Isolation of midgut agglutinin of Culex quinquefasciatus ( Diptera , Culicidae)

Tahany Ayaad

doi:10.21608/eajbsa.2009.15429

Abstract

A Lectin that agglutinates Eschrechia coli (ATCC 25922) , Staphylococcus aureus (ATCC 6538) live bacteria and various mammalian red blood cells (RBCs) was identified in Culex quinquefasciatus midgut extract ( Cqlec) by using human (three groups: A, B, and O, RH+)mouse, rat, guinea-pig, rabbit and goat erythrocytes. With the use of (NH4)2 SO4 fractionation, anion - exchange and GluNA CBr-Sepharose 6B affinity chromatography, C. quinquefasciatus mid gut lectin has been purified to homogeneity. IEF and reducing SDS/PAGE revealed that the isolated mid gut lectin have isoelectric point (PI) of 6.30, and a subunit approximate molecular weight of 34.5 KDa .The highest agglutination activity of crude and isolated Cqlec were detected against both Eschrechia coli cells and rabbit RBCs. Significant differences in hemagglutinin titers and carbohydrate inhibition were detected between sugar fed and blood fed adult female mosquitoes.Overall agglutinin levels were increased following a blood meal feeding and E. coli induction using a hypodermic needle. This study presents the first report on the occurrence of heterogeneous anti rabbit RBC agglutinins in the midgut extracts of C. quinquefasciatus from Al kharj area in Saudi Arabia. The HA of lectins are Ca 2+ independent, heat-resistant, and are strongly inhibited by D(+)-mannose and D(+) glucose followed by N-acetyl-Dglucosamine. Raffinose and N-acetyl-D-manosamine were found to be moderate inhibitors. Non of the lectins were inhibited by galactose , lactose , trehalose or fetuin (1%) but the glycosubstances mucin and laminarin showed strong inhibitition using low concentrations . 2D-NMR spectroscopy revealed a component of the corresponding residue in structure having group regions of resolution spectrum of Man9- GlcNAc2Asn.

Highlights

Lectins are defined as carbohydrate-binding proteins or glycoproteins of nonimmune origin or as carbohydrate-binding proteins other than antibodies or enzymes
Humoral immunity was induced in adult females by experimentally pricking the wing with a needle dipped in a bacterial suspension of 1.1 x l06 ml-1 viable cells Gram negative Escherichia coli (ATCC 25922), equivalent to 1 McFarland tube suspended in 1μl phosphate-buffered saline(PBS, 137mM NaCl, 2.7mM KCl, 8.1 mM Na2HPO4/ 1.3mM KH2PO4, pH 7.2) and allowed to recover for 24 h in separate laboratory cages
The present study attempted at the isolation of the culicid C. quinquefasciatus midgut lectin using a combination of (NH4)2SO4 fractionation, ion-exchange and affinity chromatographies

Summary

INTRODUCTION

Lectins are defined as carbohydrate-binding proteins or glycoproteins of nonimmune origin or as carbohydrate-binding proteins other than antibodies or enzymes. Due to the lack of acquired immune response, the host defense against microbial infection in invertebrates solely depends on innate immune systems ( Ingram,1997).This has been proposed in invertebrates by the pattern recognition proteins (PRPs) that bind conserved pathogen associated molecular patterns (PAMPs) molecules. The latter is present in the array of carbohydrate components on the surface of microorganisms. A preliminary characterization of the purified lectin was performed

MATERIAL AND METHODS

A Sepharose 6B affinity column was prepared as follows

RESULTS AND DISCUSSION