Recognition roles of the carbohydrate glycotopes of human and bovine lactoferrins in lectin–N-glycan interactions

Biochimica et Biophysica Acta 1810 (2011) 139–149

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Recognition roles of the carbohydrate glycotopes of human and bovine lactoferrins in lectin–N-glycan interactions Meng-Hsiu Yen 1, Albert M. Wu ⁎, Zhangung Yang, Yu-Ping Gong, En-Tzu Chang Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, College of Medicine, Chang Gung University, Kwei-san, Tao-yuan, 333, Taiwan

a r t i c l e

i n f o

Article history: Received 12 July 2010 Received in revised form 10 October 2010 Accepted 15 October 2010 Available online 2 November 2010 Keywords: Lactoferrin Iron-binding glycoprotein N-glycans Lectin binding Carbohydrate glycotope

a b s t r a c t Background: Lactoferrin is an iron-binding protein belonging to the transferrin family. In addition to iron homeostasis, lactoferrin is also thought to have anti-microbial, anti-inflammatory, and anticancer activities. Previous studies showed that all lactoferrins are glycosylated in the human body, but the recognition roles of their carbohydrate glycotopes have not been well addressed. Methods: The roles of human and bovine lactoferrins involved in lectin–N-glycan recognition processes were analyzed by enzyme-linked lectinosorbent assay with a panel of applied and microbial lectins. Results and conclusions: Both native and asialo human/bovine lactoferrins reacted strongly with four Manspecific lectins — Concanavalia ensiformis agglutinin, Morniga M, Pisum sativum agglutinin, and Lens culinaris lectin. They also reacted well with PA-IIL, a LFucNMan-specific lectin isolated from Pseudomonas aeruginosa. Both human and bovine lactoferrins also recognized a sialic acid specific lectin-Sambucus nigra agglutinin, but not their asialo products. Both native and asialo bovine lactoferrins, but not the human ones, exhibited strong binding with a GalNAcNGal-specific lectin-Wisteria floribunda agglutinin. Human native lactoferrins and its asialo products bound well with four GalNGalNAc-specific type-2 ribosome inactivating protein family lectinsricin, abrin-a, Ricinus communis agglutinin 1, and Abrus precatorius agglutinin (APA), while the bovine ones reacted only with APA. General significance: This study provides essential knowledge regarding the different roles of bioactive sites of lactoferrins in lectin–N-glycan recognition processes. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Lactoferrin (Lf) is an iron-binding glycoprotein and belongs to a member of the transferrin family. It is present in mammalian external secretions and also in the neutrophilic granules of leukocytes [1,2]. It is a highly basic glycoprotein with a molecular weight of 80 kDa. The polypeptide chain of Lf is 690 amino acids and is folded into two globular lobes, representing its N- and C-terminal halves [3]. Ironbinding of Lf (holo-Lf) maintains a stable structure, while release of iron (apo-Lf) induces a strong conformation and stability change [4]. In addition to iron homeostasis, lactoferrins have also been found to have many other biological functions including anti-microbial [5,6],

Abbreviations: Lf, lactoferrin (hLf, human lactoferrin and bLf, bovine lactoferrin); ELLSA, enzyme-linked lectinosorbent assay; RIP, ribosome inactivating protein; II, Galβ1 → 4GlcNAc, human blood group type II precursor sequence; B, Galα1 → 3Gal, human blood group B specific disaccharide; Lex, Galβ1 → 4[LFucα1 → 3]GlcNAc, Lewis x glycotope ⁎ Corresponding author. Tel.: + 886 3 211 8966; fax: + 886 3 211 8456. E-mail address: [email protected] (A.M. Wu). 1 Present address: Department of Pediatrics, Chang Gung Memorial Hospital and Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Kwei-san, Tao-yuan, 333, Taiwan. 0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2010.10.007

anti-inflammatory activities [7], and protecting hosts from cancer development and metastasis [8]. All lactoferrins have been found to be glycosylated [9]; however the role of glycosylation has not been well understood in the past. Decrease in immunogenicity and resistance to proteolysis are the known functions of glycosylation [10,11]. During the past three decades, several lines of evidence have demonstrated that additional functions of Lf might involve glycan moieties. Mammalian Lfs can act as receptors for C-type animal lectins, and glycan moieties of Lf play crucial role in the interaction processes [12–19]. Moreover, several bacterial lectins and lectin-like proteins were also reported to be receptors of mammalian Lfs [20–23]. However, little was available about the nature of lectin binding to these glycans [24]. Therefore, the recognition roles of N-glycans of both human and bovine Lfs (hLf and bLf) in lectin–glycan recognition processes were systemically analyzed by enzyme-linked lectinosorbent assay (ELLSA) [25,26] with a panel of applied and microbial lectins. The results indicate that both native and asialo-hLf and bLf reacted strongly with four Man-specific lectins — Concanavalia ensiformis agglutinin (Con A), Morniga M, Pisum sativum agglutinin (PSA), Lens culinaris lectin (Lentil) [27–29], and a LFucNMan-specific PA-IIL prepared from soluble fraction of Pseudomonas aeruginosa [30], and a sialic acid specific lectinSambucus nigra agglutinin (SNA) [31,32], indicating that the

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oligomannose complex can also be receptors for Man-specific microbial and animal lectins, and sialic acid residue for sialic acid specific lectins. Both native and asialo-bLf exhibited strong binding with a GalNAcNGal-specific lectin-Wisteria floribunda agglutinin (WFA) [28], but the human ones exhibited weak binding. Human Lf reacted well with four type-2 ribosome inactivating protein (type 2 RIP) family lectins-ricin [33], abrin-a [34], Ricinus communis agglutinin 1 (RCA1) [35], and Abrus precatorius agglutinin (APA) [36], while bovine ones reacted only with APA [36]. The results of this study provide a basic understanding of the roles of glycotopes in N-glycans of lactoferrins in lectin-carbohydrates recognition processes. 2. Materials and methods 2.1. Lactoferrins and glycoproteins Human and bovine lactoferrins (hLf and bLf) were purchased from Sigma-Aldrich Co. (USA). Ovine submaxillary mucin (OSM) was purified according to the method of Tettamanti and Pigman [37–39]. Hog gastric mucin#4 was isolated from crude hog gastric mucin as described previously [40]. Treatment of mucin#4 with HCl (pH 1.5, 100 °C, 5 h) results in hog gastric mucin#21 [41]. All other glycoproteins and sugar inhibitors were purchased from either Sigma-Aldrich Co. (USA) or Dextra (UK). 2.2. Desialylation of sialoglycoproteins Desialylation of Lfs and other glycoproteins was performed by mild acid hydrolysis in 0.01 N HCl at 80 °C for 90 min, followed by extensive dialysis against distilled H2O for 2 days to remove small fragments [37]. 2.3. Lectins and biotin labeling GalNAc-specific Helix pomatia agglutinin (HPA), Dolichos biflorus agglutinin (DBA) and Maclura pomifera agglutinin (MPA) were purified

by adsorption to insoluble polyleucyl hog gastric (A+ H) mucin and eluted with either melibiose or GalNAc, as described earlier [42–44]. LFuc-specific Ralstonia solanacearum lectin (RSL) was purified from extracts of the bacterium R. solanacearum (ATCC 11696), purchased from the American Type Cultural Collection [45]. LFuc-specific Pseudomonas aeruginosa lectin (PA-IIL) was purified from P. aeruginosa (ATCC 33347) cell extracts as previously described [30]. Man-specific Morniga M and GalNAc-specific Morniga G were purified from Morus nigra bark by a combination of affinity chromatography and ion exchange chromatography as previously described [29,46]. Man-specific Pisum sativum agglutinin (PSA) [28] and Lens culinaris lectin (Lentil, LCL) [28], chitin-specific wheat germ agglutinin (Triticum vulgaris agglutinin, WGA) [28], LFuc-specific Anguilla anguilla agglutinin (AAA) [47], GalNGalNAc-specific ricin (Ricinus communis toxin) [33], Ricinus communis agglutinin 1 (RCA1, RCA120) [35] and peanut agglutinin (Arachis hypogaea agglutinin, PNA) [41], GalNAcNGal-specific Vicia villosa isolectin B4 (VVL-B4) [28], Agaricus bisporus agglutinin (ABA) [48], Artocarpus lakoocha agglutinin (ALA) [49], Codium fragile subspecies tomentosoides agglutinin (CFT) [50,51], Griffonia simplicifolia lectin-I isolectin A4 (GSI-A4) [52], Artocarpus integrifolia lectin (jacalin, AIA) [53], and Psophocarpus tetragonolobus agglutinin (PTA) [28] were purchased from Sigma-Aldrich Co. (USA). Man-specific Concanavalia ensiformis agglutinin (Con A), chitin-specific Datura stramonium lectin (DSL), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) [27,28], GalNGalNAc-specific Griffonia simplicifolia lectin-I isolectin B4 (GSI-B4) [54], GalNAcNGal-specific Soy bean agglutinin (SBA, Glycine max agglutinin) [28], Amaranthus caudatus lectin (ACL) [41], Wisteria floribunda agglutinin (WFA), and sialic acid specific Sambucus nigra agglutinin (SNA) were purchased from Vector Laboratories (USA). Abrus precatorius agglutinin (APA) [36] and Abrus precatorius toxin-a (abrin-a) [34] were kindly given by Drs. L.P. Chow and J.Y. Lin, Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan. Lectins were biotin labeled with biotin-amidocaproate-N-hydroxy-succinimide ester (biotin ester; Sigma-Aldrich Co., USA) in a 2:1 (w/w) ratio as described previously [25].

Table 1 Binding intensities of human and bovine lactoferrins to Man, chitin, sialic acid and LFuc-specific lectins as analyzed by ELLSA. Panel in Fig. 1

Lectinsa

Determinantsb (carbohydrate specificities)

Amount of lectin (ng)

A. Man-specific lectins a Con A b Morniga M c PSA d Lentil

Mβ1 → 4 C Mβ1 → 4 C Mβ1 → 4 C Mβ1 → 4 C

10 20 200 200

B. Chitin-specific lectins e LEA f STA g DSL h WGA

(GlcNAcβ1 → 4)2–4 (GlcNAcβ1 → 4)2–4 (GlcNAcβ1 → 4)2–4 mGlcNAcβ1→ N mTn

100 200 100 50

C. Sialic acid specific lectin i SNA Neu5Ac-OS D. LFuc-specific lectins j PA-IIL k RSL l AAA a

ABH, Ley,a,b,x ABH, Lea,b,x H, Lea

1.5 (A405) unit (ng)

Maximum A405 absorbancec

Binding intensityc

hLf

hLf

hLf

bLf

bLf

bLf

Native

Asialo

Native

Asialo

Native

Asialo

Native

Asialo

Native

Asialo

Native

Asialo

50.0 22.0 23.0 18.0

170.0 150.0 100.0 65.0

3.5 4.0 55.0 127.0

12.5 20.0 300.0 320.0

4.4 4.6 4.4 4.4

4.3 4.4 4.3 4.4

4.5 4.4 4.3 4.1

4.3 4.4 3.6 4.3

5+ 5+ 5+ 5+

5+ 5+ 5+ 5+

5+ 5+ 5+ 5+

5+ 5+ 5+ 5+

– – – –

420.0 – – 300.0

136.0 – – 200.0

1.1 1.0 0.1 0.0

0.9 0.2 0.0 0.0

4.1 1.4 1.2 1.8

3.7 1.3 0.8 4.1

2+ 2+ − −

+ ± − −

5+ 2+ 2+ 3+

5+ 2+ + 5+

–

63.0

–

4.4

0.0

4.0

0.1

5+

–

5+

–

90.0

400.0 – –

2.2 3.9 0.0

1.9 3.7 0.0

3.6 0.2 0.0

3.1 0.5 0.1

4+ 5+ −

3+ 5+ −

5+ ± −

5+ + −

– – – –

10

45.0

200 10 12.5

125.0 38.0 –

590.0 138.0 –

– –

Abbreviation of lectins is shown in Section 2.3. Carbohydrate specificity of lectins as expressed by lectin determinants — M, the tri-mannose core structure in N-linked glycoprotein; C, GlcNAcβ1 → 4GlcNAc (chitin disaccharide); m, multivalent; Tn, GalNAcα1 → Ser/Thr; Neu5Ac-OS, Neu5Ac-terminated oligosaccharides; ABH, human blood group A, B and H active glycotopes; and Lea,b,x,y, Lewis a, b, x and y active glycotopes. c Results were interpreted according to the measured A405 after 4 h of incubation as follows: 5+ (O.D. ≥ 2.5), 4+ (2.5 N O.D. ≥ 2.0), 3+ (2.0 N O.D. ≥ 1.5), 2+ (1.5 N O.D. ≥ 1.0), 1+ (1.0 N O.D. ≥ 0.5), ± (0.5 N O.D. ≥ 0.2), and − (O.D. b 0.2). b

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Table 2 Binding intensities of human and bovine lactoferrins to Gal and GalNAc-specific lectins as analyzed by ELLSA. Panel in Fig. 2

A. Gal N GalNAc-specific lectins Strong-IIβ active lectins a b c d Weak-IIβ active lectins e f B. GalNAc N Gal-specific lectins Strong-IIβ active lectins g Weak-IIβ active lectins h

i j k l

Lectinsa

Determinantsb (carbohydrate specificities)

APA RCA1 Ricin Abrin-a

T N I/II N E N B ≫ Tn II N I N B N T ≫ Tn T N I/II, Tn II, E

GSI-B4 PNA

B N E N A ≫ II T ≫ I/II

Amount 1.5 (A405) unit (ng) Maximum A405 absorbancec Binding intensityc of lectin hLf bLf hLf bLf hLf bLf (ng) Native Asialo Native Asialo Native Asialo Native Asialo Native Asialo Native Asialo

4 5 5 25 100 50

Morniga G T/Tn, II ABA ACL AIA ALA MPA WFA SBA PTA DBA CFT GSI-A4 HPA VVL-B4

50

Tα N T N L 10 T ≫ I/II 5 T N Tn ≫ I/II 10 T N Tn 25 T, Tn 10 A (NAh), F N Tn, I/II 20 A (NAh), Tn, I/II 200 A/Ah, F, Tn N B 200 F N Ah N A N Tn 20 F, Tα N Tn N Ah 25 FNANBNINT 20 F N A (N Ah) N Tn, T 20 mTn ≫ T 5

40.0 130.0 150.0 170.0

55.0 300.0 180.0 250.0

114.0 – – –

135.0 – – –

4.3 3.8 4.2 4.2

4.3 2.3 3.9 4.3

3.4 1.2 0.4 0.9

4.1 0.2 0.3 0.8

5+ 5+ 5+ 5+

5+ 4+ 5+ 5+

5+ 2+ ± +

5+ ± ± +

– –

– –

– –

– –

0.3 0.0

0.1 0.0

0.6 0.0

1.1 0.1

± −

− −

+ −

2+ −

36.0

110.0

4.2

2.9

4.3

4.1

5+

5+

5+

5+

– – – – – 210.0 – – – – – – –

– – – – – 130.0 335.0 – – – – – –

0.1 0.0 0.0 0.0 0.2 0.2 0.1 0.4 0.1 0.0 0.0 0.0 0.0

0.1 0.0 0.0 0.0 0.1 0.7 0.0 0.1 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.1 3.5 0.4 0.3 0.1 0.0 0.0 0.0 0.0

0.2 0.1 0.0 0.0 0.2 4.3 3.1 0.5 0.2 0.1 0.0 0.0 0.0

− − − − ± ± − ± − − − − −

− − − − − + − − − − − − −

− − − − − 5+ ± ± − − − − −

± − − − ± 5+ 5+ + ± − − − −

85.0 – – – – – – – – – – – – –

300.0 – – – – – – – – – – – – –

a

Abbreviation of lectins is shown in Section 2.3. Carbohydrate specificity of lectins as expressed by lectin determinants — T, Galβ1 → 3GalNAc; Tn, GalNAcα1 → Ser/Thr; I/II, Galβ1 → 3/4GlcNAc; E, Galα1 → 4Gal; B, Galα1 → 3Gal; A, GalNAcα1 → 3Gal; L, Galβ1 → 4Glc; Ah, GalNAcα1 → 3[LFucα1 → 2]Gal; F, GalNAcα1 → 3GalNAc; and m, multivalent. c Results were interpreted according to the measured A405 after 4 h of incubation as follows: 5+ (O.D. ≥ 2.5), 4+ (2.5 N O.D. ≥ 2.0), 3+ (2.0 N O.D. ≥ 1.5), 2+ (1.5 N O.D. ≥ 1.0), 1+ (1.0 N O.D. ≥ 0.5), ± (0.5 N O.D. ≥ 0.2), and − (O.D. b 0.2). b

2.4. Microtitre plate lectin-enzyme binding assay The assay was performed according to the procedures described earlier [25,26]. The volume of each reagent solution applied to wells of the plate was 50 μl/well, and all incubations, except for coating, were performed at 20 °C. The reagents, if not indicated otherwise, were diluted with TBS containing 0.05% Tween 20 (TBS-T) and TBS-T was

also used for washing plates, in between incubations. Ninety six-well microtiter plates (Maxisorp, NUNC, Denmark) were coated with glycoproteins in 0.05 M sodium carbonate buffer (0.05 M NaHCO3/ 0.05 M Na2CO3), pH 9.6, and kept overnight at 4 °C. After washing the plate, biotin labeled lectins (as specified in Tables 1 and 2) in TBS-T were added in each well, and the plate was incubated for 30 min. The plates were carefully washed to remove free lectin, and ExtrAvidin/

Table 3 Inhibitory potencies of human and bovine lactoferrins giving 50% inhibition to the binding of Man, chitin, Gal/GalNAc and sialic acid specific lectins as analyzed by ELLSA-inhibition. Panel in Fig. 3

Lectina

Quantity giving 50% inhibition of binding (ng)b hLf Native

A. Man-specific lectins a Con A b Morniga M

N2.8 × 103 (0.0%)d N2.8 × 103 (5.5%)

B. Chitin-specific lectins c LEA d WGA

N2.8 × 103 (18.2%) N2.8 × 103 (5.1%)

C. Gal/GalNAc-specific lectins e APA f Morniga G

3.4 × 103 N2.8 × 103 (4.0%)

D. Sialic acid specific lectin g SNA

40.0

a

Mass relative potencyc bLf

Asialo 1.8 × 103 1.8 × 103

3.0 × 103 N 2.8 × 103 (5.4%)

1.0 × 102 1.2 × 103

N 1.4 × 103 (38.4%)

hLf

bLf

Native

Asialo

Native

Asialo

Native

Asialo

3.0 × 102 5.0 × 102

70.0 1.1 × 103

– –

4.4 22.2

26.7 80.0

1.1 × 102 36.3

3.0 × 103 1.9 × 103

3.7 × 102 2.7 × 102

– –

1.8 × 102 –

1.8 × 102 5.8

1.5 × 103 40.7

1.5 × 103 70.0

2.6 × 102 2.7 × 102

0.6 –

22.0 3.7

1.5 62.9

8.5 16.3

40.0

N 1.4 × 103 (30.4%)

2.0 × 103

–

2.0 × 103

–

Abbreviation of lectins is shown in Section 2.3. b The inhibitory potencies were estimated from the inhibition curve and expressed as the amount of Lfs (ng) required for 50% inhibition of binding between biotin labeled lectins and corresponding plate coated glycoproteins: Con A (5.0 ng) − asialo bovine α1 acid (50 ng); Morniga M (10 ng) − asialo bovine α1 acid (10 ng); LEA (10 ng) − hog gastric mucin#21 (10 ng); WGA (2.5 ng) − asialo ovine submaxillary mucin (200 ng); APA (0.5 ng) − asialo fetuin (60 ng); Morniga G (5.0 ng) − asialo ovine submaxillary mucin (50 ng); and SNA (5.0 ng) − porcine thyroglobulin (25 ng). c Mass relative potency = mass of monosaccharide inhibitor for 50% inhibition is taken as 1.0/mass of Lfs required for 50% inhibition. For Con A and Morniga M (Man — 8.0 × 103 and 4 × 104 ng, respectively); LEA and WGA (GlcNAc — 5.5 × 105 and 1.1 × 104 ng); APA and Morniga G (Gal — 2.2 × 103 and 4.4 × 103 ng); and SNA (Gal — 8.1 × 104 ng). d The inhibitory potencies of inactive Lfs are expressed as the maximum amount (ng) tested that yield inhibition (in parenthesis) below 50%.

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Fig. 1. Recognition profiles of Man (a–d), chitin (e–h), sialic acid (i) and LFuc (j–l) specific lectins to human and bovine lactoferrins (hLf and bLf), and their asialo derivatives by ELLSA [25,26]. The amount of biotin labeled lectins added in each well were ranged from 10.0 ng to 200.0 ng, as indicated in Table 1, in a total volume of 50 μl. Absorbance at 405 nm (A405) was recorded after 4 h of incubation.

alkaline phosphatase solution (diluted 1:10,000; Sigma-Aldrich Co.) was added to detect the specifically bound probes. After 1 h, the plates were washed to remove the free enzyme and then incubated with pnitrophenylphosphate (phosphatase substrate, 5 mg tablet; SigmaAldrich Co.) in 0.05 M carbonate buffer, pH 9.6, containing 1 mM MgCl2 (1 tablet/5 ml). The resulting absorbance was monitored at 405 nm after 4 h of incubation at 20 °C in the dark with the substratecontaining solution. For inhibition studies, serially diluted inhibitor samples were mixed with fixed amount of biotin labeled lectins (as indicated in Table 3). The control lectin sample was diluted 2-fold with TBS-T. After 30 min at 20 °C, lectin-inhibitor mixtures were subjected to binding assay, as described previously. The inhibitory activity was estimated from the inhibition curve and is expressed as the amount of inhibitor (ng per well) giving 50% inhibition of the control lectin binding. All the experiments were performed in duplicates or triplicates, and the data represents are mean values of the results. The standard deviations did not exceed 10% and in most experiments were less than 5% of the mean values. Blank wells (only buffer) gave low absorbance

values (below 0.1). The use of Tween 20 negated the need to block wells before lectin addition. 3. Results and discussion 3.1. Intensities of N-glycans of lactoferrin–lectin interactions The recognition profiles of Lfs with lectins as studied by the enzyme-linked lectinosorbent (ELLSA) and inhibition assays [25] are shown in Figs. 1–3. The binding data are expressed as amounts of Lf (ng) required for binding that corresponds to 1.5 absorbance units at 405 nm (A405) and as maximum absorbance after 4 h of incubation, and are summarized in Tables 1 and 2. The ability of lactoferrins to inhibit the binding of lectins to glycoproteins and the amounts of ligands required for 50% inhibition of the lectin–glycan interaction are listed in Table 3. 3.1.1. Lactoferrin–Man-specific lectin interactions All native/asialo-Lfs reacted strongly with all four Man-specific lectins tested (Con A, Morniga M, PSA and Lentil) [27–29], and

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Fig. 2. Recognition profiles of Gal (a–f) and GalNAc (g–l) specific lectins to human and bovine lactoferrins (hLf and bLf), and their asialo derivatives by ELLSA [25,26]. The amount of biotin labeled lectins added in each well were ranged from 4.0 ng to 200.0 ng, as indicated in Table 2, in a total volume of 50 μl. Absorbance at 405 nm (A405) was recorded after 4 h of incubation.

reached over 2.5 unit of A405 within 4 h, in which native were more reactive than asialo ones as they required more amount of asialo-Lfs to reach 1.5 unit of A405 (Fig. 1, panel a–d; Table 1). It is possible that trimannose core structure (Manα1 → 3[Manα1 → 6]Manβ1 → ; M) of both complex type and high mannose type N-glycans of Lfs should be the major recognition factors in binding. The effect of sialic acid on binding remains to be further clarified. The abilities of various Lfs to inhibit the interaction of Man-specific lectins (Con A and Morniga M) and asialo bovine α1 acid glycoprotein (mIIβ, M) [55,56] by ELLSA-inhibition assay [25] were also determined (Table 3 and Fig. 3). It is shown that most of Lfs inhibited the Con A

and Morniga M-carbohydrate interactions (Fig. 3, panels a and b; Table 3). These results further confirmed that Lf N-glycans may be the most important recognition factor for Man-specific lectins (Table 4). 3.1.2. Binding of lactoferrin N-glycans with chitin-specific lectins Among the Lfs tested with four chitin-specific lectins (LEA, STA, DSL and WGA) [28] by ELLSA [25], native and asialo-bLfs reacted strongly with LEA and asialo-bLf with WGA. They reached over 2.5 unit of A405 within 4 h (Fig. 1, panel e–h; Table 1). Native-hLf reacted moderately with LEA and STA; the others were either nonreactive or reacted negligibly (Fig. 1; Table 1). These results suggested

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Fig. 3. Inhibition profiles of Man (a and b), chitin (c and d), Gal/GalNAc (e and f) and sialic acid specific lectin (g) binding by human and bovine lactoferrins (hLf and bLf), and their asialo derivatives. The amount of biotin labeled lectins added in each well and coated glycoproteins were indicated in Table 3, in a total volume of 50 μl. Absorbance at 405 nm (A405) was recorded after 2 h of incubation. The amount (ng) of Lfs required for 50% inhibition of binding was determined and compared with corresponding most active monosaccharide inhibitors.

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Table 4 Proposed active glycotopes of human and bovine lactoferrins toward Man, LFuc, sialic acid, Gal/GalNAc and chitin-specific lectins. Lectina

Active glycotopesb hLf

bLf

M, (GlcNAcβ1 → 2)2M M N (GlcNAcβ1 → 2)2M M, (IIβ1 → 2)2M with LFucα1 → 6GlcNAc at reducing end M, (IIβ1 → 2)2M with LFucα1 → 6GlcNAc at reducing end and sialic acids in non-reducing end

M, (GlcNAcβ1 → 2)2M, oligomannose chains M, oligomannose chains N (GlcNAcβ1 → 2)2M M, (IIβ1 → 2)2M with LFucα1 → 6GlcNAc at reducing end M, (IIβ1 → 2)2M with LFucα1 → 6GlcNAc at reducing end and sialic acids in non-reducing end

LFucα1 → 6GlcNAc

PA-IIL AAA

x LFucα1 → 3GlcNAc (Le ) in terminal non-reducing end and LFucα1 → 6GlcNAc in reducing end N M x LFucα1 → 3GlcNAc (Le ), LFucα1 → 6GlcNAc, M –

LFucα1 → 6GlcNAc, M, oligomannose chains –

(C) Sialic acid specific lectin SNA

Neu5Acα2 → 6Galβ1 → 4GlcNAc

Neu5Acα2 → 6Galβ1 → 4GlcNAc

(A) Man-specific lectins Con A Morniga M Lentil PSA

(B) LFuc-specific lectins RSL

(D) Gal/GalNAc-specific lectins (i) Gal N GalNAc APA RCA1 Ricin Abrin-a GSI-B4 PNA (ii) GalNAc N Gal Morniga G ABA, ACL, AIA, ALA and MPA WFA and SBA PTA, DBA, CFT, GSI-A4, HPA and VVL-B4 (E) Chitin-specific lectins LEA and WGA STA and DSL

Exposed Exposed Exposed Exposed – –

and/or and/or and/or and/or

crypto crypto crypto crypto

polyvalent polyvalent polyvalent polyvalent

IIβ IIβ IIβ IIβ

in reducing end, M, oligomannose chains

Exposed and/or crypto polyvalent IIβ – – – Exposed Bβ –

Exposed and/or crypto polyvalent IIβ – – –

GalNAcβ1→, exposed and/or crypto polyvalent IIβ – GalNAcβ1→ –

Exposed and/or crypto polyvalent IIβ and GlcNAcβ1→ ≫ crypto C Divalent-crypto C

Exposed and/or crypto polyvalent IIβ and GlcNAcβ1→ ≫ crypto C Tetravalent-crypto C

a

Abbreviation of lectins is shown in Section 2.3. b Carbohydrate specificity of lectins as expressed by lectin determinants — Bβ, Galα1 → 3Galβ1→; C, GlcNAcβ1 → 4GlcNAc; Lex, Galβ1 → 4[LFucα1 → 3]GlcNAc; IIβ, Galβ1 → 4GlcNAcβ1→; M, Manα1 → 3[Manα1 → 6]Manβ1→; (GlcNAcβ1 → 2)2M, GlcNAcβ1 → 2Manα1 → 3[GlcNAcβ1 → 2Manα1 → 6]Manβ1→; (IIβ1 → 2)2M, Galβ1 → 4GlcNAcβ1 → 2Manα1 → 3[Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6]Manβ1→; and (IIβ1 → 2)2 Mβ1 →C, Galβ1 → 4GlcNAcβ1 → 2Manα1 → 3[Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6] Manβ1 → 4GlcNAcβ1 → 4GlcNAc.

that the effect of polyvalent GlcNAcβ1→ and GlcNAcβ1 → 4GlcNAcβ1 → (C) glycotopes of bLf are strong enough to interact with LEA and WGA, but not with STA and DSL. The overall number of GlcNAcβ1→ and GlcNAcβ1 → 4GlcNAc residues in N-glycans or hLf was insufficient to interact with the lectins (Table 4). They were also further confirmed by the inhibition assay (Fig. 3, panel c and d; Table 3). This property can be used to differentiate between bLf and hLf. 3.1.3. Roles of the sialic acid of lactoferrin in Lectin–N-glycan recognition process Removal of sialic acids is one of the most efficient methods to study the role of sialic acid in lectin-recognition process. Native-hLf and bLf reacted very strongly with sialic acid specific lectin (SNA) [31,32], and reached over 2.5 unit of A405 within 4 h whereas the asialo derivatives were non-reactive (Fig. 1, panel i; Table 1). This result indicated the importance of terminal sialic acids in Lfs N-glycans (Table 4). This was further confirmed by the strong inhibitory potency of native-Lfs; 40.0 ng of hLf and bLf were sufficient for the 50% inhibition of the lectin–glycan interaction between SNA and porcine thyroglobulin (sialyl IIβ) [57], whereas asialo-Lfs reduced the inhibitory power (Fig. 3, panel g; Table 3). 3.1.4. Interaction profiles of N-glycans of lactoferrins with three LFucspecific lectins When the Lfs tested with three LFuc-specific lectins (PA-IIL, RSL and AAA) [30,45,47] by ELLSA [25] for recognition intensities, native/

asialo-hLfs reacted best with RSL [45], and native/asialo-bLf with PAIIL [30]. They reached over 2.5 unit of A405 within 4 h, in which native were more reactive than asialo ones as they required more amount of asialo-Lfs to reach 1.5 unit of A405 (Fig. 1, panel j–k; Table 1). Native/ asialo-hLfs were reacted moderately with PA-IIL (Fig. 1, panel j) and all Lfs were non-reactive towards AAA (Fig. 1, panel l) [47]. These results demonstrated that RSL can be used to differentiate between bovine and human Lfs, and the major glycotopes for LFuc-specific lectins could be LFucα1 → 6GlcNAc at the reducing end of N-glycans for both Lfs (Table 4).

3.1.5. Recognition profiles of hLf and bLf N-glycans–Gal/GalNAc-specific lectin interactions When twenty Gal and/or GalNAc-specific lectins were tested, native/asialo-hLfs reacted strongly with four GalNGalNAc-specific lectins (ricin, abrin-a, RCA1 and APA) [33–36], and one GalNAcNGalspecific Morniga G [46] and reached over 2.5 unit of A405 within 4 h (Fig. 2, panel a–d and g; Table 2). Native/asialo-bLf reacted strongly only with one GalNGalNAc-specific lectin (APA) [36] and two GalNAcNGal-specific lectins (Morniga G and WFA) [28,46], and reached over 2.5 unit of A405 within 4 h (Fig. 2, panel a, g and i; Table 2). SBA [28] reacted strongly only with asialo-bLf (Fig. 2, panel j; Table 2). Others were non-reactive or weak with Lfs (Table 2). By mapping the intensities of interaction and the carbohydrate specificities of lectins, it can be concluded that IIβ in the complex type Nglycan of Lfs should be the major recognition factor for these lectins

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Fig. 4. Primary structures of N-linked oligosaccharides of human lactoferrin. They are complex type N-glycans with α-1,6-fucosylated GlcNAc at reducing end, and mono- and disialylated di-antennary Galβ1 → 4GlcNAcβ1 → (di-IIβ) at the non-reducing end [(i) to (iv)] [58–60]. In the non-reducing terminal IIβ, GlcNAc can be glycosylated with α-1,3-LFuc as Lex glycotope (Galβ1 → 4[LFucα1 → 3]GlcNAc) [(ii)] and Gal with α-2,6-Neu5Ac [(ii) and (iv)] [58–60].

(Table 4). This was further confirmed by inhibition assay (Fig. 3, panel e and f; Table 3). Due to the unavailability of exclusive Galα1→3Galβ1→(Bβ) and GalNAcβ1→4GlcNAcβ→glycotope specific lectins, their recognition roles were not studied here. 3.2. Biological implication of human and bovine lactoferrin glyco-moieties It has been well established that both hLf and bLf contain 6.4% and 11.2% of carbohydrates respectively with LFuc, Gal, Man, GlcNAc, Neu5Ac and GalNAc in a molar ratio of 1.8, 2.3, 3.0, 4.2, 1.1, and 0 in hLf and 0.7, 1.6, 3.0, 3.3, 0.9, and 0.8 in bLf [9,58]. hLf has two complex type Nglycan chains per molecule whereas its bovine counterpart has one to two complex type and two to three high mannose type, a total of four Nglycan chains per molecule [58]. In addition to higher sugar content, bLf is also rich in mannose residue than that of hLf (4.84% vs. 1.35%). Complex type N-glycans in both hLf and bLf are of di-antennary type sharing a common core structure mostly with α-1,6-fucosylated GlcNAc at reducing end, and contain both mono- and di-sialylated di-antennary Galβ1→ 4GlcNAcβ1→ (di-IIβ) at the non-reducing end [Fig. 4: (i) to (iv); Fig. 5: (i) and (ii)] [58–60]. These di-antennary complex type Nglycan structures are found in many other members of transferrins [9,58] and B chain of porcine thyroglobulins [57]. In the non-reducing terminal IIβ of hLf, GlcNAc can be glycosylated with α-1,3-LFuc (Galβ1 →4[LFucα1 →3]GlcNAc as Lex glycotope) [Fig. 4: (ii) to (iv)] and Gal can be sialylated by α2 →6 linkage [Fig. 4: (ii) and (iv)] [58–60]. bLf complex type N-glycan branches are more complicated. They contain Galα1→ 3 [Fig. 5: (ii)] and Neu5Acα2 →6 to the Gal of IIβdisaccharide at non-reducing ends [Fig. 5: (i) and (ii)]. An unusual glycotope, GalNAcβ1→ 4GlcNAc [Fig. 5: (iii) and (iv)] [58,61] that has not been found in humans is also present, and its biological function

remains to be characterized. The high mannose type N-glycans in bLf increased their molecular size by linear elongation or tri-antennary oligomannose formation [Fig. 5: (v) to (vii)]. They do not contain α-1,6fucosylated GlcNAc at the reducing end [58,62]. Similar high mannose type N-glycans have also been found in soybean agglutinin [63], the A chain of calf thyroglobulin [64], human IgM [65] and ovalbumin [66]. When their glycan recognition profiles are identified, their biological functions can be elucidated at the molecular and submolecular levels. Furthermore, the well-characterized Lf can be used as important reagents to analyze the binding properties of lectins. Thus the theme of this study is to establish the relationship between the recognition role of carbohydrate moieties of Lfs and the glycotopes by our established ELLSA and inhibition of ELLSA [25,26]. Studies on the differential lectin-recognition abilities of Lfs and their N-glycan structures could be of value in interpreting the different biological functions of Lfs. For example, both native and asialo-hLfs and bLfs reacted with rat hepatic lectin-1 (RHL-1) [18,19], and isolated rat hepatocyte receptor bound strongly with synthetic oligosaccharide with multivalent-IIβ (Galβ1 → 4GlcNAcβ1 →) compositions [67]. Therefore, multivalent-IIβ of complex type N-glycans of Lfs are likely to have one of the key roles in the glycan recognition process since strong polyvalent-IIβ active lectins (APA, RCA1, abrin-a, ricin and Morniga G) [33–36,46] reacted well with hLfs and bLfs (Fig. 2, panel a–d and g; Table 2). The importance of glycotopes of Lfs identified in the present study is also reflected in the differential binding abilities of Lfs to type 1 fimbriae of E. coli, which preferentially recognizes the core structure of oligomannose type N-glycopeptides [68]. Type 1 fimbriae-mediated hemagglutination of E. coli had been shown to be inhibited efficiently by bLf and its glycopeptide fragments but not by hLf [20], and it is likely due to the absence of oligomannose type N-glycans in hLf. The glycotope GalNAcβ1 → 4GlcNAcβ1 → of bLf

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147

Fig. 5. Primary structures of N-linked oligosaccharides of bovine lactoferrin. Complex type N-glycans (a) are with [(i), (ii) and (iv)] and without [(iii)] α-1,6-fucosylated GlcNAc at reducing end, contain mono- and di-sialylated di-antennary Galβ1 → 4GlcNAcβ1 → (di-IIβ) at the non-reducing end [(i) to (iv)]. In non-reducing terminals, Gal of IIβ-disaccharide (Galβ1 → 4GlcNAcβ1→) are substituted with either α-1,3-Gal [(ii)] or α-2,6-Neu5Ac [(i) and (ii)], and partly replaced by GalNAc [(iii) and (iv)] [58,61]. High mannose type Nglycans (b) contain linear to tri-antennary oligomannose chains [(v) to (vii)] [58,62].

[Fig. 5: (iii) and (iv)], which is not found in human, has to be further characterized. However, this is currently hampered by the lack of availability of the corresponding lectins and monoclonal antibodies. In conclusion, the present study indicates that N-glycans of lactoferrin react strongly with applied lectins and possess several active glycotopes including a tri-mannose core structure, an oligomannose chain (in bLf only), an (IIβ1 → 2)2M structure, LFuc and sialic acid residues. These results again suggest that N-glycans are important factors for the biological functions of lactoferrin and demonstrate that it is possible for lactoferrin to be used in analyzing binding properties of lectins. The recognition profiles mapped from this study can be used to predict the biological function of lactoferrins and provide an essential background for their applications, especially its anti-microbial and anti-inflammatory aspects. Acknowledgements This work was supported by Grants from the Chang Gung Medical Research Project (CMRP Nos. 180481 and 170442), Kwei-san, Tao-

yuan, Taiwan, and the National Science Council (NSC 97-2628-B-182002-MY3, and 97-2320-B-182-020-MY3), Taipei, Taiwan.

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