Purification And Characterization Of Mannan-binding Lectin

Background and Objective: Mannan-binding lectin (syn: mannose-binding lectin, MBL) is a Ca2+-dependent (C-type) serum lectin. It has an important role in the innate immune system against pathogenic microorganisms, especially for children of 6 months to 2 years old.MBL is an oligomer comprised of several identical subunits. Each peptide chain has a C-terminal lectin domain (carbohydrate recognition domain, CRD ) and a collagenous region near the N-terminal. These two regions are critical to the full function of the protein. The CRD can bind specifically to several sugars, especially to the sugar groups on microbial surfaces. After the binding, MBL can activate the complement system through a kind of protease桵BL-associated serine protease (MASP). This route is called the lectin pathway. It also can interact directly with cell surface receptors and thereby promote opsonophagocytosis and other immune processes.Although the concentration of MBL in serum is normally rather low (1.5 mg/L), it can increase 2-3-fold during the early stage of infection. It has been reported that either low MBL concentration or the abnormal protein structure dueto the MBL gene mutations has a close relationship with increased infection susceptibilities in children, whose immune system is immature.This study aims to purify MBL from sheep serum and human plasma, detect its binding activities to specific sugars and several clinically relevant bacteria to explore its role in immune reactions against infections in children.Methods: Prepare the affinity resin by coupling mannan to CNBr activated Sepharose 4B. Purify MBL from sheep serum and human plasma by affinity chromatography on mannan-Sepharose 4B in the presence of Ca2+. Then elute the protein with the presence of EDTA, which can take Ca2+ away from MBL so that MBL is eluted from the affinity column. Detect the molecular weight of MBL on 10% SDS-PAGE under reducing conditions. After the reaction with Collagenase VII, sheep serum MBL is analyzed again on 10% SDS-PAGE under reducing conditions. Detect the binding activities of MBL to several monosaccharides and bacteria with enzyme-linked lectin assay (ELLA).Results:(1)Purification of MBL: MBL is purified from sheep serum and human plasma byaffinity chromatography. Under reducing conditions there appears two bands of29kD and 33kD and other higher molecular weight bands on SDS-PAGE of sheepMBL while there appears two bands of 30kD and 90kD on SDS-PAGE of humanMBL.(2)Identification of the collagen-like region of sheep serum MBL: Under reducingconditions it appears bands of 22.4kD and 21.6kD on SDS-PAGE after thedigestion with Collagenase VII.(3)Binding activities of sheep MBL to monosaccharides: The affinities of MBL tomonosaccharides are different. The selectivity from high to low is as follows:N-Acetylglucosamine>L-Fucose>D-Glucosamine>N-Acetylgalactosamine.(4)Binding activities of sheep MBL to bacteria: The binding abilities of MBL tobacteria are different. It shows a strong binding ability to Klebsiella omithinolytica and Escherichia coli. while it shows a relatively lower binding ability to Staphylococcus haemolyticus, Enterobacter cloacae and Staphylococcus epitermidis. To different isolates of Klebsiella pneumoniae, Haemophilus influenzae, and Staphylococcus aureus, MBL shows quite different binding abilities.Conclusion: MBL can be purified from the serum or plasma of mammals by affinity chromatography on mannan-Sepharose 4B. The molecular weight of one peptide chain of human MBL is 30kD, while sheep MBL has two kinds of peptide chains with the molecular weight of 29kD and 33kD respectively. Sheep serum MBL has a collagen-like region, which can be digested by Collagenase VII. The binding abilities of sheep MBL to different monosaccharides are different. The selectivity is similar to that of human MBL. Sheep MBL shows different bindings to different bacteria with a relatively stronger binding to Gram-negative ones.