The 'involution' of mannose-binding lectin

Mannose-binding lectin (MBL) acts as a serum opsonin in innate immune defense and induces complement activation by the lectin pathway. In humans, low levels of functional serum MBL are caused by the dominant action of three single nucleotide substitutions in exon 1 that disrupt the glycine-rich backbone structure of the protein. The presence of common MBL variant alleles is associated with both infectious and autoimmune diseases. Conversely, it has also been suggested that MBL variants are maintained because of selective advantages for the host. In man, the MBL genetic system comprises one functional gene (MBL2) and one expressed pseudogene (MBL1P1), whereas the lower primate, the rhesus monkey resembles rodents with two functional MBL genes. We have investigated the molecular mechanisms behind the evolutionary loss of MBL expression from lower primates to man, including silencing of the MBL1P1 gene and the generation of MBL2 variant structural alleles and promoter polymorphisms leading to the present human MBL2 haplotypes. We present data showing that the MBL1P1 gene has been repeatedly hit throughout evolution and silenced eventually by mutations in the glycine residues of the collagen-like region. Our results indicate that the MBL1P1 gene has been selectively turned off during evolution through the same molecular mechanisms causing the MBL2 variant alleles in man, suggesting an evolutionary selection for low-producing MBL genes.     

Hepatitis C virus infection and mutations of mannose-binding lectin gene MBL

Summary.  We assessed the genetic polymorphism of mannose-binding lectin (MBL) in 93 patients with chronic hepatitis C (45 responders and 48 nonresponders to interferon) and 218 healthy controls. Mutant allele was identified only at codon 54 (Gly→Asp), leading to three genotypes (54 m/m, 54 W/m, and 54 W/W). Frequency of 54 m/m was significantly lower in interferon-responders (2.2%) compared to those in nonresponders (14.6%) and controls (10.6%): p<0.05. Our results suggest that homozygous carriage of the variant allele of codon 54 of MBL may predict poor response to interferon in chronic hepatitis C patients. Accepted December 4, 1997 Received November 25, 1997     

DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cell infection

Fusion of the human immunodeficiency virus (HIV) to the plasma membrane of target cells is mediated by interaction of its envelope glycoprotein, gp120, with CD4 and appropriate chemokine receptors. gp120 additionally binds to DC-SIGN, a C-type lectin expressed on immature dendritic cells. This interaction does not result in viral fusion, but instead contributes to enhanced infection in trans of target cells that express CD4 and chemokine receptors. Here we show that DC-SIGN mediates rapid internalization of intact HIV into a low pH nonlysosomal compartment. Internalized virus retains competence to infect target cells. Removal of the DC-SIGN cytoplasmic tail reduced viral uptake and abrogated the trans-enhancement of T cell infection. We propose that HIV binds to DC-SIGN to gain access to an intracellular compartment that contributes to augmentation or retention of viral infectivity.     

Human mannose-binding lectin inhibits human cytomegalovirus infection in human embryonic pulmonary fibroblast

A limited number of drugs have been used for treatment of human cytomegalovirus (HCMV), all sharing the similar antiviral mechanism of inhibiting virus replication. This study investigates the anti-HCMV activities of mannose-binding lectin (MBL) from blocking virus entry and inhibiting virus spread. Recombinant human MBL was produced in CHO cells and native human MBL was isolated from human serum. A HCMV neutralization test was performed by pre-treating HCMV with each diluted MBL solution. Then the treated HCMV was inoculated onto the human embryonic pulmonary fibroblasts (HELF), which was followed by HCMV-DNA detection, PP65 positivity examination and confocal imaging of the infected cells. To test the activity of MBL in inhibiting viral spreading after viral invasion, HCMV growth inhibition test was performed. The infected cells were incubated with each diluted MBL, every 24 h, the supernatant was tested for HCMV-DNA. After 72 h, cells were collected for HCMV-DNA and PP65 examination. Then the cytopathic effect was observed and cell viability was measured at the 5 days after infection. HCMV neutralization test revealed 10 μg/mL MBL significantly decreased the HCMV invasion in HELF and the anti-HCMV activity can be blocked by 20 mg/mL mannan. HCMV growth inhibition test indicated that at 48 h after HCMV invasion, the HCMV-DNA level in the culture supernatant with 10 μg/mL MBL was lower than the control. After 72 h, both the HCMV-DNA levels and PP65 positivity in cells incubated with MBL were reduced. This is the first to report on the anti-HCMV activities of MBL by in vitro studies.     

Interactions between mannose-binding lectin and MASPs during complement activation by the lectin pathway

The lectin pathway of complement performs a key role within the immune system by recognising pathogens through patterns of sugar moieties displayed on their cell surfaces and neutralising them via an antibody-independent reaction cascade. While particularly important during early childhood before the adaptive immune system is established, or when adaptive immunity is compromised, it has a protective function throughout life, neutralising invading pathogens directly and helping to stimulate and direct an effective immune response. Complement activation is initiated when complexes comprising mannose-binding lectin (MBL) or serum ficolins and MBL-associated serine protease-2 (MASP-2) bind to pathogens. Binding induces conformational changes in these complexes, leading to autoactivation of the MASPs, which in turn activate the downstream reaction cascade. A major goal in complement research is to understand the molecular events that trigger complement activation. Over the last few years, structure-function studies have improved our knowledge of the way in which MBL binds to MASPs by defining the portions of these proteins that interact and by solving the structures of key protein fragments. In this review, I will summarise the main findings of these studies and describe current theories to explain how the components combine to initiate the reaction cascade.     

Influence of mannose-binding lectin on HIV infection and tuberculosis in a Western-European population

Mannose-binding lectin (MBL) is a serum lectin that mediates phagocytosis and activates complement. Its deficiency has been associated with increased susceptibility to infectious diseases, mainly in childhood. However, non-producer mbl-2 alleles are common in most populations, suggesting a selective advantage of these alleles. We have analysed the association of mbl-2 structural and promoter polymorphisms with HIV infection and tuberculosis (TBC) in a white Spanish population, including 615 HIV patients with and without TBC, 127 no-HIV TBC patients, 142 TBC household contacts and 344 controls. The frequency of low or non-producer mbl-2 genotypes was lower in HIV patients than in controls. HIV-TBC patients presented lower frequencies of low or non-producer alleles and genotypes than HIV no-TBC patients and controls. Additionally, we found a significantly positive correlation between the incidence of TBC and the frequency of non-producer mbl-2 alleles in Western Europe. Therefore, MBL deficiency may be associated with a lower risk of HIV infection, and also of active TBC, at least in HIV patients. The protective role of low-producer mbl-2 genotypes against TBC together with the positive correlation observed between non-producer mbl-2 alleles and TBC incidence, suggest a balancing selection: in spite of an increased susceptibility to respiratory infections associated with MBL deficiency, mbl-2 deficient alleles would have been selected along different populations as a consequence of its selective advantage against intracellular pathogens, such as M. tuberculosis.     

Association between combined properdin and mannose-binding lectin deficiency and infection with Neisseria meningitidis

BACKGROUND: Individuals genetically deficient of properdin are more susceptible to meningococcal disease. Likewise low concentration or decreased biological activity of mannose-binding lectin (MBL) is associated with higher incidence of bacterial infections during childhood. In this study we report our findings in a Danish family with a remarkably high incidence of meningococcal meningitis-in total four cases, one of them fatal. METHODS: Properdin and MBL were quantified by ELISA and the properdin gene was screened for sequence variations using denaturing high-performance liquid chromatography (DHPLC) and subsequent sequencing of abnormal patterns. The MBL gene was genotyped for the three known variant alleles (B, C and D) as well as three promoter polymorphisms (-221Y/X, -550H/L and +4P/Q). RESULTS: Two out of six males with undetectable properdin activity had meningitis. They had also low MBL serum levels or carried an MBL variant allele, whereas high MBL concentrations were measured in three out of four properdin deficient males--without meningitis. A splice site mutation in exon 10 (c.1487-2A>G) was found in the properdin gene and co segregated with biochemically measured properdin deficiency. CONCLUSION: Our results indicate that a combined deficiency of both properdin and MBL increases the risk of infection with Neisseria meningitidis and stress the importance of epistatic genetic interactions in disease susceptibility.     

Relative roles of complement factor 3 and mannose-binding lectin in host defense against infection

Staphylococcus aureus is a major cause of severe nosocomial and community-acquired infections. Phagocytes and humoral molecules, including complement, have been proposed to cooperate in host defense against gram-positive bacteria. Circumstantial evidence indicates a role for complement, but this has not been formally defined. Complement activation is initiated by the classical, alternative, or lectin pathway, with the latter requiring mannose-binding lectin (MBL, also known as mannose-binding protein). MBL is an oligomeric serum protein that recognizes carbohydrates decorating a broad range of infectious agents, including S. aureus. We previously reported that MBL null mice were highly susceptible to S. aureus infection, confirming that MBL plays a key role in first-line host defense. In this study, we evaluated the relative roles of C3 and MBL against S. aureus infection by generating MBL x C3 null mice to compare with C3 single null mice. C3 deficiency alone significantly reduced survival to 19% from 97% of wild-type mice (P < 0.0001). Surprisingly, an additional MBL deficiency reduced the survival further to 7% (P < 0.0001). However, the MBL deficiency alone had a smaller though significant effect on survival, which was 77% (P = 0.018 versus wild-type mice). These results confirm an essential function for complement in host resistance against S. aureus infection but also identify an MBL-dependent mechanism that is C3 independent.     

Absence of mannose-binding lectin prevents hyperglycemic cardiovascular complications

Diabetes, stress, pharmaceuticals, surgery, and physical trauma can lead to hyperglycemic conditions. A consistent relationship has been found between chronic inflammation and the cardiovascular complications of hyperglycemia. We hypothesized that cardiomyopathy and vasculopathy resulting from acute hyperglycemia are dependent on mannose-binding lectin (MBL) and lectin complement pathway activation. Hyperglycemia was induced in wild-type (WT) C57BL/6 and MBL-null mice after streptozotocin administration. Echocardiographic data and tissue samples were collected after 4, 7, or 14 days of acute hyperglycemia. Hyperglycemic WT mice demonstrated dilated cardiomyopathy with significantly increased short and long axis area measurements during systole and diastole compared to hyperglycemic MBL-null mice. The EC(50) for acetylcholine-induced relaxation of mesenteric arterioles in WT mice after 4 days of hyperglycemia demonstrated a significant loss of nitric oxide-mediated relaxation compared to normoglycemic WT or hyperglycemic MBL-null mice. Myocardial histochemistry and Western blot analysis revealed a significant influx of macrophages, altered morphology, and increased elastin and collagen deposition in hyperglycemic WT hearts compared to MBL-null hearts. Serum transforming growth factor-β1 levels were significantly lower in hyperglycemic MBL-null compared to WT mice, suggesting decreased profibrotic signaling. Together, these data suggest that MBL and the lectin complement pathway play a significant role in vascular dysfunction and cardiomyopathy after acute hyperglycemia.     

Differential recognition of obligate anaerobic bacteria by human mannose-binding lectin.

Deficiency of the innate, humoral immune component mannose-binding lectin (MBL) predisposes individuals to a variety of infections, but the importance of MBL in infection by anaerobes has not been addressed. The attachment of MBL to a wide range of anaerobic bacteria associated with human disease and colonization was surveyed. The results suggest that for the species we examined, resistance to MBL binding may be associated with organisms that are more commonly pathogenic and that MBL binding to some bacteria may be phase variable.     

Evolution of the mannose-binding lectin gene in primates

The mannose-binding lectin MBL2 plays an important role in the innate immune system. It binds carbohydrates surface, acts as an opsonin and activates the complement system. With the aim of studying the evolution of the MBL2 gene in primates, we sequenced its coding region in 12 non-human primate species and compared them with the human sequence. We demonstrated that nucleotide and amino-acidic sequences of the MBL2 among primates are highly homologous, underlining the importance of this molecule in the defense system against pathogen invasions. In particular, in the collagen-like domain that confers the characteristic structure to MBL2 protein, the identity among primates is really high. In the carbohydrate recognition domain, we evidenced some primates' group-specific amino-acidic mutations not resulting in changes of the structure or function of this MBL2 domain. Phylogenetic analysis did not evidence any positive selective pressure in MBL2 gene among non-human primates. Our findings indicate that MBL2 is well conserved in agreement with its important role in the immune system: in non-human primates, we did not observe the same 'plasticity' of the MBL2 human gene, where a frequency of more than 1% of nucleotide variations was described in the coding and promoter regions.     

人甘露糖结合凝集素在小鼠肝脏细胞内表达

目的：在小鼠肝脏组织中表达人甘露糖结合凝集素（mannose-binding lectin，MBL）。方法：采用PCR方法从pGEMT-MBL质粒中扩增人MBLcDNA序列，克隆人pUC19载体中，限制性酶切鉴定及测序正确后，将MBLcDNA克隆人真核表达载体pcDNA3质粒中，构建成pcDNA3-MBL质粒，经尾静脉大容量快速注射pcDNA3-MBL质粒20μg，8h后处死小鼠，Western Blot方法检测小鼠肝脏组织和血清中人MBL，免疫组织化学方法检测小鼠肝脏组织中人MBL的表达情况。结果：成功扩增出人MBLcDNA序列并构建克隆载体pUC19-MBL，测序结果正确，成功构建pcDNA3-MBL真核表达载体。小鼠尾静脉注射pcDNA3-MBL质粒后8h，免疫组化检测证实肝细胞中有人MBL表达，Western blot方法检测发现血清和肝脏组织中出现人MBL蛋白。结论：真核表达载体pcDNA3-MBL裸质粒DNA静脉注射可以在小鼠肝脏中大量表达人MBL，并分泌人血，为临床纠正先天性低MBL患者的易感染状态提供了新的思路。     

Modulation of HIV and SIV neutralization sensitivity by DC-SIGN and mannose-binding lectin

The C-type lectin DC-SIGN binds to oligosaccharides on the human and simian immunodeficiency virus (HIV, SIV) envelope glycoproteins and promotes infection of susceptible cells. Here, we show that DC-SIGN recognizes glycans involved in SIV sensitivity to neutralizing antibodies and that binding to DC-SIGN confers neutralization resistance to an otherwise sensitive SIV variant. Moreover, we provide evidence that mannose-binding lectin (MBL) can interfere with HIV-1 neutralization by the carbohydrate-specific antibody 2G12.     

Chlamydia trachomatis infection, Fallopian tube damage and a mannose-binding lectin codon 54 gene polymorphism

BACKGROUND: Mannose-binding lectin (MBL), a component of the innate immune system, provides a first-line defense against invading microorganisms. Polymorphisms in the MBL gene have been associated with increased risk of infection. Chlamydia trachomatis genital tract infections are a major cause of Fallopian tube occlusion. Our objective was to test whether an MBL codon 54 polymorphism might contribute to development of C. trachomatis-associated tubal damage. METHODS: In a case-control study, 97 women with occluded and 104 women with patent Fallopian tubes were tested for a history of chlamydial infection by serology and for their MBL codon 54 genotype by PCR and restriction fragment length polymorphism analysis. Clinical data were blinded to those performing all laboratory analyses. RESULTS: Women with tubal occlusion who also had a positive chlamydial serology had the highest rate of variant MBL B allele carriage (P<0.001). Among women who were chlamydial antibody negative, allele B carriage was also more frequent in those with blocked, as opposed to patent, Fallopian tubes (P<0.01). CONCLUSIONS: Wild-type allele A homozygosity is protective against, while carriage of the variant allele B is a risk factor for, Fallopian tube occlusion in women who are seropositive or seronegative for C. trachomatis.     

The role of mannose-binding lectin in health and disease

Mannose-binding lectin (MBL) is a pattern recognition molecule of the innate immune system. It belongs to the collectin family of proteins in which lectin (carbohydrate-recognition) domains are found in association with collagenous structures. In man, these proteins include serum MBL, lung surfactant protein A (SP-A) and lung surfactant protein D (SP-D). MBL binds to a range of sugars including N-acetyl-D-glucosamine, mannose, N-acetyl-mannosamine, fucose and glucose. This permits the protein to interact with a wide selection of viruses, bacteria, yeasts, fungi and protozoa decorated with such sugars. Unlike the other collectins, MBL bound to microbial surfaces is able to activate the complement system in an antibody and C1-independent manner. This activation is mediated by complexes of MBL with a serine protease called MBL-associated serine protease 2 (MASP-2), which specifically cleaves C4 and C2 to create a C3 convertase enzyme. MBL may also interact directly with cell surface receptors and thereby promote opsonophagocytosis by a complement-independent pathway. It has been suggested that MBL plays an important role in the first hours/days of any primary immune response to a sugar decorated pathogen. This provides the host with a first-line of defence before the adaptive immune system becomes operative and in humans may be particularly important between 6 and 18 months of age when the adaptive system is still immature. MBL deficiency is one of the most common human immunodeficiencies and arises primarily from three single point mutations in exon 1 of the MBL-2 gene. These mutations result in a failure to assemble fully functional multimeric protein. Several studies have shown that deficiency of MBL increases the overall susceptibility of an individual to infectious disease. The most striking example of this is the association of acute respiratory tract infections with MBL deficiency in early childhood. In contrast, there is evidence that for some intracellular parasites MBL deficiency may be protective and this might explain the high frequency of MBL mutations in sub-Saharan Africa and South America. Increasingly, there is evidence that the association between MBL levels and disease is complex. For example, the protein appears to influence the severity of several diseases. The mechanism whereby MBL exerts such effects is unclear but one possibility is through a dose-dependent modulation of pro-inflammatory cytokines.     

Calcium signals in single T cells on activation by lectin

Succinylation of concanavalin A (Con A) reduces its oligomer size while retaining its mitogenicity, and provides a probe of T cell activation. We have observed responses of cytosolic ionized calcium to succinyl Con A in suspensions of Jurkat and rat lymph node (LN) cells, using a fluorimeter, and in single cells settled on glass, using a dual wavelength video imaging system. In the fluorimeter a mitogenic level of succinyl Con A (30 micrograms/ml) produced only a 15-30 nM rise in average cell calcium in the suspended Jurkat or rat cells whereas the use of quantitative video imaging produced asynchronous 250-1000 nM pulses of free calcium in 35% of Jurkat cells and 300-850 nM pulses in 45% of rat LN cells. In Jurkat cells these pulses were sometimes repetitive, giving rise to apparent oscillations. In the fluorimeter 30 micrograms/ml of native Con A (a supra-mitogenic concentration) produced a 300 nM rise in average cell calcium in suspended Jurkat cells, and a 100 nM rise in rat LN cells; when major histocompatibility complex class II-bearing cells were removed the response rose. Mitogenic Con A (3 micrograms/ml) produced a much lower rise in calcium. With video imaging the response seen was greater. Levels greater than 30 micrograms/ml Con A caused 700-5000 nM pulses synchronously in 94% of Jurkat cells and 250-1000 nM pulses in 73% of rat LN cells. At 3 micrograms/ml Con A produced asynchronous 300-1100 nM pulses in 36% of rat LN cells. We conclude that the absence of a calcium signal in the fluorimeter can conceal asynchronous calcium responses in individual cells and that brief asynchronous cytosolic calcium pulses are sufficient for lectin to activate rat T cells.     

Lack of association between hepatitis B virus infection and polymorphism of mannose-binding lectin gene in Korean population

Mannose-binding lectin (MBL) plays an important role in immune defense. This study was undertaken to investigate the association between hepatitis B virus infection and polymorphisms of MBL gene. We assessed the single nucleotide polymorphism at codon 54 in exon 1 of MBL in patients with hepatitis B virus infection and HBsAg negative controls in Korean population. A total of 498 enrolled subjects was classified into four groups. Group 1; Clearance, Group 2; Inactive healthy carrier, Group 3; Chronic hepatitis, Group 4; Liver cirrhosis. MBL gene polymorphisms at codon 54 led to three genotypes (G/G, G/A, A/A). When we divided subjects into clearance group (group 1) and persistence group (group 2-4), G/G genotype and A-allele carrier were observed in 55.6% and 44.4% in clearance group, 64.8% and 35.2% in persistence group (p=0.081), respectively. When hepatitis B virus persistent cases were divided into inactive healthy carrier (group 2) and disease progression group (group 3 and 4), MBL gene polymorphisms at codon 54 were not related to disease progression (p=0.166). MBL gene polymorphism at codon 54 was not associated with the clearance of hepatitis B virus infection nor progression of disease in chronic hepatitis B virus infection.     

Restricted polymorphism of the mannose-binding lectin gene of indigenous Australians

Mannose-binding lectin (MBL) is an important complement-activating protein of the human innate immune system. Deficiency of MBL is associated with an increased risk of various infections and arises from three structural gene mutations in exon 1 (variants B, C and D) and/or the presence of a low efficiency promoter. The C allele is found in sub-Saharan Africa whereas the B allele is found elsewhere, suggesting that these mutations occurred after the suggested hominid migration out of Africa [100-150 000 years before present (BP)]. Paradoxically, these alleles may have a selective advantage in protection against intracellular pathogens and occur at particularly high frequencies in sub-Saharan Africa (C variant) and South America (B variant). Since hominids reached Australia at least 50 000 years ago, a study of MBL polymorphisms in the indigenous population was of interest. Using heteroduplex technology we found a paucity of MBL structural gene mutations in two population groups from geographically distinct regions. Of 293 individuals tested, 289 were wild-type and four were heterozygous for either the B or D allele. In each individual with an MBL mutation the HLA haplotype profile suggested some Caucasian admixture. We also found a restricted range of MBL promoter haplotypes and the serum MBL levels were higher than those of any other ethnic group studied to date (median 3.07 microg/ml). Our data suggest that the B mutation probably arose between 50 000 and 20 000 BP. Its absence from the founder gene pool of indigenous Australians may also partly explain their vulnerability to intracellular infections such as tuberculosis.