Characterization of a novel O-acetyl sialic acid specific lectin from the hemolymph of the marine crab, Atergatis integerrimus (Lamarck, 1818)

Highlights

• The marine crab, Atergatis integerrimus is the potent source of lectin.

• A novel O-acetyl sialic acid specific lectin was isolated from this crab.

• The molecular weight of the purified lectin was 216 kDa with three sub units.

• The purified lectin agglutinated various erythrocytes with greater avidity.

• It has wide application in determining sialic acid moieties on the malignant cells.

Abstract

An O-acetyl sialic acid specific lectin was purified from the hemolymph of the marine crab Atergatis integerrimus by affinity chromatography using BSM (Bovine Submaxillary Mucin) coupled to cyanogen bromide activated Sepharose 4B and biospecific adsorption using formalinized buffalo erythrocytes. The purified AiL (Atergatis integerrimus lectin) showed an 1218 fold increase in specific activity when compared to the crude hemolymph agglutinin. The lectin, on non - denaturing PAGE showed a single band of 216 kDa and when subjected to SDS - PAGE, the lectin resolved into three subunits of molecular weight 70, 72 and 74 kDa. Physico chemical characterization revealed the lectin as pH and temperature sensitive, calcium dependent and sensitive to calcium chelators. Based on the calcium dependency of the lectin, AiL could be classified as a C-type lectin. The purified lectin agglutinated buffalo erythrocytes with greater avidity and was inhibited by the glycoproteins BSM, thyroglobulin, fetuin, PSM, and sugars raffinose, trehalose, l - fucose, α - Lactose, melibiose and GluNAc suggesting the affinity of the lectin to sialic acid. Reduction in HA with asialo buffalo erythrocytes and HAI titer with desialylated BSM, confirms the sialic acid specificity of the lectin. The reduction in HAI following de - O - acetylation confirms the specificity of the lectin for O - acetyl sialic acid. FTIR analysis confirms the purified lectin as a glycoprotein with spectral bands corresponding to amide bands and saccharides. Thus this study paves way to assess the therapeutic application of this lectin that could be targeted to modified sialic acid moieties that are expressed on the malignant cells and pathogenic microbes and also deduce the crystal structure of the lectin.

Introduction

Lectins are ubiquitous glycoproteins of non - immune origin that recognize specific carbohydrate structures and agglutinate a variety of animal cells by binding to cell surface glycoproteins and glycolipids [1]. Lectin - carbohydrate interaction represents a ligand - receptor interaction that is universal in all living organisms [2] and such interactions aid in different biological roles like cellular and tissue transport of carbohydrates, glycoproteins and calcium [3], cytolytic and cytotoxic [4] and cell adhesion, migration and apoptosis [5]. They are capable of inducing cell proliferation, cell arrest or apoptosis and have been implicated in organ morphogenesis, tumor cell metastasis, leukocyte trafficking, immune response and inflammation, as well as recognition of extracellular matrix [6]. Lectins isolated from animal tissues were investigated as apoptotic agents, immunomodulatory, antiviral and anticancer drug targets [7]. Among the different classes of lectins isolated from invertebrates, sialic acid specific lectins have gained much importance owing to its function which includes induction of apoptosis, negative regulation of B cell signaling, induction of cytokine secretion [8] and inhibition of bacterial and viral sialidases by altering the immunopotency of sialoconjugates expressed on the microbial surface [9]. Sialic acids are a family of sugars with more than twenty derivatives which differs only in the acyl substitution of the C-5 amino group and O-substituted sialic acids exhibit species and tissue specific distribution [10]. O-acetylation of sialic acids may change with transformation or alteration in the environment of the cell [11] and modified sialic acids such as 9-O-acetyl sialic acid, N-glycolyl neuraminic acid and α-2,6 sialic acids have been detected in human malignant cells [12]. Among arthropods, crustaceans were found to be rich source of sialic acid specific lectins. Lectins have been purified from many brachyuran crabs that are sialic acid specific [13,14]. A 9-O-acetyl and 4-O acetyl sialic acid specific lectin was purified from the hemolymph of marine crab Cancer antennarius which can recognize the modified sugar moieties expressed on tumour cells [15]. A sialic acid specific lectin having a unique specificity for N-glycolyl neuraminic acid has been isolated from marine crab Scylla serrata [16] and multiple agglutinins were detected form S. serrata that agglutinated bacteria [17]. Lectin highly specific to O-acetyl sialic acid and mannose residues expressed on bacterial pathogens was purified from marine crab Erimacrus isenbeckii [18]. A calcium dependent lectin has been isolated from the hemolymph of marine crab Trichopeltarion nobile [19], and Atergatis ocyroe [20] and antimicrobial property of lectin has been demonstrated in marine crabs Portunus pelagicus [21,22]. Lectin that recognizes N-glycolyl neuraminic acid and exhibits antiproliferative property against cancer cells was isolated from the Korean marine crab Philyra pisum and is used as a diagnostic and an anticancer agent [23,24]. The information on isolation and characterization of sialic acid specific lectins from marine crabs is limited. The present investigation reveals purification of novel sialic acid specific lectin from marine crab A. integerrimus that can be used in targeted delivery against microbes and malignant cells.