Article

Identification of a Site on Mannan-binding Lectin Critical for Enhancement of Phagocytosis

Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2001; 276(46):43087-94. DOI: 10.1074/jbc.M105455200
Source: PubMed

ABSTRACT

Mannan-binding lectin (MBL) constitutes an important part of the human innate immune defense system. It has been shown to mediate the activation of complement upon binding to specific microbial carbohydrate motifs, to directly opsonize organisms, and to enhance the phagocytosis of targets suboptimally opsonized with IgG or complement components C3b or C4b. This enhancement of phagocytic activity induced by MBL and other molecules that contain a collagen-like region contiguous with a pattern recognition domain is mediated by a 126,000 M(r) surface glycoprotein, designated C1qR(P). Although it has been known that the collagen-like domain of these "defense collagens" contains the interaction site(s) that triggers this enhancement of uptake, the specific interaction site has not been identified. To address this issue, wild type and mutant MBL constructs were generated, inserted into baculovirus, expressed in Sf9 cells, and the recombinant MBL (rMBL) proteins purified by mannan affinity chromatography. The effect of wild type and mutant rMBL on the phagocytosis of targets suboptimally opsonized with IgG or with IgM and C4b by human peripheral blood monocytes was then assessed. Two mutants, one of which has five GXY triplets deleted below the kink region of MBL and the other one having only two of the GXY triplets deleted below the kink, failed to enhance phagocytosis, suggesting the importance of the specific sequence GEKGEP in stimulating phagocytic activity. Similar sequences were detected in other defense collagens, implicating the consensus motif GE(K/Q/R)GEP as critical in mediating the enhancement of phagocytosis through C1qR(P.) Clarification of specific ligand-C1qR(P) interactions should facilitate the investigation of the signal transduction processes involved in the cell activation, as well as provide the basis for the design of specific modulators of the functions mediated by this receptor.

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    • "Upon recognition, C1q, MBL, and ficolin may initiate complement via the classical (C1q) or the lectin pathway (MBL and ficolin). C1q, MBL, and SPA have also been shown to enhance monocyte phagocytosis of targets suboptimally opsonized with immunoglobulin G (IgG) or complement receptor type 1 ligands, C4b and C3b (Bobak et al. 1987Bobak et al. , 1988a, b), in a collagen-like domain-dependent manner (Arora et al. 2001). It is also becoming increasingly evident that C1q and MBL play an important role in the recognition and removal of apoptotic cells (Ogden et al. 2001; Vandivier et al. 2002; Nauta et al. 2003a, b). "
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    • "Within this domain, several structural features are thought to contribute to the many distinct functions attributed to the collagen-like domain of mannan-binding lectin. The N-terminus of the collagen-like domain , including a conserved GEKGEP motif involved in interactions with the C1q receptor, is implicated in opsonophagocytosis (Arora et al., 2001). Meanwhile, within the C-terminus of the collagen-like domain are several conserved amino acid residues that form a putative MASP-binding motif (Wallis et al., 2004) and the assembly of the MBL trimer (Larsen et al., 2004). "
    [Show abstract] [Hide abstract] ABSTRACT: The mannan-binding lectin gene (MBL) participates as an opsonin in the innate immune system of mammals, and single nucleotide polymorphisms (SNPs) in MBL cause various immune dysfunctions. In this study, we detected SNPs in MBL2 at exon 1 using polymerase chain reaction single-strand conformation polymorphism analysis and DNA sequencing techniques in 825 Chinese Holstein cows. Four new SNPs with various allele frequencies were also found. The g.1164 G>A SNP was predicted to substitute arginine with glutamine at the N-terminus of the cysteine-rich domain. In the collagen-like domain, SNPs g.1197 C>A and g.1198 G>A changed proline to glutamine, whereas SNP g.1207 T>C was identified as a synonymous mutation. Correlation analysis showed that the g.1197 C>A marker was significantly correlated to somatic cell score (SCS), and the g.1164 G>A locus had significant effects on SCS, fat content, and protein content (P < 0.05), suggesting possible roles of these SNPs in the host response against mastitis. Nine haplotypes and nine haplotype pairs corresponding to the loci of the 4 novel SNPs were found in Chinese Holsteins. Haplotype pairs MM, MN, and BQ were correlated with the lowest SCS; MN with the highest protein yield; MM with the highest protein rate, and MN with the highest 305- day milk yield. Thus, MM, MN, and BQ are possible candidates for marker-assisted selection in dairy cattle breeding programs.
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    • "Lectin pathway activation leads to opsonization of the target with complement components, C3b and C4b, and to the formation of the membrane attack complex that helps kill bacteria (Ihara et al., 1982; Kawasaki et al., 1989; Schweinle et al., 1989). MBL also promotes phagocytosis of various microorganisms (Ihara et al., 1982; Turner et al., 1986; Kuhlman et al., 1989; Schweinle et al., 1989; Tenner et al., 1995; Nepomuceno et al., 1999; Arora et al., 2001; Neth et al., 2002; Ip et al., 2004; Jack et al., 2005; Ono et al., 2006; Shiratsuchi et al., 2008; Brouwer et al., 2008; Van Asbeck et al., 2008), which may depend on the direct opsonic activity of MBL (Kuhlman et al., 1989; Jack et al., 2005; Ono et al., 2006; Shiratsuchi et al., 2008) or require complement activation (Neth et al., 2002; Ip et al., 2004; Brouwer et al., 2008; Van Asbeck et al., 2008). Studies have also reported that MBL enhances phagocytosis and mediates complement-dependent neutralization of influenza viruses (Hartshorn et al., 1993; Anders et al., 1994). "
    [Show abstract] [Hide abstract] ABSTRACT: Mannan-binding lectin (MBL) mediates innate immune responses, such as activation of the complement lectin pathway and phagocytosis, to help fight infections. In the present study, employing recombinant forms of human MBL (rMBL), the role of wild type MBL (rMBL/A) and its structural variant rMBL/C in mediating THP-1 phagocytosis of fluorescent-labeled zymosan was examined and compared to MBL purified from human plasma (pMBL/A). Flow cytometric analyses revealed that opsonization of zymosan with rMBL/A and pMBL/A (0.5-30microg/ml) resulted in a 1.9- and 2.7-fold enhancement in its uptake by THP-1 cells in the presence of serum that was depleted of both MBL and the classical pathway component, C1q (MBL/C1q Dpl serum). In contrast, no enhancement in phagocytosis was observed when zymosan was opsonized with rMBL/C. Addition of MBL monoclonal antibody, EDTA, or mannan to the opsonization reaction mixture inhibited THP-1 phagocytosis of pMBL/A opsonized zymosan. Heat inactivation of MBL/C1q Dpl serum abolished the 2-fold increase in phagocytosis and in the absence of serum the direct opsonic activity of MBL did not contribute significantly to the uptake of zymosan into THP-1 cells. Activation products of complement components C3 and C4 were deposited on zymosan opsonized with pMBL/A and rMBL/A but not rMBL/C indicating that MBL-mediated phagocytosis of zymosan requires activation of the complement lectin pathway. The findings imply that impaired MBL-mediated phagocytosis may put individuals homozygous for the mutant allele MBL/C but not wild type MBL/A at increased risk to infections such as yeast.
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