Isolation, Cloning, and Characterization of a Novel Phosphomannan-binding Lectin from Porcine Serum

Bruce Yong Ma,Natsuko Nakamura,Vladimir Dlabac,Haruna Naito,Shinsuke Yamaguchi,Makiko Ishikawa,Motohiro Nonaka,Masaji Ishiguro,Nobuko Kawasaki,Shogo Oka,Toshisuke Kawasaki

doi:10.1074/jbc.m611820200

Abstract

Mannan-binding protein (MBP) is a C-type serum lectin that is an important constituent of the innate immune defense because it activates the complement system via the lectin pathway. While the pig has been proposed to be an attractive source of xenotransplantable tissues and organs, little is known about porcine MBP. In our previous studies, phosphomannan, but not mannan, was found to be an effective inhibitor of the C1q-independent bactericidal activity of newborn piglet serum against some rough strains of Gram-negative bacteria. In contrast, the inhibitory activities of phosphomannan and mannan were very similar in the case of MBP-dependent bactericidal activity against rough strains of Escherichia coli K-12 and S-16. Based on these findings, we inferred that an MBP-like lectin with slightly or completely different carbohydrate binding specificity might exist in newborn piglet serum and be responsible for the C1q-independent bactericidal activity. Herein we report that a novel phosphomannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from both newborn and adult porcine serum and characterized. Porcine PMBL functionally activated the complement system via the lectin pathway triggered by binding with both phosphomannan (P-mannan) and mannan, which, unlike MBP, was effectively inhibited by mannose 6-phosphate- or galatose-containing oligosaccharides. Our observations suggest that porcine PMBL plays a critical role in the innate immune defense from the newborn stage to adult-hood, and the establishment of a newborn piglet experimental model for the innate immune system studies is a valuable step toward elucidation of the physiological function and molecular mechanism of lectin pathway.

Highlights

Innate immunity was formerly thought to be a nonspecific immune response characterized by phagocytosis
As an extension of these studies, here we report that a novel P-mannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from newborn piglet serum, by P-mannan-Sepharose 4B affinity chromatography, and characterized
It is a well known fact that the serum of newborn piglets is extremely deficient in all classes of immunoglobulins, which results from the special type of placentation in this animal species that prevents the transfer of antibodies from the sow to the fetus

Summary

EXPERIMENTAL PROCEDURES

Newborn and adult porcine sera were supplied by Japan SLC, Inc. (Shizuoka, Japan) and Invitrogen/Invitrogen (Copenhagen, Denmark), respectively. A standard calibration kit for gel filtration, Epoxy-activated Sepharose 6B resins, and Hitrap Q Sepharose HP and Superose 4B columns were purchased from Amersham Biosciences. Newborn and adult porcine lectins were purified from newborn and adult porcine sera, respectively, by affinity chromatography on a P-mannan-Sepharose 4B column as described previously [1]. Gel filtration of the purified adult porcine serum lectins, 20 ␮g each, was performed with a FPLC system (Amersham Biosciences) on a Superose 6 column (Amersham Biosciences), which had been equilibrated with buffer D (TBS containing 10 mM EDTA, pH 8.0). The purified newborn and adult porcine serum lectins were resolved on a 5–20% Tris-HCl gradient gel (ATTO) under reducing conditions, respectively, and stained with Coomassie Brilliant Blue (CBB). Bands were visualized with a Fuji film LAS 3000 (FUJIFILM, Tokyo, Japan)

Protein Sequencing of Purified Newborn and Adult Porcine Serum Lectins

Carbohydrate Binding and Inhibition Specificity of Porcine Serum Lectins

RESULTS

Porcine PMBL was assayed in a

DISCUSSION