Multifunctional role of β-1, 3 glucan binding protein purified from the haemocytes of blue swimmer crab Portunus pelagicus and in vitro antibacterial activity of its reaction product

Accepted Manuscript Multifunctional role of β-1, 3 glucan binding protein purified from the haemocytes of blue swimmer crab Portunus pelagicus and in vitro antibacterial activity of its reaction product Mahalingam Anjugam, Arokiadhas Iswarya, Baskaralingam Vaseeharan PII:

S1050-4648(15)30242-4

DOI:

10.1016/j.fsi.2015.11.023

Reference:

YFSIM 3705

To appear in:

Fish and Shellfish Immunology

Received Date: 1 August 2015 Revised Date:

28 October 2015

Accepted Date: 16 November 2015

Please cite this article as: Anjugam M, Iswarya A, Vaseeharan B, Multifunctional role of β-1, 3 glucan binding protein purified from the haemocytes of blue swimmer crab Portunus pelagicus and in vitro antibacterial activity of its reaction product, Fish and Shellfish Immunology (2015), doi: 10.1016/ j.fsi.2015.11.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Multifunctional role of β-1, 3 glucan binding protein purified from the haemocytes of blue

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swimmer crab Portunus pelagicus and in vitro antibacterial activity of its reaction product

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Mahalingam Anjugam, Arokiadhas Iswarya, Baskaralingam Vaseeharan *

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Crustacean Molecular Biology and Genomics Lab, Department of Animal Health and

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Management, Alagappa University, Science Block 4th floor, Burma colony, Karaikudi-630004,

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Tamil Nadu, India.

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* Corresponding author

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Dr. B. Vaseeharan,

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Professor& Head

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Crustacean Molecular Biology and Genomics Lab,

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Department of Animal Health and Management,

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Alagappa University, Science Block 4th Floor, Burma Colony,

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Karaikudi- 630 004, Tamil Nadu, India.

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Tel: + 91 4565 225682

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Fax: + 91 4565 225202.

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E-mail: [email protected]

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ABSTRACT

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β- 1, 3 glucan binding protein (β-GBP) was isolated from the haemocytes of blue swimmer crab,

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Portunus pelagicus and purified by laminarin coupled Sephadex G-100 affinity column

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chromatography. The purified β-GBP has the molecular mass of 100 kDa, confirmed by SDS-

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PAGE. The X-ray diffraction analysis of purified β-GBP indicates the crystalline nature of the

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protein and also the presence of single peak confirming the existence of β -glucan molecule. The

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results of agglutination assay showed that the purified β-GBP had the ability to agglutinate with

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yeast cell, Saccharomyces cerevisiae and mammalian erythrocytes. β-GBP can agglutinate with

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yeast cells at the concentration of 50 µg/ml. The phagocytic and encapsulation activity of

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purified β-GBP from P. pelagicus was determined with yeast cell Saccharomyces cerevisiae and

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sepharose bead suspension respectively. This reveals that, β-GBP have the ability to detect the

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pathogen associated molecular patterns (PAMP) found on the surface of fungi and bacteria. The

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recognition of invading foreign substances and in the involvement of functional activities

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induces the activation of prophenoloxidase. This revealed that β-GBP play a major role in the

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innate immune system of crustaceans by stimulating the prophenoloxidase system. Moreover, it

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was obvious to note that β-GBP exhibited antibacterial and antibiofilm activity against Gram

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positive and Gram negative bacteria. This study concludes the functional aspects of β-GBP

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purified from P. pelagicus and its vital role in the stimulation of prophenoloxidase cascade

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during the pathogenic infection.

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Keywords: β-1, 3 glucan binding protein, Prophenoloxidase, Antibiofilm activity, Agglutination

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1. Introduction

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Practically all multicellular organisms possess various defense systems against invading

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microorganisms. These defense systems are crucial for their survival and perpetuity.

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Invertebrates, which do not have immunoglobulin, have developed unique modalities to detect

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and respond to microbial surface antigens, such as lipopolysaccharide (LPS), peptidoglycan and

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β-glucan. These surface antigens are collectively known as Pathogen associated molecular

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patterns (PAMPs). Invertebrates lack immune systems that involve antigen-antibody reactions

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and do not have an immune memory, therefore most invertebrate species show no evidence of

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acquired immunity. Hence, the protection mechanism of crustaceans depend exclusively on the

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innate immune system that is stimulated by PAMPs found on the surface of bacteria and fungi

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recognized by pattern recognition proteins (PRPs) such as β-glucan binding proteins (β-GBP),

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Gram negative binding protein (GNBP), peptidoglycan recognition proteins (PGRPs),

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lipopolysaccharide and β-glucan binding proteins (LGBP), and which in turn, elicit cellular or

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humoral effectors mechanisms to devastate invading pathogens [1]. Each PRPs can bind

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specifically to microbe/pathogen-associated molecular patterns (M/PAMPs), which are

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molecules shared by groups of related microbes [2]. Due to the lack of a true adaptive immune

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system, many invertebrates like crab rely on their innate immune system initiated by pattern

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recognition proteins or pattern recognition receptors, to combat the invading pathogens [3-5]. To

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date, the major pattern recognition proteins β-GBP, GNBPs, also known as β-1, 3-glucan

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recognition proteins (βGRPs), PGRPs, and LGBPs were characterized from various crustaceans

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[4, 6-8]. Among PRPs, β-glucan binding proteins (β-GBP) are the key molecule present in the

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innate immune system of crustaceans which can be activated by foreign invaders and plays the

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vital role in the immune system of invertebrates.

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β-GBP is the component of the crustacean immune system and it was purified and

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characterized from many invertebrates. β-GBP was formerly purified from two insects, Blaberus

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craniifer [9] and Bombyx mori [10] and then from several crustaceans including freshwater

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crayfish, Pacifastacus leniusculus and Procambarus clarkii with the molecular mass of 100 kDa

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[11]. β-GBP purified from freshwater crayfish, Astacus astacus had two bands which were

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recognized with the molecular mass of 95 kDa and 105 kDa [6]. β-GBP purified from the marine

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crustacean, Carcinus maenas is a 110-kDa protein, capable of inducing direct phagocytic

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stimulation [12]. In the same way, Penaeus californiensis plasma β-GBP was purified by using

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anti-crayfish β-GBP antibodies as a 100-kDa monomeric protein [13] that extends the proPO

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activating system. Similar molecular mass has been described for Penaeus stylirostris and

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Penaeus vannamei ß-GBP, by immobilized laminarin and low ionic strength precipitation

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respectively [14]. Such as in other crustacean β-GBPs, the protein isolated from Penaeus

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californiensis, Penaeus vannamei and Penaeus stylirostris are glycosylated with sugar residues

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containing mannose or glucose and N-acetyl glucosamine as determined by a positive reaction

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with concanavalin A and wheat germ agglutinin. We previously purified the β-GBP from the

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green tiger shrimp, Penaeus semisulcatus and Episesarma tetragonum with the molecular mass

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of 112 kDa and 100 kDa respectively [15,16].

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In the marine or freshwater habitats, crustaceans live in an environment often rich in

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different parasites and pathogens. Therefore, crustaceans must be able to mount an efficient

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defense against invading pathogenic organisms. Although the hard cuticle forms a structural and

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chemical barrier to parasites, there is still a need for an efficient internal immune defense

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network to deal with opportunistic or pathogenic microorganisms that can gain entry into the

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body cavity through wounds or during the molt. Now a day, understanding the immune system

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of crustaceans and their defense mechanisms has become a primary concern for better production

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of crustacean products. To decrease the occurrence of disease in crustacean aquaculture, great

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efforts have been done to study the innate immune system of crustaceans. Furthermore,

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crustacean β-GBP has been proved to be involved in different biological functions such as

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transport of lipids and immune functions [17]. Eventhough, the purification, characterization and

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functional analysis of β-GBP in various invertebrates have been reported in literature, the exact

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molecular functional response of β-GBP in prophenoloxidase system has not elucidated in detail.

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In addition, definite molecular size of the β-GBP protein was not reported in crustaceans. Hence,

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more studies are needed to confirm the β-GBP molecule size and its involvement in immune

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response in various crustaceans. In the present study, we purified β-GBP from the haemocytes of

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blue swimmer crab, Portunus pelagicus by laminarin coupled Sephadex G-100 column

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chromatography. The function of purified β-GBP was analyzed by yeast agglutination,

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haemagglutination, phagocytosis, encapsulation and PO enhancing activity. In addition, the

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antibacterial and antibiofilm property of β-GBP reaction products was also tested against the

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Gram positive (Bacillus cereus and Listeria monocytogens) and Gram negative (Vibrio

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parahaemolyticus and Proteus vulgaris) bacteria.

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2. Materials and Methods

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2.1. Experimental animal Fresh and live blue swimmer crab, Portunus pelagicus were collected from the coastal

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area of Thondi, Ramanathapuram district, Tamilnadu, India. The collected animals were

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carefully brought to the laboratory, maintained in FRP tanks and acclimatized for 3 days before

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the experiments. The animals were fed with shrimp and fish meat at 10% of body weight till the

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haemolymph collection.

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2.2. Haemocyte lysate supernatant preparation

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Haemolymph was withdrawn from the chelate leg using 25-guage needle attached to a 10

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ml syringe containing precooled anticoagulant solution. Then, the haemolymph was centrifuged

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at 500 x g at 4 °C for 20 min to obtain plasma [16]. The plasma was diluted with an equal

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volume of TBS-II (50 mM Tris HCl, 100 mM NaCl, 100 mM CaCl2; pH-7.5) and centrifuged at

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5000 x g for 30 min at 4 °C. The resultant haemocyte lysate supernatant (HLS) was stored at -80

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°C for further use.

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2.3. Purification of β-GBP from the haemocytes of P. pelagicus

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β-GBP was purified by affinity chromatography using laminarin coupled

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Sephadex G-100 matrix column (10 x 1.5 cm), which had been previously equilibrated with

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TBS-II. β-GBP was isolated from the haemocyte lysate supernatant by laminarin precipitation

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following the method described previously [11]. Briefly, the HLS was mixed with 10 mg of

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laminarin (Sigma), stirred (4 hrs at 4 °C) and centrifuged (25,000 x g, 15 min, 4 °C). Then, the

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mixure was dialysed (Mw exclusion limit <14,000 Da) extensively against the same buffer and

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the supernatant was passed through laminarin-Sephadex column (0.8 -x1.6 cm) at a flow rate of

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3ml h-1. The collected fractions were loaded onto polyacrylamide gel electrophoresis (SDS-

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PAGE- 12% separating gel, 4% stacking gel) [18]. The electrophoresis was performed at

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constant electricity. After electrophoresis, the gels were stained with Coomassie brilliant blue

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(CBB) R-250.

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2.4. High Performance Liquid Chromatography analysis The homogeneity of the purified P. pelagicus β-GBP was analyzed using a reversed-

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phase HPLC C18 column (Zorbax Bio-series GF-250, Du Pont, Willington, DE, USA) with a

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linear gradient between 0.05% trifluoroacetic acid in water and 0.052% trifluoroacetic acid in

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80% acetonitrile. The column is calibrated with reference proteins for molecular weight

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estimation under identical conditions.

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2.5. Circular Dichroism (CD)-Spectra analysis

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CD is defined as the difference in absorption of left and right circularly polarized light

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(Circular Dichroism (CD) Jasco J-720 spectropolarimeter). Spectral scans were performed from

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200 to 250 nm, with a step resolution of 0.1 nm and a bandwidth of 1.0 nm and at a speed of 50

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nm/min. Values from three scans were averaged per sample. A 1-mm-path-length quartz cuvette

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was used for the measurements. P. pelagicus β-GBP was measured to detect the protein

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concentration by using 30 to 40 µM in 20 mM Tris-HCl-20 mM NaCl, pH 7.4, with or without

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20 mM sodium dodecyl sulfate.

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2.6. XRD analysis

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In order to determine the spatial distribution, atomic coordinates and arrangement of

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atoms, P. pelagicus purified β-GBP was coated on clean glass slide, freeze dried and used for

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XRD analysis (XRD, Scintag-SDS 2000) at 40 kV/20mA using continuous scanning 2 theta

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modes. Average grain size and shape of the P. pelagicus purified β-GBP was determined using

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Scherrer’s formula[d=(0.9 λ/ßcos, where d is the mean diameter of P. pelagicus β-GBP, λ is the

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wavelength of the X-ray radiation source and ß is the angular FWHM of the XRD peak at the

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diffraction angle).

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2.7. Functional analysis of P. pelagicus purified β-GBP

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2.7.1. Yeast agglutination assay

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Yeast cells (Saccharomyces cerevisiae) were used to evaluate the agglutination response

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of purified β-GBP. Fifty microlitre of purified β-GBP was added to the U bottomed 96 well

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microtitre plate and a 2 fold serial dilution was prepared using Tris buffer. Equal volume of yeast

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suspension was then added to each well and incubated for 4 hrs at 25 °C in a humid chamber. To

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compare the purified β-GBP aggregation with yeast cell, 50µg of purified β-GBP and the same

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volume of laminarin were added and prepared the serial dilution as described above. In control,

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instead of purified β-GBP and laminarin, Bovine serum albumin (BSA) was added. The

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agglutination of yeast cells by β-GBP and control was monitored through inverted light

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microscopy at the magnification of 20 X.

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2.7.2. Haemagglutination assay Heparinized blood was collected from human, goat and chicken, stored at 4 °C and used

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within 4 days. The erythrocytes were washed 3 times in PBS (0.02 M K-phosphate buffer (pH

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7.4) containing 0.9% NaCl) and then resuspended in PBS to give 2% (v/v) suspensions for the

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agglutination assays. The agglutinating activity was assayed by the serial dilution method in V

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bottomed microtitre plates by adding 50 µl of the erythocyte suspension to 30 µg/ml and 50

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µg/ml of the serially diluted preparation of the P. pelagicus purified β-GBP (dialyzed against

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PBS) and PBS for the controls. The plates were left at room temperature for 24 hrs or overnight

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at 37 °C before to recording results.

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2.7.3. Phagocytosis assay

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In vitro phagocytic action of P. pelagicus purified β-GBP was ensure with yeast cell

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suspensions. Yeast cell, Saccaramyces cerevisiae (106 cells/ml) at the concentration of 50 µg/ml

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was added to 20 µg/ml of TBS-II (10 mM Tris HCl; 145 mM NaCl; 500 ml D.H2O). Then, 50

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and 100 µg/ml of P. pelagicus purified β-GBP was added to the yeast cells. After 10 min, the

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ingestion of yeast cells by P. pelagicus β-GBP was observed under light microscopy.

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2.7.4. Encapsulation assay

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Purified β-GBP was mixed with an equal volume TBS-II (Tris HCl-50 mM, NaCl-10

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mM, CaCl2), and 500 µl of double distilled water. To this mixture, sepharose bead suspension

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was added and incubated for 45 min at 23 °C with gentle mixing at 15 min interval. The entire

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volume from each suspension was spread on a glass slide and left in a moist chamber for 10 min

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at 23 °C to allow the beads to settle on the glass slide. After placing a coverslip, the samples

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were examined under light microscopy.

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2.7.5. Phenoloxidase activity of P. pelagicus purified β-GBP

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The ability of P. pelagicus β-GBP to activate the proPO system was determined

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following the method described by [19] with slight modifications. In brief, P. pelagicus purified

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β-GBP (10, 30, and 50 µg/ml) was pre-incubated with a same volume of laminarin (soluble β-1,

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3-glucan from the algae, L. digitata; 1 mg ml-1) at 25 °C for 1hr. After the formation of β-G- β-

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GBP complex, 50 µl of HLS was added and re-incubated with 5 mM CaCl2. To this mixture,

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50µl of L-DOPA (3mg ml-1) was added. The purified β-GBP and laminarin at different

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concentration were independently incubated with HLS and L-DOPA. In controls, P. pelagicus

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β-GBP and laminarin was replaced by Tris buffer. The activity of PO was measured

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spectrophotometrically at 490 nm and expressed as units/min-1/mg protein-1. Each assay was

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repeated three times at different interval periods. The dose-dependent change was observed when

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the concentration of β-GBP and laminarin was increased.

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2.8. Antibacterial activity of reaction product of P. pelagicus β-GBP

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2.8.1. Bacterial strains

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Biofilm forming Gram positive (B. cereus and L. monocytogens) and Gram negative [(V.

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parahaemolyticus (Accession. No. HQ693275.1) and P. vulgaris (Accession. No. HQ640434.1)]

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bacteria were used in the experiment to evaluate the antibacterial potential of reaction product of

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P. pelagicus β-GBP. Both the Gram positive bacteria were procured from Microbial Type

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Culture Collection (MTCC), Chandigarh, India and maintained in our laboratory, while both the

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Gram negative bacteria were used from our laboratory culture collection. For experiments, active

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cultures were prepared by transferring a loopful of culture to the test tubes containing Nutrient

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Broth (NB) incubated without agitation for 24 hrs or at 37 °C.

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Standard well diffusion assay was practiced to each culture. Muller Hinton Agar (MHA)

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plates were prepared and the inoculums (18-24 hrs old broth culture) of each bacterium were

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spread on them using sterile swabs. The well is made on the plate by cork borer. To compare the

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bactericidal activity of purified β-GBP, the experiments were performed as follows: PBS

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(Control); PBS + substrate (laminarin); PBS + β-GBP; PBS (10 µl) + substrate (10 µl) + β-GBP

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(10 µl) (reaction product). The reaction products were prepared as per the protocols reported in

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[20, 21] with minor modifications. The substances were loaded on each well at the concentration

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of 30 µg/ml. The plates were then incubated at 37 °C for 24 hrs. After 24 hrs, the zone of

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inhibition was clearly observed and measured.

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2.8.2. Antibiofilm activity of reaction product of P. pelagicus β-GBP

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For determining the effect of reaction products of P. pelagicus β-GBP on biofilm

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inhibition against the Gram positive (B. cereus and L. monocytogens) and Gram negative (V.

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parahaemolyticus and P. vulgaris) bacteria, 1.5 ml of nutrient broth was poured in each well of

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24 well polystyrene plates. Then, small sterile glass pieces (1x1cm) were placed into polystyrene

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plates and incubated at 37 °C for 48 hrs. After 2 days incubation, reaction product of P.

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pelagicus β-GBP was added to each well (50 µg/ml) and incubated the plate for another 24 hrs.

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After the incubation, glass pieces were removed and stained with acridine orange and crystal

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violet for observation under confocal laser scanning microscopy (CLSM) and light microscopy

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respectively. Then, the glass pieces were washed with acetone to remove the loosely attached

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bacteria and excess of stains and the biofilm inhibition was inspected by CLSM and light

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microscopy (NIKON ECLIPSETS 100) at magnification of 20 X.

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3. Results

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3.1. Purification of β-GBP from P. pelagicus haemocytes

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The pattern recognition molecule β-GBP was isolated from the plasma of P. pelagicus.

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The plasma (HLS) was subjected to laminarin-conjugated epoxy-activated Sephadex G-100

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matrix, and the matrix-bound β-GBP was purified by affinity chromatography. The purity of β-

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GBP in the different fractions was assessed by SDS-PAGE. The purified P. pelagicus β-GBP ran

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as a single band (Fig. 1) of approximately 100 kDa in 12% SDS-PAGE.

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3.2. HPLC and Circular Dichroism (CD)-Spectra analysis of P. pelagicus purified β-GBP

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Homogeneity of purified β-GBP was proven by HPLC analysis. Purified β-GBP shows

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sharp symmetrical peak with an absorbance of 280 nm in absorbance spectra and a peak

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retention time 3.537 of min in HPLC on a Bio-series C18 column (Fig. 2). CD scanning

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predicted the secondary structure of P. pelagicus β-GBP at the range of 200-250 nm. Signals

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were obtained between 200 and 240 nm due to peptide bond, a weak bond, but broad n-π*

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transition was centered around 215nm and a more intense π-π* transition around 210 nm. CD

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spectra of P. pelagicus β-GBP was recorded in TBS-I buffer (pH 7.5), spectrum showed a broad

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negative minimum at 210 nm and a cross-over at 225 nm. Negative ellipticity values presented at

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below 225 nm, which is the signature for the formation of a right-handed helix and the activity of

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the peptide depending on the presence of helicity (Fig. 3). The low positive ellipiticity values

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below 215 nm clearly suggest the presence of unordered segments in this protein. The broad

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negative minimum extending from 200 to 240 nm can be attributed to the presence of β-sheet

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structure.

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3.3. XRD analysis of P. pelagicus purified β-GBP An XRD analysis of the P. pelagicus purified β-GBP showed a high diffraction peak at

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31.6° which indexed the (1 1 1), planes in glucan’s hexagonal structure. The peak obtained at

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31.6° shows the purity of the protein and crystalline nature of the β-GBP. The lattice constant

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calculated from this pattern is given by a = 1.54060Å, and the data obtained are matched with

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database of Joint Committee on Powder Diffraction Standards (JCPDS) file No #492332 for ß-

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glucan molecule (Fig. 4). This result indicates that the purified β-GBP has the crystalline

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molecule glucan, due to the existence of the ligand crystalline peaks that occurred.

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3.4. Functional analysis of P. pelagicus purified β-GBP

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3.4.1. Yeast agglutination assay

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Agglutination assay revealed that P. pelagicus purified β-GBP was a βG binding protein

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and has the potential to agglutinate with fungal cells. P. pelagicus purified β-GBP effectively

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agglutinate the yeast cell, Saccharomyces cerevisiae in vitro. The maximum agglutination was

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observed in yeast cells with purified β-GBP (Fig. 5). Meanwhile, the agglutination was occurred

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gradually by the presence of β-GBP coupled with laminarin, this is due to the formation of βG-

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β-GBP complex. Extremely, there was no reaction experiential on control which is replaced by

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BSA.

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3.4.2. Haemagglutination activity

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The P. pelagicus purified β-GBP was agglutinated with mammalian and chicken RBCs.

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The highest agglutination was occurred with human and goat RBCs. Meantime, lowest

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agglutination was occurred with chicken RBCs (Fig. 6 F). When the plasma of P. pelagicus was

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added to human and goat RBCs, agglutination occurred rapidly (Fig. 6 B). Eventhough, human

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RBCs agglutinate quickly with β-GBP of P. pelagicus when compared to goat RBC (Fig. 6 D).

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This is due to the presence of sialic acid on the surface of RBC cells. β-GBP of P. pelagicus

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binds to the sialic acid and then agglutination occurs. But chicken RBCs did not show

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agglutination like human and goat RBCs. This agglutination reaction indicates that β-GBP of P.

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pelagicus have the ability to recognize the foreign invaders by PRPs.

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3.4.3. In vitro phagocytic assay

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The results suggest that, P. pelagicus β-GBP involved in the phagocytic activity with

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yeast cells. Moreover, the haemocytes had the clear cut protecting activity against the foreign

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particle enter in the host (data not shown). Upon extension of the incubation time up to 30 min

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with yeast cells, it was vividly notable that several haemocytes contained up to 5 yeast cells

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intracellular, thereby demonstrating phagocytic activity of P. pelagicus haemocytes in vitro.

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While testing with P. pelagicus purified β-GBP, the intracellular (Ingested) yeast cells appeared

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darker and markedly lost their surrounding bright rings as compared with free or extra cellularly

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attached yeast cells (Fig. 7). Thus, the apparent differences enabled easy and unambiguous

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determination of the ingestion (like melanisation) of yeast cells by β-GBP.

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3.4.4. Encapsulation assay

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Encapsulation ability of P. pelagicus β-GBP was tested against the fixed sepharose bead

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suspension. The beads are surrounded by P. pelagicus β-GBP and form black ring around the

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cell. Number of beads which are encapsulated by P. pelagicus β-GBP is based on the

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concentration of β-GBP (Fig. 8). When the concentration was increased, the number of beads

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encapsulated by P. pelagicus β-GBP was also increased. This noted that P. pelagicus β-GBP

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have the skill to encapsulate and kill the foreign invaders.

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3.4.5. Phenoloxidase enhancing activity of P. pelagicus purified β-GBP

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The complex of P. pelagicus purified β-GBP and laminarin (10, 30 & 50 µg/ml) showed

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that the PO activity increases with increase in concentration. The highest PO activity was

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measured when using the highest concentration of purified P. pelagicus β-GBP. While the effect

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of PO activity was tested with laminarin, purified β-GBP at different concentrations, the

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enhancement of PO activity was increased in a dose-dependent manner (Fig. 9). At the same

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time enhancement of PO activity was lower in β-GBP and laminarin alone. This shows that the

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laminarin and β-GBP complex (βG + β-GBP) are the potential activators to enumerate the PO

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activity and involved in the triggering of innate immune system.

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3.4.6. Antibacterial activity of reaction product of P. pelagicus ß-GBP

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Reaction products of P. pelagicus β-GBP were evaluated for their antibacterial activity

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by well diffusion assay. Reaction products of β-GBP showed higher antibacterial activity against

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Gram positive and Gram negative bacteria when compared to substrate and purified β-GBP

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unaccompanied. This was further confirmed by spectrophotometric readings and the P. pelagicus

327

β-GBP reaction products showed the antibacterial activity which was evident from the growth

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curve analysis (data not shown). After the addition of β-GBP reaction products, the zone of

329

inhibition was observed in B. cereus, L. monocytogens V. parahaemolyticus and P. vulgaris

330

(Supplementary Fig. 1) when compared to other combination. The zone of inhibition was

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increased at the concentration of 30 µl of reaction product of β-GBP, whereas, there was minor

332

inhibition occurred in PBS + β-GBP combination (30 µl) and there was no inhibition occured at

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PBS + substrate alone. These results confirm that the reaction products of β-GBP have the ability

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to kill the bacteria at the concentration of 30 µl.

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3.4.7. Biofilm inhibition assay

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Biofilm are known to be the assemblages of microorganisms that are irreversibly

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associated with a surface and enclosed in a matrix of extracellular materials and environmental

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niches. The impact of reaction product of P. pelagicus β-GBP (PBS + laminarin + β-GBP)

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against the biofilm forming ability of Gram positive and Gram negative bacteria was assessed

340

through light microscopic and CLSM image analysis. From the antibacterial analysis, we

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confirmed that 30 µl of reaction product β-GBP could inhibit the bacterial growth. Based on this,

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we choose 50 µl of β-GBP reaction product for antibiofilm analysis. In light microscopic

343

analysis, a visible reduction in the biofilm formation of Gram positive (Fig. 10A) and Gram

344

negative bacteria was observed (Fig. 10B), in the presence of β-GBP reaction product at 50 µl

345

compared to that of control. Further, it was also confirmed by CLSM studies (Fig. 10C, D). It

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indicates that, the disruption of biofilm architecture was occurred after treatment with β-GBP

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reaction product for 24 hrs and resulted in a decrease of biofilm formation at 50 µl concentration.

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An increase in the incubation period resulted in the complete removal of the bacterial cells from

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glass substratum. These data indicates that reaction product of P.pelagicus β-GBP effectively

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inhibit the biofilm formation of both Gram positive and Gram negative bacteria.

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4. Discussion

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In this study, we purified the pattern recognition protein molecule β-GBP from the

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haemocytes of blue swimmer crab, P. pelagicus and its functional aspects were proved by

354

agglutination assays. In addition, we first report the antibacterial and antibiofilm activity of

355

reaction product of P. pelagicus β-GBP against Gram positive and Gram negative bacteria. β-

356

GBP were first purified from two insects, Blaberus craniifer [9] and Bombyx mori [10] and then

357

from several crustaceans including freshwater crayfish Pacifastacus leniusculus [11], yellow leg

358

shrimp Penaeus californiensis [13], green tiger shrimp Penaeus semisulcatus [15] mangrove

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crab Episesarma tetragonum [16] and white shrimp Litopenaeus vannamei [14]. Further, β-GBP

360

was also purified from molluscan form Perna viridis [22]. Recently, β-GBP was purified from

361

the freshwater prawn, Macrobrachium rosenbergii at the molecular weight of 113 kDa on SDS-

362

PAGE [23]. In our study, P. pelagicus purified β-GBP shows the molecular weight of 100 kDa

363

on SDS-PAGE. In crustaceans, the β-GBP has been isolated as monomeric with the molecular

364

mass of approximately 100 kDa [24]. The 100 kDa of β-GBP was purified from various

365

crustaceans such as crayfish Astacus astacus and red swamp crayfish Procmbarus clarkii [6],

366

brown shrimp Penaeus californiensis [7], white shrimp Penaeus vannamei [14], Brazilian shrimp

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Far-fantepenaeus paulensis and Litopenaeus schmitti [25] and from the insect cockroach

368

Blaberus craniifer. In crustaceans, β-GBP with 95-112 kDa have been isolated including several

369

species of penaeids: yellowleg shrimp Farfantepenaeus californiensis [13, 17], and blue shrimp

370

Litopenaeus stylirostris [26]. Furthermore, the high molecular weight of β-GBP was purified

371

from insect Blaberus discoidalis (520 kDa) [27] and marine mussel Perna viridis (510 kDa) [22].

372

A β-GBP having the molecular weight 31 kDa was isolated and purified from the haemocytes of

373

black tiger shrimp Penaeus monodon [28]. The result of the present study revealed that the β-

374

GBP is a monomeric protein with moderate size of molecular weight. The molecular weight

375

deviation of the protein is possibly due to the species -specific difference. Further, structural

376

elucidation and crystallographic study were needed to know the exact molecular mass of the β-

377

GBP protein from crustaceans. HPLC and CD analysis showed the β-GBP’s purity and later

378

confirmed the β-sheets present in its structure. The low positive ellipticity values below 215 nm

379

clearly suggest the presence of unordered segments in this protein. Our preface studies on the CD

380

spectra of purified β-GBP, suggest that, the presence of predominantly β-sheets than α-helices

381

and coils in the protein structure [15]. β-GBP predominantly have β- sheets in its secondary

382

structure, which helps in protein molecule’s stability, folding, target recognition and shows their

383

level of maturity in their biological functions. This result coincides with the secondary structure

384

of β-GBP reported by [22] from marine mussel P. viridis, mangrove crab E. tetragonum [16]

385

and green tiger shrimp P. semisulcatus [15]. Moreover, the role of β-sheet in target recognition

386

of the protein may be strongly preserved to ensure the maintenance of biological function and

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this is achieved through the high level of conservation of key residues in the β-sheets [29].

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In XRD analysis, the single peak confirms the presence of β-glucan in the haemocytes of

390

P. pelagicus. The peak has been matched with the JCPDS PDF no #492332. These result

391

revealed that the purified β-GBP has the crystalline molecule glucan, due to the presence of the

392

ligand crystalline peaks that occurred. This may be helpful to understand the crystalline nature of

393

the protein binding with the ligand molecule, which was matched with our previous report of

394

XRD pattern of β-GBP molecule purified from E. tetragonum [16].

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Besides, we account the support for β-GBP’s major role in the immune system of cr

396

ustaceans based on the agglutination reactions and antibiofilm activity. Our study recorded that,

397

P. pelagicus purified β-GBP showed agglutination reaction with the yeast cell Saccharomyces

398

cerevisiae and mammalian and chicken RBCs. Earlier studies have shown the presence of yeast

399

agglutinating molecules in the blood of insects and crustaceans by affinity precipitation

400

technique using laminarin [10-12] or curdlan [30, 31]. Agglutination with yeast and erythrocytes

401

were already reported in the purified β-GBP from marine mussel, Perna viridis [22]. Similar

402

agglutination reaction of purified β-GBP from green tiger shrimp P.semisulcatus and mangrove

403

crab E. tetragonum were previously reported [15, 16]. This result suggests that, crustacean’s β-

404

GBP may possess at least two sugar-recognition sites in its structure, which is responsible for

405

yeast agglutination. On the other hand, in other shrimp species, β-GBPs were only able to bind to

406

β-1,3-glucans and not to other sugar complexes such as lipopolysaccharide (LPS) [28]. β-GBP

407

involved in the activation of phagocytic activity by the formation of circle around the yeast cells

408

which correlate with the phagocytic activity of haemocytes isolated from E. tetragonum and

409

Macrobrachium rosenbergii [32, 33].

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P. pelagicus, β-GBP showed enhanced PO activity in the presence of laminarin. PO

411

enhancement of β-GBP by laminarin was previously described in Bombyx mori [10], Blaberus

412

cranifer [9], and Pacifastacus lenisculus [34]. These results imply that the activation of proPO

413

system was induced by laminarin which seems to be involved in the binding of laminarin with β-

414

GBP. P. pelagicus purified β-GBP enhances the proPO system and it is well established that

415

yeast β-glucans (βG) are known to induce different defense responses in crustaceans. Moreover,

416

β-GBPs role in the activation of proPO system in P. pelagicus β-GBP was determined through

417

in vitro assay by incubation of a complex with laminarin and β-GBPs. The activation of proPO

418

system by the yeast cells with P. pelagicus β-GBP highlights the importance of β-GBP role in

419

antimicrobial defense mechanism and also involved in the elimination of yeast pathogens. β-

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GBPs react with β-glucans and formed β-glucan- β-GBP complex induces degranulation of

421

hemocytes subsequently enhances the activation of proPO system which brings about synthesis

422

of melanin through oxidation of phenols [35, 24]. For instance, the activation of proPO system

423

by βG- β-GBP complexes was shown to trigger proPO-activating enzyme (PPAE) activity into

424

the active phenoloxidase (PO) [24]. Agglutinin alone does not induce haemocyte agglutination,

425

but when the agglutinin reacts with an LPS-containing particle; it is capable of reacting with the

426

haemocyte surface and increases the phagocytic activity [36]. Likewise, β-GBP by itself is

427

unable to induce, release and activation of the proPO system, but the β-GBP– β-glucan complex

428

is able to react with the circulating cells and increase the effect of glucans on the proPO system

429

[36-38, 13]. Thus, β-GBPs are capable of activating cellular activities only after reaction with the

430

microbial carbohydrates (LPS) peptidoglycans or glucans.

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In the present study, reaction product of β-GBP showed inhibition on the biofilm

432

growth of both Gram positive and Gram negative bacteria. Antibacterial activity of β-GBP

433

reaction product revealed that β-GBP or substrate alone could not effectively inhibit the bacterial

434

growth, when β-GBP was combined with substrate laminarin, it might be an efficient in the

435

killing and inhibition of Gram positive and Gram negative bacteria. Furthermore, the killing

436

effect may be due the action of β-GBP together with laminarin which reacts on the surface of

437

bacteria and form a polymer membrane that prevents the nutrients entering into the cell. To the

438

best of our knowledge, this is the first report to elucidate the antibacterial activity of β-GBP

439

reaction product from P.pelagicus.

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However, the immune molecule, phenoloxidase reaction product of Apostichopus

441

japonicas showed antibacterial activity against Vibrio splendidus and Vibrio harveyi [20].

442

Correspondingly, the reaction products (dopamine as substrate) generated by Crassostrea gigas

443

and Chlamys farreri had the greatest antibacterial activity against Vibrio splendidus and Vibrio

444

harveyi [21, 39]. Our findings supports the above proposition and suggests that, immune related

445

P. pelagicus β-GBP protein is not restricted to the recognition and agglutination of microbes, but

446

also to boost up the destruction of pathogens through enhancement of phagocytosis by β-GBP

447

and the activation of antimicrobial proPO system. Moreover, in the present study, we confirmed

448

the antimicrobial activity of β-GBP reaction products. Above all, the present study brings new

449

evidences that P. pelagicus β-GBP plays distinct roles in the pathogen recognition and initiation

450

of immune responses in crustaceans. In brief, crustacean β-GBP has been shown to be involved

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in: (i) recognition and binding of fungal cell wall components [40,11], (ii) enhancement of

452

phagocytosis by hemocytes [14], (iii) activation of the proPO system [11, 13,22], (iv) transport

453

of lipids (41,17], (v) agglutination of fungal cells [16] and (vi) antibacterial and antibiofilm

454

activity against Gram positive and Gram negative by β-GBP reaction product (present study).

455

Our study will help to understand the multifunctional role of β-GBP in crustacean immune

456

system and explore the knowledge in protection mechanism of invertebrates.

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In conclusion, we report the purified β-GBP (100 kDa) from the haemocytes of Portunus

458

pelagicus. The purified β-GBP was confirmed by SDS-PAGE, HPLC, CD and XRD spectra. For

459

the first time, this study reports the in vitro antibacterial and antibiofilm activity of the reaction

460

product of β-GBP against Gram positive (B. cereus and L. monocytogens) and Gram negative (V.

461

parahaemolyticus and P. vulgaris) bacteria. In this experiment, we evidenced that purified β-

462

GBP of P. pelagicus showed multifunctional activity such as agglutination reaction, PO

463

enhancing activity, phagocytosis, encapsulation and in vitro antibacterial activity.

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Acknowledgements

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This work was supported by the Department of Biotechnology (DBT), Government of India,

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New Delhi, India, under the Project grants code BT/PR7903/AAQ/3/638/2013.

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References

469

[1] Vazquez

470

Immunity mechanisms in crustaceans. Innate Immun. 2009; 15:179-188.

471 472

[2]Medzhitov R, Janeway Jr, CA. Innate immunity: the virtues of a nonclonal system of recognition. Cell 1997; 91:295–298

473

[3]Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RAB. 1999. Phylogenetic perspectives

474

in innate immunity. 1999: Science 284; 1313-1318.

475

[4]Lee S, Söderhäll K. Early events in crustacean innate immunity. Fish Shellfish Immunol

476

2002; 12: 421-437.

477

[5]Iwanaga S, Lee BL. Recent advances in the innate immunity of invertebrate animals. Biochem

478

Mol Biol 2005; 38:128-150.

TE D

465

AC C

EP

L, Alpuche J, Maldonado G, Agundis C, Pereyra-Morales A, Zenteno E.

ACCEPTED MANUSCRIPT

[6]Duvic B, Soderhall K. β-1, 3-Glucan-binding proteins from plasma of the freshwater

480

crayfishes Astacus astacus and Procambarus clarkii. J Crust Biol 1993; 13:403-408.6

481

[7]Vargas-Albores F, Guzman MA, Ocha JL. A lipopolysaccharide-binding agglutinin isolated

482

from brown shrimp (Penaeus californiensis Holmes) haemolymph. Comp Biochem Physiol B

483

1993; 104: 407-13.

484

[8]Lee SY, Wang R. So¨ derha¨ ll K. A lipopolysaccharide- and β-1,3-glucan-binding protein

485

from hemocytes of

486

characterization, and cDNA cloning. J Biol Chem 2000; 275: 1337-1343.

487

[9]Soderhall K, Rogener W, Soderhall I, Newton RP, Ratcliffe NA. The properties and

488

purification of a Blaberus craniifer plasma protein which enhances the activation of haemocyte

489

prophenoloxidase by a β-1,3-glucan. Insect Biochem 1988:18; 323-330.

490

[10]Ochiai M, Ashida M. Purification of a β-1, 3- glucan recognition protein in the

491

prophenoloxidase activating system from haemolymph of the silkworm, Bombyx mori. J Biol

492

Chem 1988; 263:12056-12062.

493

[11]Duvic B, Söderhäll K. Purification and characterization of a βeta-1,3-glucan binding protein

494

from plasma of the crayfish Pacifastacus leniusculus. J Biol Chem 1990; 5:9327-9332.

495

[12]Thornqvist PO, Johansson MW, Soderhall K. Opsonic activity of cell adhesion proteins and

496

ß-1,3-glucan-binding proteins from two crustaceans. Dev Comp Immunol 1994; 18: 3-12.

497

[13]Vargas-Albores F, Jimenez-Vega F, Soderhall K. A plasma protein isolated from brown

498

shrimp (Penaeus californiensis) which enhances the activation of prophenoloxidase system by β-

499

1, 3-glucan. Dev Comp Immunol 1996; 20: 299-306.

500

[14]Vargas-Albores F, Jime´nez-Vega F, Yepiz-Plascencia GM. Purification and comparison of

501

β -1, 3-glucan binding protein from white shrimp (Penaeus vannamei). Comp Biochem Physiol

502

B 1997; 116: 453-458.

503

[15]Sivakamavalli J, Vaseeharan B. Purification, Characterization and functional analysis of a

504

novel ß-1, 3- glucan binding protein from green tiger shrimp Penaeus semisulcatus. Fish

505

Shellfish Immunol 2013; 35: 689-696.

RI PT

479

AC C

EP

TE D

M AN U

SC

the freshwater crayfish Pacifastacus leninusculus. Purification,

ACCEPTED MANUSCRIPT

[16]Sivakamavalli J, Vaseeharan B. Bifunctional role of a pattern recognition molecule β-1, 3-

507

glucan binding protein purified from mangrove crab Episesarma tetragonum. Fish Shellfish

508

Immunol 2014; 119, 25-31.

509

[17]Yepiz-Plascencia G, Vargas-Albores F, Jimenz-Vega F, Ruiz-Verdugo LM, Romo-Figueroa

510

G. Shrimp plasma HDL and ß-glucan binding protein (ßGBP): comparison of biochemical

511

characteristics. Comp Biochem Physiol B 1998; 121:309-314.

512

[18]Laemmli UK. Cleavage of structural proteins during the assembly of the head of

513

bacteriophage T4. Nature 1970; 227: 680-685.

514

[19]Asokan R, Arumugam M, Mullainadhan P. Functional analysis of plasma prophenoloxidase

515

system in the marine mussel Perna viridis. Comp Biochem Physiol A Mol Integ Physiol 1998;

516

120: 753-762.

517

[20]Jiang JW, Zhou ZC, Dong Y, Cong C, Guan XY, Wang B, Jiang B, et al. Invitro

518

antibacterial analysis of phenoloxidase reaction products from sea cucumber Apostichopus

519

japonicas. Fish Shellfish Immunol 2014; 39: 458-463.

520

[21]Xing J, Jiang JW, Zhan WB. Phenoloxidase in the scallop Chlamys farreri: purification and

521

antibacterial activity of its reaction products generated in vitro. Fish Shellfish Immunol 2012; 32:

522

89-93.

523

[22]Jayaraj SS, Thiagarajan R., Arumugam M, Mullainadhan P. Isolation, purification and

524

characterization of β-1, 3-glucan binding protein from the plasma of marine mussel Perna

525

viridis. Fish Shellfish Immunol 2008; 24: 715-725.

526

[23]Mohanty J, Sahoo PK, Pillai BR, Mohnaty S, Garnayak SK, Kumar S. Purification and

527

characterization of a β-glucan binding protein from the haemolymph of freshwater prawn

528

Macrobranchium rosenbergii. Aqua 2015; 46: 95-104.

529

[24]Vargas-Albores, F., Yepiz-Plascencia, G., 2000. β- glucan binding protein and its role in

530

shrimp immune response. Aquaculture 191, 13-21.

AC C

EP

TE D

M AN U

SC

RI PT

506

ACCEPTED MANUSCRIPT

[25]Goncalves P, Vernal J, Rosa RD, Yepiz-Plascencia G, Batista de Souza CR, Barracco MA,

532

et al. Evidence for a novel biological role for the multifunctional β-1, 3-glucan binding protein in

533

shrimp. Mol Immunol 2013; 51: 363-7.

534

[26]Vargas-Albores F, Guzman-Murillo MA, Ochoa JL. Size dependent haemagglutinating

535

activity in the haemolymph from sub-adult blue shrimp (Penaeus stylirostris Stimpson). Comp

536

Biochem Physiol 1992; 103A: 487-491.

537

[27]Chen C, Rowley AF, Newton RP, Ratcliffe NA. Identification, purification and properties of

538

a β-1, 3-glucan-specific lectin from the serum of the cockroach (Blaberus discoidalis) which is

539

implicated in immune defence reactions. Comp. Biochem. Physiol B Biochem Mol Biol 1999;

540

122: 309-319.

541

[28]Sritunyalucksana K, Lee SY, So¨ derha¨ ll K. A β-1, 3-glucan binding protein from the

542

black tiger shrimp, Penaeus monodon. Dev Comp Immunol 2002; 26: 237-45.

543

[29]Browne JP, Strom M,. Martin SR, Bayley PM. The Role of β-Sheet Interactions in Domain

544

Stability, Folding, and Target Recognition Reactions of Calmodulin. Biochem 1997; 36: 9550-

545

9561.

546

[30]Ochiai M, Ashida M.. A pattern recognition protein for β-1,3- glucan. J Biol Chem 2000;

547

275: 4995-5002.

548

[31]Ma C, Kanost MR. A βeta 1, 3-glucan recognition protein from an insect, Manduca sexta,

549

agglutinates microorganisms and activates the phenoloxidase cascade. J Biol Chem 2000: 275;

550

7505-7514.

551

[32] Sivakamavalli J, Rajakumaran P, Vaseeharan, B. In vitro Studies on Cellular Mediated

552

Immune Response in Haemocytes of Crab-Episesarma tetragonum. Int J Mol Zoo 2013: 3: 24-

553

31.

554

[33] Sung HH, Hwang SF, Tasi FM. Responses of giant freshwater prawn (Macrobrachium

555

rosenbergii) to challenge by two strains of Aeromonas spp. J Invertebr Pathol 2000; 76: 278-84.

AC C

EP

TE D

M AN U

SC

RI PT

531

ACCEPTED MANUSCRIPT

[34]Lee MH, Osakis T, Lee JY, Baek MJ, Zhang R., Park JW. et al, Peptidoglycan recognition

557

proteins involved in 1,3- β-D-glucan dependent prophenoloxidase activation system of insect. J

558

Biochem 2004; 279: 3218-3227.

559

[35]So¨ derha¨ ll K, Cerenius L, Johansson MW. 1996. The prophenoloxidase activating system

560

in invertebrates. In: So¨ derha¨ ll K, Iwanaga S, Vasta GR, editors. New directions in invertebrate

561

immunology. Fair Haven, NJ: SOS Publications; p. 229-253.

562

[36]Barracco MA, Duvic B, So¨derh¨all K. The β-1, 3-glucan-binding protein from the crayfish

563

Pacifastacus leniusculus, when reacted with a β-1, 3-glucan, induces spreading and

564

degranulation of crayfish granular cells. Cell Tissue Res 1991; 266:491–497.

565

[37]Johansson MW, So¨derh¨all K. Cellular defence and cell adhesion in crustacean. Anim Biol

566

1992; 1: 97-107.

567

[38]Vargas-Albores F. The defense system of brown shrimp Penaeus californiensis.: humoral

568

recognition and cellular responses. J Mar Biotechnol 1995; 3: 153–156.

569

[39]Luna-Acosta A, Saulnier D, Pommier M, Haffner P, Decker SD, Renault T, et al. First

570

evidence of a potential antibacterial activity involving a laccase-type enzyme of the

571

phenoloxidase system in Pacific oyster Crassostrea gigas haemocytes. Fish Shellfish Immunol

572

2011; 31: 795-800.

573

[40]Cerenius L, Liang Z, Duvic B, Keyser P, Hellman U, Palva ET, Iwanaga S, Söderhäll K,

574

Structure and biological activity of a 1,3-βeta-d-glucan-binding protein in crustacean blood. J

575

Biol Chem 1994: 269; 29462-29467.

576

[41] Hall M, Vanheusden MC, Söderhäll K. Identification of the major lipoproteins in Crayfish

577

Haemolymph as proteins involved in immune recognition and clotting. Biochem Biophys Res

578

Commun 1995; 216: 939-946.

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Figures

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Counts A1

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Figure legends Fig.1 Analysis of β-GBP purified from the haemocytes of P. pelagicus on SDS-PAGE, 12% as separating gel and 4% polyacrylamide as stacking gel. Lane I: Purified β-GBP (100 kDa), Lane II: protein molecular weight marker.

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Fig.2 HPLC analysis of purified P.pelagicus β-GBP from the plasma, using a reversed phase C18 column and the protein emerged as a single peak with retention time of 3.537 min. Fig.3 Secondary structure prediction of P. pelagicus β-GBP using Circular Dichroism

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Fig. 4 Crystalline surface and Lattice arrangement of molecules in the P. pelagicus purified βGBP were analyzed through X-ray crystallography (XRD).

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Fig.5 Yeast agglutination activity of P. pelagicus purified β-GBP with yeast (S cerevisiae,106 cells/ml). Yeast cells as control (A), Agglutination of yeast cells with β-GBP + laminarin (B), Agglutination with β-GBP (C). Fig.6 Haemagglutination activity of P. pelagicus purified β-GBP with mammalian and chicken RBCs. Normal RBC of human (A), goat (C) and chicken (E), agglutination of P. pelagicus β-GBP with 20 µl of human RBCs (B) goat RBC (D) and Chicken RBC (E).

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Fig.7 Light microscopy images of P. pelagicus purified β-GBP showing the phagocytic activity against the yeast cells. Fig.8 Light microscopic view of P. pelagicus purified β-GBP encapsulation against sepharose bead suspension. Fig.9 Enhancement of PO activity by βG- β-GBP complex

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Fig.10 In situ microscopy visualization of biofilm inhibition in Gram positive B. cereus, L. monocytogens and Gram negative V. parahaemolyticus , P. vulgaris when treated with the 50 µl of reaction product of P. pelagicus β-GBP. Light microscopy views (A & B),

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Confocal laser scanning microscopy views (C & D).

Supplementary figure

Fig.1 Reaction product of P. pelagicus β-GBP showed the antibacterial activity against the Gram positive B. cereus, L. monocytogens and Gram negative V. parahaemolyticus , P. vulgaris at 30 µg/ml. (a) PBS + substrate; (b) PBS + β-GBP (c) PBS (10µl) + substrate (10 µl) + β-GBP (10 µl) (reaction product).

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Highlights β-1,3 glucan binding protein was purified from the haemocytes of blue swimmer crab Portunus pelagicus by laminarin coupled Sephadex G-100 column chromatography.

•

An immune function of β-GBP was reported through agglutination, phagocytosis and encapsulation studies.

•

Enhancement of PO activity by β-GBP was discussed

•

Antibacterial and antibiofilm activity of β-GBP reaction product was reported.

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