) ) + )

5 ABA T/Tn, IIRat ++ ++ ++ + + + + ++ + + ++

Dog ++ +++ ++ + + + + ++ + + ++

Pig ++ +++ ++ + + + + ++ + + ++

6 ACL T, Tn >I/IIRat ) ++ ++ ) ) ) ) ) ) ) +

Dog ) +++ ++ ) ) ) ) ) ) ) +

Pig ) +++ +++ ) ) ) ) ) ) ) +

7 PNA TRat ) + + ) ) ) ) ) ) ) )Dog ) + + ) ) ) ) ) ) ) )Pig ) + + ) ) ) ) ) ) ) )

8 Ricin T, Tn, I/IIRat ) + + ) ) ) ) ) ) ) )Dog ) ++ ++ ) ) ) ) ) ) ) +

Pig ) +++ ++ ) ) ) ) ) ) ) +

9 APA T/IIRat + + + ) ) ) ) + ) ) +

Dog + +++ ++ ) ) ) ) + ) ) +

Pig + +++ ++ ) ) ) ) + ) ) +

10 WGA I/IIRat + ++ + + + + ++ ++ + + ++

Dog + +++ ++ + + + ++ ++ + + ++

Pig + +++ ++ + + + ++ ++ + + ++

11 ECL I/IIRat ) ) ) ) ) ) ) ) ) ) +

Dog ) ) ) ) ) ) ) ) ) ) +

Pig ) ) ) ) ) ) ) ) ) ) +

12 RCA1 I/IIRat ) ) ) ) ) ) ) ) ) ) ++

Dog ) ) ) ) ) ) ) ) ) ) +++

Pig ) ) ) ) ) ) ) ) ) ) +++

13 Abrin-a E, IIRat + + + ) ) ) ) ) ) ) ++

Dog + +++ +++ ) ) ) ) ) ) ) +++

Pig + +++ +++ ) ) ) ) ) ) ) +++

14 GSI-A4 F/ARat ) ) ) ) ) ) ) ) ) ) )Dog ) ) ) ) ) ) ) ) ) ) –Pig ) ) ) ) ) ) ) ) ) ) –

15 GSI-B4 BRat ) ) ) ) ) ) ) ) ) ) )Dog ) ) ) ) ) ) ) ) ) ) )

622 Neurochem Res (2006) 31:619–628

123

staining to ILM in the pig retina. There was no visible

binding in other layers of the retina.

Maclura pomifera lectin (MPL) (Figs. 13–15)

MPL bound weakly to the OS and IS of photoreceptor

layer of rat retina. However, in the retinas of dog and

pig, MPL showed strong binding at the photoreceptor OS

and IS. There was weak MPL staining at the ILM in the

pig retina. No reactions were found by MPL to other

retinal layers of rat, dog, or pig was found.

Agaricus bisporus agglutinin (ABA) (Figs. 16–18)

Photoreceptor OS and IS were moderately stained by

ABA in rat retina. In the retinas of dog and pig, pho-

toreceptor OS were heavily stained and photoreceptor IS

were moderately stained by ABA. Agaricus bisporus

Table 2 continued

No. Lectina Specificityb RPEc OSd ISe ONLf OPLg INLh IPLi GCLj NFLk ILMl BVm

Pig ) ) ) ) ) ) ) ) ) ) )16 AAA ABH

Rat ) ) ) ) ) ) ) ) ) ) )Dog ) ) ) ) ) ) ) ) ) ) )Pig ) ) ) ) ) ) ) ) ) ) )

a,bAbbreviations of lectins and glycotope are shown in Table 1cRPE=retinal pigment epithelium; dOS=outer segment; eIS=inner segment; fONL=outer nuclear layer; gOPL=outer plexiform layer; hINL=inner

nuclear layer; iIPL=inner plexiform layer; jGCL=ganglion cell layer; kNFL= nerve fiber layer; lILM=internal limiting layer; mBV=blood vessels

Lectin staining intensity: n++, moderate; o+++, strong binding; p+, weak; q), invisible

Figs. 1–15 (1–3) These photos reveal well-preserved retina in

hematoxylin and eosin stain retinal specimen in rat, dog, and pig.

(4–6) Morniga M, a mannose-specific lectin from mulberry tree

(Morus nigra), labels all layers of retina. There is no apparent

difference in the binding pattern of Morniga M among the retinas of

rat, dog, and pig. (7–9) Vicia villosa isolectin-B4 (VVL-B4) intensely

labels photoreceptor outer segments. Photoreceptor inner segments

react with VVL-B4 moderately in the retinas of rat and dog, and

intensely in the retina of pig. (10–12) The outer segments of

photoreceptor layer are moderately stained by Jacalin in the retina of

rat and pig, and strongly stained in pig retina. The inner segments are

weakly stained by Jacalin in the three mammalian retinas. The arrow

in figure 12 indicates weak Jacalin staining to internal limiting

membrane in pig retina. (13–15) Maclura pomifera lectin (MPL)

binds weakly to the outer segments and inner segments of

photoreceptors in rat retina and binds strongly to the outer segments

and inner segments of photoreceptor layer in dog and pig retina. The

internal limiting membrane of pig retina is also weakly stained by

MPL (arrow). Scale bar in figure 15 applies to all figures. Scale

bar=50 lm

Neurochem Res (2006) 31:619–628 623

123

agglutinin labeled RPE, ganglion cells, and blood vessels

moderately. It also weakly labeled cells of the ONL,

OPL, INL, IPL, NFL, and ILM. There was no clear

difference in the binding pattern of ABA among the

retina of rat, dog, and pig.

Amaranthus caudatus lectin (ACL) (Figs. 19–21)

ACL exhibited moderate staining in the OS and IS of the

photoreceptor layer in the rat retina. In the dog retina, ACL

showed strong binding to photoreceptor OS, and moderate

binding to photoreceptor IS. However, ACL bound

strongly to photoreceptor OS and IS in the retina of pig.

ACL also weakly stained the blood vessel wall of retina in

these mammalian retinas. There was no staining in other

retinal layers.

Interaction of retinal glycans with Galb1–3GalNAca1-(Ta)

specific lectin—peanut agglutinin (PNA) (Figs. 22–24)

The PNA lectin preferentially bound to the region of cone

IS and OS. The binding pattern was basically the same in

the retina of rat, dog, and pig. There was no binding of

PNA in the other layers of the retina.

Interaction of retinal glycans with Galb1–3GalNAca1-

(Ta) and Galb1–4GlcNAcb1-(II) specific lectins

Ricinus communis toxin (Ricin) (Figs. 25–27)

Ricin stained weakly photoreceptor IS and OS in the rat

retina. In canine retina, there was moderate staining both in

the region of the photoreceptor OS and IS. In pig retina,

there was strong binding in photoreceptor OS, and mod-

erate staining at the IS region. Ricin labeled weakly blood

vessel walls in the retinas of dog and pig. However, no

Ricin binding to other layers of retina was found.

Abrus precatorius agglutinin (APA) (Figs. 28–30)

APA reacted weakly with RPE, photoreceptor OS and IS in

rat retina. APA also weakly labeled ganglion cell bodies,

and blood vessels in rat retina. There was stronger labeling

in the photoreceptor IS and OS in dog and pig retinas. RPE,

Figs. 16–30 (16–18) Agaricus bisporus agglutinin (ABA) binds to

retinal pigment epithelium, the outer segments and inner segments of

the photoreceptor layer, outer nuclear layer, outer plexiform layer,

inner nuclear layer, inner plexiform layer, ganglion cells layer, blood

vessel walls, nerve fiber layer, and internal limiting membrane. (19–

21) Amaranthus caudatus lectin (ACL) binds preferentially to

photoreceptor outer and inner segments. ACL also faintly stains

blood vessel wall (arrows). (22–24) Peanut agglutinin (PNA)

preferentially binds to the region of cone inner and outer segment

in the retinas of rat, dog, and pig (arrows). (25–27) Ricinus communis

toxin (Ricin) stains photoreceptor inner and outer segments. Ricin

also stains blood vessel walls weakly in the retinas of dog and pig

(arrows). (28–30) Abrus precatorius agglutinin (APA) reacts with

retinal pigment epithelium, photoreceptor inner and outer segments,

ganglion cells (arrows), and blood vessels (asterisk) in the three

mammalian retinas. Scale bar in figure 30 applies to all figures. Scale

bar=50 lm

624 Neurochem Res (2006) 31:619–628

123

ganglion cell bodies, and vascular endothelium cells were

also weakly labeled by APA in dog and pig retinas.

Interaction of retinal glycans with Galb1–3/

4GlcNAcb1-(I/II) specific lectins

Wheat germ agglutinin (WGA) (Figs. 31–33)

WGA bound strongly to the OS region of photoreceptor

cells, and moderately to IPL, ganglion cell bodies, and

blood vessels. WGA also bound weakly to RPE, ONL,

OPL, INL, NFL, and ILM. Except for weaker WGA binding

of photoreceptor OS and IS, the binding pattern of WGA in

rat retina was similar to dog and pig retina.

Erythrina cristagalli lectin (ECL) (Figs. 34–36)

ECL reacted weakly with blood vessel walls in the three

mammalian retinas. There was no ECL binding in other

layers of the retina.

Ricinus communis agglutinin (RCA1) (Figs. 37–39)

Retinal vessels were moderately stained by RCA1 in rat,

and intensely stained in dog and pig. There was no staining

in other structures of the retinas.

Interaction of retinal glycans with Gala1–4Gal (E)

and II specific lectin—Abrus precatorius toxin-a

(Abrin-a) (Figs. 40–42)

Abrin-a weakly labeled RPE and photoreceptor OS and IS,

and moderately labeled blood vessels walls of rat retina. In

the retinas of dog and pig, Abrin-a weakly labeled RPE, but

strongly labeled photoreceptor IS and OS and blood vessels

walls. The binding pattern of Abrin-a among rat, dog, and

pig was quite similar.

Interaction of retinal glycans with GalNAca1–3Gal/

GalNAc (A/F) specific lectin-Griffonia simplicifolia

I-A4 (GSI-A4), Gala1–3Gal (B) specific lectin-G.

simplicifolia I-B4 (GSI-B4) and LFuca1–2Gal (H)

specific lectin-Anguilla anguilla agglutinin (AAA)

GSI-A4 labeled blood vessel walls weakly to none in the

retinas of dog, pig and other layers of the three mammalian

retinas. Similarly, GSI-B4 and AAA did not react with any

structures of the three mammalian retinas (figure not

shown).

Inhibition of lectin–retinal glycan interactions

by glycoproteins and monosaccharides

To confirm that the observed binding of the lectins was not

due to non-specific adsorption or simply tissue entrapment

of a smaller molecular weight lectin, various glycoproteins

with known polyvalent carbohydrate structures and termi-

nal glycotopes were used to block lectin–carbohydrate

interactions. With all of the lectins, binding was totally

inhibited or markedly reduced (WGA) by polyvalent gly-

coproteins. For instance, in the case of Galb1–3GalNAca1

(T)-specific lectin ACL, the addition of T/Tn-containing

glycoprotein, such as asialo PSM, completely abolished its

binding. Nevertheless, inhibition with glycoproteins con-

taining other glycotopes, for example blood group A+H

[GalNAca1–3(LFuca1–2)Gal]-active glycoprotein from

hog gastric mucin #4 or cyst Beach phenol insoluble (blood

group B active gp) did not diminish the binding intensity of

Figs. 31–42 (31–33) Wheat

germ agglutinin (WGA) labels

all layers of retina and the blood

vessel walls. (34–36) Erythrina

cristagalli (ECL). ECL reacted

weakly with blood vessel walls

(arrows) in the retina. (37–39)

Ricinus communis (RCA1)

heavily stains retinal vessels in

rat, dot, and pig retina (arrows).

(40–42) Abrus precatorius

toxin-a (Abrin-a) labels retinal

pigment epithelium and

photoreceptor outer and inner

segments, and blood vessel

walls (arrows) obviously in the

three mammalian retinas

Neurochem Res (2006) 31:619–628 625

123

ACL. In addition, adding monosaccharides (Gal, GalNAc,

methyl a/b-Gal) or disaccharide (Galb1–4Glc) as inhibitors

did not abolish ACL staining. These results confirmed the

specific interaction of each lectin with the carbohydrates

within the retinal tissue.

Discussion

Lectins are carbohydrate-binding proteins of non-immune

origin [11, 12]. Those lectins that can be used as tools to

study glycobiological systems are defined as ‘applied’

lectins [9, 11–15]. Many have been used to study the dis-

tribution of glycoconjugates in normal [2, 12, 14, 16–23]

and diseased retinas [24–29]. However, most of these

studies used only one animal species and focused only on

part of the retina, such as the interphotoreceptor matrix and

photoreceptors. Information on the lectin binding patterns

of other retinal layers was not well organized and limited in

scope. Moreover, interpretation of these results was based

on monosaccharides, rather than on disaccharide structural

units or polyvalent glycotopes [30, 31], which may lead to

a false-conclusion [4]. Therefore, it would be much more

relevant to use both oligosaccharide and polyvalent gly-

cotopes present in macromolecules rather than monosac-

charides to confirm carbohydrate–lectin interactions,

especially for Agaricus bisporus agglutinin (ABA), which

is not easily inhibited by monosaccharides. In this study,

three representative mammalian retinal neurons were

studied.

The possible, distinct carbohydrate sequences and their

specified distribution in retinal layers were defined in this

study. The knowledge of carbohydrates has important

implications on the future retinal biology and pathology.

Retinal detachment (RD), for example, is an important

cause of severe visual loss and is a common cause of

blindness after cataract surgery [32, 33]. The formation of

RD is due to separation of photoreceptors from the

underlying RPE. Carbohydrate structures in the junction of

retina and RPE may play valuable role in the maintenance

of the retinal adhesion to its underlying RPE structure. In

previous study, glycoconjugates are identified in IPM, of

which the major structural component is chondroitin 6-

sulfate glycosaminoglycan, a form of proteoglycan [34].

Glycans on IPM closely adhere to cone photoreceptors and

to the RPE and sustain the retinal attachment [35, 36], and

inhibition of proteoglycan synthesis in IPM results in RD

[36]. Therefore, glycoconjugates on the IPM-related

structures and their exfoliate substances which undergoes

glycosylation change during RD could be a good marker

predicting the prognosis of the disease or the mediators

facilitating the progression of this disease. In addition,

carbohydrate also plays some role during retinal develop-

ment in the embryonic stage [37], and also involves path-

ologic conditions of the eye such as age-related macular

degeneration [38], and retinoblastoma [39]. The knowledge

of the carbohydrate in the retina may help elucidate the

pathogenesis of those diseases.

Our study finds retinal binding patterns of lectins with

similar specificities are not completely identical. For

instance, Jacalin and Peanut lectin have similar carbo-

hydrate specificities, but they show different binding

patterns in retina. One possible explanation is that Jac-

alin binds more strongly with T/Tn glycotopes present in

glycoproteins, while PNA shows high affinity for T and

II antigens. Moreover, for the proportional contributions

of individual residues of T disaccharides (Gal and Gal-

NAc of Galb1–3GalNAc), it has been reported that Gal

> GalNAc in Peanut, but GalNAc > Gal in Jacalin [5,

40]. This could be the key reason for the differences in

binding pattern between Jacalin and Peanut.

In most cases, the concentration of lectin can affect

the binding of lectins toward small amounts and low

affinity types of glycotopes present on macromolecules

[10]. The lectin binding profile in the retina at a very

high concentration of lectin can be different from those

at a low lectin concentration (unpublished data). When

the lectin concentration was increased, binding of lectin

to small quantities and poor affinity types of glycotopes

present on the macromolecules were shown. For differ-

ential purposes, optimal amounts of lectin were selected.

Only a major or dominant carbohydrate glycotope was

demonstrated in the retinal neurons. An area without

lectin staining reflects the fact that there is no or very

little carbohydrate present in that area in comparison to

an area with strong binding. Under this condition, the

binding of lectins in the retinal neurons could be com-

pletely inhibited by polyvalent inhibitory glycotopes

present on the macromolecules, which rules out non-

specific interactions.

In conclusion, our study demonstrates that: (i) the retina

of rat, dog, and pig are specifically stained by different

lectins, indicating that binding to the retinal structures

depends on the sugar-binding specificities of the different

lectins, showing the presence of different glycoconjugates

in different parts of the retina i.e. each layer had its own

binding specificity; (ii) all sections of the retinas reacted

well with Morniga M, indicating N-linked glycans are

present in all retinal layers; (iii) no detectable human blood

group ABH active glycotopes were found in the layers of

the retinas; (iv) outer and inner segments contained glyco-

conjugates rich in ligands reacting with Ta (Galb1–3Gal-

NAca1-Ser/Thr) and Tn (GalNAca1-Ser/Thr) specific

lectins; (v) the cone cells of the retina bound specifically to

peanut agglutinin (PNA) which mainly recognizes Ta

residues could be used as a specific marker for these

626 Neurochem Res (2006) 31:619–628

123

photoreceptor cells; and (vi) the binding intensity of retina

in the rat, dog and pig was similar, but with different

intensity. This information might be useful to understand

the structural and functional role of carbohydrates in the

retina of the eye and its related pathogenesis.

Acknowledgements This study was supported by grants from Na-

tional Science Council, Taiwan (NSC 94-2320-B-182-044, NSC 94-

2320-B-182-053). This work was also supported by grants from the

Chang-Gung Medical Research Project (CMRPD no. 33022, Kwei-

san, Tao-yuan, Taiwan) and grants from the Fund for Scientific

Research-Flanders (to E.J.M. Van Damme).

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