Identification of a Site on Mannan-binding Lectin Critical for Enhancement of Phagocytosis
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.
- SourceAvailable from: Nenoo Rawal
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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). "
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.Molecular Immunology 09/2010; 47(15):2505-14. DOI:10.1016/j.molimm.2010.05.292 · 2.97 Impact Factor
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- "). Amino acid consensus sequence GEKGEP, which is involved in C1q receptor (C1qRp) interaction (Arora et al., 2001) is present in both porcine MBL. This suggests that porcine MBL has the same ability as C1q to stimulate phagocytosis in the complement system (Holmskov et al., 2003). "
ABSTRACT: Mannose-binding lectin (MBL) mediates activation of the complement system via the lectin pathway. Two forms of MBL, MBL-A and MBL-C, were characterized in rodents, rabbits, bovine and rhesus monkeys, whereas only one form was identified in humans, chimpanzees and chickens. The two forms are encoded by two distinct genes named MBL1 and MBL2, which have been identified in many species including the pig. In this report, we studied the two porcine genes MBL1 and MBL2. The porcine MBL genes had higher identities to bovine rather than primate and rodent sequences. Both genes were assigned to chromosome 14 by radiation hybrid panel and linkage mapping. Both MBL genes were highly expressed in liver. MBL1 was also found to be expressed in the lung, testis and brain, whereas low expression of MBL2 was detected in the testis and kidney. New single nucleotide polymorphisms of porcine MBL2 gene were found and genotyped in an experimental F2 pig population, together with a previously reported SNP of MBL1. MBL1 genotypes differed in C3c serum concentration, i.e. in vivo complement activity, at P < 0.1. Correspondingly, linkage analysis revealed a quantitative trait locus for C3c serum level close to the position of the MBL genes. The study thus promotes the porcine MBL genes as functional and positional candidate gene for complement activity.International Journal of Immunogenetics 02/2007; 34(1):55-63. DOI:10.1111/j.1744-313X.2007.00656.x · 1.25 Impact Factor
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- "Such receptor is localized on human cells of myeloid lineage (monocytes or neutrophils), the U937, THP-1, and K562 cell lines and platelets (Nepomuceno and Tenner 1998). Experiments conducted with purified fragments of C1q and MBL suggest that the phagocytosis enhancement signal resides in the collagen-like tail domain of the molecules (Bobak et al. 1987, Guan et al. 1994, Arora et al. 2001), so it is very probable that bovine collectins also show affinity to C1qRp. "
ABSTRACT: Conglutinin, collectin-43 (CL-43) and collectin-46 (CL-46) are serum proteins characteristic for Bovidae. They belong to collectins--family of oligomeric proteins composed of trimeric subunits containing collagen-like sequences joined to C-type lectin domains. The genes encoding conglutinin, CL-43 and CL-46 are located on the bovine chromosome 28, and phylogenetic analysis indicates their common origin--from the lung surfactant protein D gene. Northern blot or immunocytochemical analysis confirm biosynthesis of bovine collectins mainly in the liver (conglutinin, CL-43) and in the thymus (CL-46). The level of conglutinin in the serum of dairy cows depends on many factors such as breeding, the season of the year, the stage of the reproductive cycle and infection. The collectins are involved in the innate immune defense. They bind to microbial surface carbohydrates inducing aggregation and, thereby, impeding infectivity. On the other hand the destruction of pathogens occurs due to stimulation of effector cells. CL-43 as well as conglutinin, binds to the collectin receptor (C1qR) localized on many types of cells identified as a surface variant of calreticulin. Conglutinin and CL-43 show antiviral activities towards influenza A virus and rotaviruses. Conglutinin also displays protective activity against bacterial infections.Polish journal of veterinary sciences 02/2006; 9(4):265-75. · 0.60 Impact Factor