Tài liệu Báo cáo khoa học: Purification and characterization of a sialic acid specific lectin from the

Purification and characterization of a sialic acid specific lectin from

the hemolymph of the freshwater crab Paratelphusa jacquemontii

Maghil Denis, P. D. Mercy Palatty, N. Renuka Bai and S. Jeya Suriya

Department of Zoology, Holy Cross College, Rochnagar, Nagercoil Tamil Nadu, India

A naturally occurring hemagglutinin was detected in the

serum of the freshwater crab, Paratelphusa jacquemontii

(Rathbun). Hemagglutination activity with different mam￾malian erythrocytes suggested a strong affinity of the serum

agglutinin for horse and rabbit erythrocytes. The most

potent inhibitor of hemagglutination proved to be bovine

submaxillary mucin. The lectin was purified by affinity

chromatography using bovine submaxillary mucin-coupled

agarose. The molecular mass of the purified lectin was

34 kDa as determined by SDS/PAGE. The hemagglutina￾tion of purified lectin was inhibited by N-acetylneuraminic

acid but not by N-glycolylneuraminic acid, even at a

concentration of 100 mM. Bovine submaxillary mucin,

which contains mainly 9-O-acetyl- and 8,9 di-O-acety-N￾acetyl neuraminic acid was the most potent inhibitor of the

lectin. Sialidase treatment and de-O-acetylation of bovine

submaxillary mucin abolished its inhibitory capacity com￾pletely. Also, asialo-rabbit erythrocytes lost there binding

specificity towards the lectin. The findings indicated an

O-acetyl neuraminic acid specificity of the lectin.

Keywords: Paratelphusa jacquemontii; hemolymph; lectin;

sialic acid; O-acetylsialic acid.

Lectins are sugar-specific proteins with multiple combining

sites capable of agglutinating cells or precipitating glyco￾conjugates [1]. Lectins may recognize specifically the whole

sugar [2], a specific site in a sugar [3], a sequence of sugars [4]

or their glycosidic linkages [5] on cell-surface glycocon￾jugates, namely glycoproteins and glycolipids, or in bacter￾ial polysaccharides. Sialic acids are a family of sugars,

N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic

acid (NeuGc), with more than 20 derivatives, which differs

only in the acyl substitution of the C-5 amino group[6].

Most of the other sialic acids contain one or more O-acetyl

substitutions of the hydroxyl groups at C-4, C-7, or C-9.

The derivatives of sialic acid are very important constit￾uents of the cell surface. They are found at the outermost

ends of the sugar chain of animal glycoconjugate. They act

as an important component of the ligands recognized by the

lectins. Recognition can be affected by specific structural

variations and modifications of sialic acids and their linkage

to the underlying sugar [7]. The most common sialic acid

found in sialoproteins and gangliosides of human tissues is

NeuAc. Modified sialic acids are found in transformed or

neoplastic cells [8–10]. The type of sialic acid and the

glycosidic linkages with the adjacent sugars contribute to a

remarkable diversity of sialyl epitopes in the sialoconjugates

on the cell surface of neoplastic cells [11]. Human malignant

melanoma contains 9-O-acetyl-NeuAc [8,12] and in human

colon carcinoma tissues N-glycolylneuraminic acid [10,13]

as well as a2,6-linked sialic acids [11,14] were detected. Thus,

sialic acid-recognizing lectins would be of immense value in

identifying and discriminating sialic acids on the surface of

cancer cells. The sialic acid-specific lectins are also useful in

distinguishing highly pathogenic strains of bacteria [15–19].

Of the known lectins that have been purified and

characterized few bind sialic acid [20–24]. Sialic acid specific

lectins have been found in several species of crustaceans,

namely Homarus americanus [25], Macrobrachium rosen￾bergii [26], Cancer antennarius [12], Scylla serrata [24] and

Penaeus monodon [27]. The lectin from Scylla serrata

was NeuGc specific [24] and that from Cancer antennarius

was 9-O-NeuAc and 4-O-NeuAc specific [12,28]. Lectins

with defined specificity for different kinds of sialic acids and

their glycosidic linkages could form a library of potential

diagnostic tools for identifying sialyl epitopes in pathogenic

bacteria [29] and malignant tumor cells.

Here we report purification of the lectin from the

hemolymph of P. jacquemontii by affinity chromatography

on bovine submaxillary mucin (BSM) agarose. The binding

specificity of the lectin was also studied.

Experimental procedures

Materials

Polypropylene Econo Columns were purchased from Bio￾Rad. CNBr-activated Sepharose 4B, bovine submaxillary

mucin, porcine stomach mucin, bovine and porcine thyro￾globulin, fetuin, transferrin, N-acetyl mannosamine, gluco￾samine and galactosamine, lactose, glucose-6-phosphate,

sucrose, fucose, glucose, fructose, xylose, raffinose, treha￾lose, melibiose, N-glycolyl- and N-acetylneuraminic acids,

Correspondence to M. Denis, Department of Zoology, Holy Cross

College, Rochnagar, Nagercoil )629001, Tamil Nadu, India.

2Tel.: +91 98421279184, E-mail: [email protected]

Abbreviations: HA, hemagglutination; HAI, hemagglutination

inhibition; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolyl￾neuraminic acid.

(Received 15 July 2003, revised 30 August 2003,

accepted 10 September 2003)

Eur. J. Biochem. 270, 4348–4355 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03828.x