High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells

We have developed a high-expression system of recombinant human mannan-binding lectin (MBL) with CHO cells. Geneticin-resistant transformants harboring human MBL cDNA in the expression vector pNOW/CMV-A were screened by immunoblot analysis for secretion of recombinant MBL. Cloning and selection by both geneticin and methotrexate resulted in the production of recombinant MBL to a final concentration of 128.8 microg/ml in media after four days of culture. SDS-PAGE and gel-filtration analyses showed that recombinant MBL is characterized by two lower-order oligomeric structures (apparent molecular weights: 1150 kDa and 300 kDa) compared to native MBL (apparent molecular weight: 1300 kDa). The recombinant human MBL has both sugar-binding and complement activation activity and, like native MBL, can inhibit hemagglutination of influenza A virus. Lectin blots with recombinant MBL indicate that it can bind such microorganisms as HIV and influenza virus suggesting that it might inhibit their infection of hosts. This high-level expression of human MBL with the full range of biological activity will be useful for studies on the immunological role of MBL in humans.     

Serum levels, ontogeny and heritability of chicken mannan-binding lectin (MBL).

Mannan-binding lectin (MBL) is a serum lectin found in mammals and recently also in birds. It is thought to play an important role in the innate immune defence through binding to surface carbohydrates on micro-organisms followed by complement activation via the MBL pathway. This results in opsonization or direct complement-mediated killing. To gain further knowledge about the physiology and function of the protein, we developed an enzyme-linked immunosorbent assay for chicken MBL and used this to investigate the level of MBL in different chicken strains during embryogenesis, early and adult life. The MBL concentrations in 308 chickens, representing 14 different strains, showed a non-Gaussian, unimodal distribution profile with a mean concentration of 5.8 micrograms/ml (range 0.4-37.8 micrograms/ml). No difference between the strains could be demonstrated and no chickens were found deficient in MBL. Ontogenetic studies showed that MBL is already detectable in embryos at a gestational age of 10 days (11 days before hatching). At hatching, the level is comparable to the level found in adult chickens. This level is fairly stable during the first weeks of life, but a deficiency state develops at 4 weeks of age, whereafter the level is normalized again at 5 weeks of age. Chickens with relatively low or high MBL levels were bred with cockerels having similar MBL levels and this resulted in F1 generations with significantly different MBL levels, suggesting that the protein level is genetically influenced.     

甘露聚糖结合凝集素(MBL)对3T3-L1前脂肪细胞成脂分化的调节作用及机制

目的研究甘露聚糖结合凝集素(mannan-binding lectin,MBL)对3T3-L1前脂肪细胞成脂分化的调节及其机制。方法体外诱导3T3-L1前脂肪细胞成脂分化,同时给予不同浓度的MBL(0、1、10、20μg/ml)干预。CCK-8法检测细胞增殖能力变化,油红O染色和细胞内甘油三酯含量测定法分析脂质积累情况。Western blot及qRT-PCR检测脂肪细胞成脂分化相关因子PPARγ及C/EBPα的蛋白质及mRNA表达水平。Western blot分析脂肪合成调控信号分子Akt的表达及磷酸化。结果实验组各浓度MBL(0、1、10、20μg/ml)对3T3-L1前脂肪细胞增殖都无影响。3T3-L1前脂肪细胞诱导分化3 d,甘油三酯检测发现MBL处理组细胞内甘油三酯水平下降,并呈剂量依赖关系;油红O染色结果进一步显示,MBL处理组的脂滴数量显著减少,吸光度值也显著降低,同样呈现浓度依赖关系。Western blot及qRT-PCR检测结果证实,MBL处理组PPARγ和C/EBPα的蛋白质及mRNA表达水平均显著下降,呈剂量依赖性。在MBL干预下,Akt的磷酸化水平也明显下调。结论MBL通过Akt信号通路调控3T3-L1前脂肪细胞的成脂分化。     

Level of mannose-binding lectin (MBL) in patients with tuberculosis

Mannose-binding lectin (MBL) constitutes an important element in innate immune response against invading microorganisms. On the other hand, it has also been suggested that MBL, due their opsonic activity, might facilitate the uptake of certain intracellular pathogens. The aim of this study was to investigate whether MBL plays a role in tuberculosis. MBL serum concentration was determined in 68 patients with tuberculosis (TB), 30 patients with non-TB lung diseases and in 64 healthy controls. Serum MBL level was tested by an ELISA test with S. cerevisiae mannan-coated microtiter plates. Generally, TB patients had significantly higher MBL levels than both control groups, and in TB group percentage of individuals with highest MBL level was significantly higher (39.7%). These results indicate that high levels of MBL may be involved in the pathogenesis of tuberculosis and to be a relative disadvantage in mycobacterial infection.     

Functional polymorphisms in the mannan-binding lectin 2 gene: effect on MBL levels and otitis media

BACKGROUND: Mannan-binding lectin (MBL) can bind to microorganisms, initiating the lectin pathway of complement activation. Aberrant MBL serum levels, caused by MBL2 gene polymorphisms, are a possible risk factor for recurrent infections. Within the 7 common MBL haplotypes, still considerable variation in MBL serum levels exists. OBJECTIVE: To investigate functional MBL levels and MBL2 polymorphisms in a large cohort of children with recurrent acute otitis media. METHODS: Twelve genetic variants in the MBL2 gene and functional MBL serum levels were determined in a cohort of children with recurrent acute otitis media. Haplotypes were constructed and associated with functional MBL serum levels and the number of otitis episodes in the previous year. RESULTS: The 7 common MBL2 haplotypes mainly determine the level of functional MBL in serum. In addition, the 3130G>C single nucleotide polymorphism, located in exon 4, further significantly influenced functional MBL levels within the LXPA haplotype. LXPA carriers with 3130G showed a significantly lower geometric mean functional MBL serum level of 0.19 mug/mL compared with 0.70 mug/mL in 3130C carriers (P = .026). Nonwild-type MBL2 carriers between 12 and 24 months had a significantly increased number of otitis episodes (5.1/y) compared with wild-type MBL2 carriers (4.1/y; P = .027). In older children, this association was not found anymore. CONCLUSION: Additional single nucleotide polymorphisms within the 7 common haplotypes can further explain the observed variation in functional MBL serum levels. MBL seems to be of particular clinical importance during early childhood, when maternally derived antibodies have waned, and protective adaptive immunity is not well developed yet. CLINICAL IMPLICATIONS: Single nucleotide polymorphisms in the promoter region, in exon 1, and in exon 4 of MBL2 contribute to increased risk for otitis media in children younger than 2 years.     

The significance of IgG subclasses and mannan-binding lectin (MBL) for susceptibility to infection in apparently healthy adults with IgA deficiency.

The aim of this study was to investigate the significance of IgG subclasses and MBL for susceptibility to infection in association with IgA deficiency. The study population consisted of 139 apparently healthy adult blood donors with IgA deficiency and normal serum levels of IgG and IgM, and an increased susceptibility to infection demonstrated at a population level. Additionally, 216 controls matched for age and sex were investigated. IgG4 deficiency was more common and the mean level of IgG4 lower in persons with IgA deficiency than in the controls. No significant associations could be demonstrated between overt IgG subclass deficiencies and increased susceptibility to infection. However, when the mean concentrations of IgG subclasses were analysed with regard to medical history, that of IgG1 was lower in persons who reported recurrent viral respiratory infections, that of IgG3 in persons who had episodes of severe infection in their history, and that of IgG4 in persons who had recurrent mild respiratory infections, compared with those who had no particular history of infections. In contrast, MBL deficiency-alone or combined with that of the IgG subclass-was not associated with increased susceptibility to infection in persons with IgA deficiency. The results indicate that the proneness to infections observed in a population of otherwise healthy persons with IgA deficiency can only for a small part be accounted for by concomitant deficiencies of IgG subclasses. Contrary to expectations, no synergism between the deficiencies of IgA and MBL could be demonstrated.     

Mannose binding lectin (MBL) and HIV

The envelope protein (gp120/gp41) of HIV-1 is highly glycosylated with about half of the molecular mass of gp120 consisting of N-linked carbohydrates. While glycosylation of HIV gp120/gp41 provides a formidable barrier for development of strong antibody responses to the virus, it also provides a potential site of attack by the innate immune system through the C-type lectin mannose binding lectin (MBL) (also called mannan binding lectin or mannan binding protein). A number of studies have clearly shown that MBL binds to HIV. Binding of MBL to HIV is dependent on the high-mannose glycans on gp120 while host cell glycans incorporated into virions do not contribute substantially to this interaction. It is notable that MBL, due to its specificity for the types of glycans that are abundant on gp120, has been shown to interact with all tested HIV strains. While direct neutralization of HIV produced in T cell lines by MBL has been reported, neutralization is relatively low for HIV primary isolates. However, drugs that alter processing of carbohydrates enhance neutralization of HIV primary isolates by MBL. Complement activation on gp120 and opsonization of HIV due to MBL binding have also been observed but these immune mechanisms have not been studied in detail. MBL has also been shown to block the interaction between HIV and DC-SIGN. Clinical studies show that levels of MBL, an acute-phase protein, increase during HIV disease. The effects of MBL on HIV disease progression and transmission are equivocal with some studies showing positive effects and other showing no effect or negative effects. Because of apparently universal reactivity with HIV strains, MBL clearly represents an important mechanism for recognition of HIV by the immune system. However, further studies are needed to define the in vivo contribution of MBL to clearance and destruction of HIV, the reasons for low neutralization by MBL and ways that MBL anti-viral effects can be augmented.     

Detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (MBL) gene by polymerase chain reaction with sequence-specific primers

This study describes a new approach to the determination of all known mannan-binding lectin (MBL) mutations. The distribution of known variants of the MBL gene in a population of healthy unrelated Danes was determined and the genotype was correlated with the plasma MBL concentrations. The following genetic polymorphisms were studied: three point mutations in the promoter region at position -550 (H/L variants), -221 (X/Y variants), -70 (nt C or T), one point mutation in the 5' untranslated (UT) region at position +4 (P/Q variants) and three point mutations located at codons 52, 54 and 57 in exon 1 of the MBL gene, at nucleotide positions 223, 230 and 239, respectively. To perform genotyping, we designed sequence specific primers for a polymerase chain reaction (PCR-SSP). PCR-SSP is a powerful technique for the discrimination of alleles resulting from single base substitutions and is a widely used technique. Another major advantage of the PCR-SSP method is its ability to determine whether sequence motifs are in cis or trans. The frequencies of variants in exon 1 obtained by PCR-SSP were completely comparable to results obtained by previously described PCR methods, restriction fragment length polymorphism (RFLP) and site-directed mutagenesis (SDM). This PCR-SSP method is performed with standard laboratory equipment and has the capacity to detect all genetic variants in 100 samples in 2 days at an estimated total cost of GBP 11 per sample. Analysing the correlation between MBL haplotype and plasma MBL levels, we confirmed that three different structural variants, B, C and D and the promoter haplotypes HY, LY and LX have a dominant effect on the concentration of MBL. The HY haplotype is associated with the highest plasma concentration, the LY haplotype with intermediate levels and the LX haplotype with the lowest levels. The LX haplotype was found to be associated with very low levels of MBL similar to those found in association with the structural B genotype. The gene frequencies of variants in the MBL gene in the Danish population studied correspond to previous reports on Caucasian populations.     

儿童巨细胞病毒感染与甘露聚糖结合凝集素表达相关性研究

目的探讨甘露聚糖结合凝集素（MBL）基因多态性及血浆蛋白水平与儿童巨细胞病毒（HCMV）感染的相关性。方法收集2007年3～11月在浙江大学医学院附属儿童医院诊断为HCMV感染的患儿作为HCMV感染组,选择同期行健康体检的儿童作为对照组。对两组行MBL基因检测和分型,并对HCMV感染组进行随访,分别测定其急性期和恢复期血浆MBL蛋白水平。分别比较两组基因变异频率和血浆MBL蛋白水平。结果HCMV感染组纳入104例,对照组纳入105例;HCMV感染组有50例患儿进行随访,HCMV感染组MBL基因启动子区-550位点的L型变异频率明显高于对照组（56.7%vs34.3%,P=0.001）,野生单体基因型HYPA的频率明显低于对照组（47.6%vs62.8%,P=0.002）,完整基因型中高水平表达基因型（YA/YA）的频率低于对照组（41.3%vs60.0%,P=0.007）,而低水平表达基因型（YA/XA,YA/YB,XA/XA）的频率高于对照组（52.9%vs29.5%,P=0.001）。HCMV感染组急性期和恢复期血浆MBL蛋白水平均低于对照组（P=0.019和0.000）。HCMV感染组急性期血浆MBL蛋白水平高于恢复期（P=0.000）。结论MBL基因多态性导致的血浆MBL蛋白水平低下与儿童HCMV感染相关,提示MBL可能对儿童HCMV感染具有保护作用。     

Mannose-binding lectin (MBL) in adult patients with inflammatory bowel disease

Inflammatory bowel disease (IBD) refers to disorders associated with progressive inflammatory processes in the gastrointestinal system. IBD consists of two major forms, Crohn’s disease (CD), and ulcerative colitis (UC). IBD became a global disease in the 21^st century. Its pathogenesis is still not fully understood. Mannose-binding lectin (MBL) is a pattern-recognising molecule, involved in anti-microbial and anti-cancer immunity. It is able to opsonize microorganisms and abnormal host cells, and to initiate complement activation. The aim of this study was to investigate possible involvement of MBL in inflammatory bowel disease in adults. Forty persons diagnosed with CD and 28 with ulcerative colitis were recruited. The control group consisted of 136 healthy persons. Single nucleotide polymorphisms of the MBL2 gene (localised to both promoter and exon 1) were determined as were serum MBL concentrations. The exon 1 variant alleles and MBL deficiency-associated genotypes were more frequent among patients compared with controls, although this difference was not statistically significant. No differences of MBL levels were found between the major groups. However in MBL2 A/A homozygous IBD patients, the median was significantly higher than in corresponding healthy subjects. That was particularly evident in the case of active Crohn’s disease (1493 ng/ml vs. 800 ng/ml, p = 0.021). It may suggest that MBL and MBL-dependent complement activation contributes to excessive inflammation and its adverse effects in the course of CD. It cannot also be excluded that high MBL activity constitutes in some cases part of a multifactorial network conducing to development of CD.     

Elevated levels of mannan-binding lectin [corrected] (MBL) and eosinophilia in patients of bronchial asthma with allergic rhinitis and allergic bronchopulmonary aspergillosis associate with a novel intronic polymorphism in MBL

Mannan-binding lectin (MBL), an important component of innate immunity, binds to a range of foreign antigens and initiates the lectin complement pathway. Earlier studies have reported high plasma MBL levels in allergic patients in comparison to healthy controls. In view of varied plasma MBL levels being determined by genetic polymorphisms in its collagen region, we investigated the association of single nucleotide polymorphisms (SNPs) in the collagen region of human MBL with respiratory allergic diseases. The study groups comprised patients of bronchial asthma with allergic rhinitis (n = 49) and allergic bronchopulmonary aspergillosis (APBA) (n = 11) and unrelated age-matched healthy controls of Indian origin (n = 84). A novel intronic SNP, G1011A of MBL, showed a significant association with both the patient groups in comparison to the controls (P < 0.01). Patients homozygous for the 1011A allele showed significantly higher plasma MBL levels and activity than those homozygous for the 1011G allele (P < 0.05). The 1011A allele also showed a significant correlation with high peripheral blood eosinophilia (P < 0.05) and low forced expiratory volume in 1 s (FEV(1)) (P < 0.05) of the patients. We conclude that the 1011A allele of MBL may contribute to elevated plasma MBL levels and activity and to increased severity of the disease markers in patients of bronchial asthma with allergic rhinitis and ABPA.     

Low mannose-binding lectin (MBL) levels in neonates with pneumonia and sepsis

We investigated whether deficiency of mannose-binding lectin (MBL), a component of innate immunity, is associated with neonatal pneumonia and sepsis during the first 72 h, i.e. early onset, and during the first month after birth. In 88 neonatal intensive care patients (71 premature), MBL2 genotype and MBL plasma levels at birth were determined prospectively by Taqman analysis and enzyme-linked immunosorbent assay, respectively. Thirty-five neonates (40%) had low, i.e. 相似文献   

Severe fibrosis in hepatitis C virus-infected patients is associated with increased activity of the mannan-binding lectin (MBL)/MBL-associated serine protease 1 (MASP-1) complex

Mannan-binding lectin (MBL) binds microorganisms via interactions with glycans on the target surface. Bound MBL subsequently activates MBL-associated serine protease proenzymes (MASPs). A role for MBL in hepatitis C virus (HCV) infection had been indicated by previous studies examining MBL levels and polymorphisms in relation to disease progression and response to treatment. We undertook this study to investigate a possible relationship between disease progression and functional MBL/MASP-1 complex activity. A functional assay for MBL/MASP-1 complex activity was employed to examine serum samples from patients with chronic HCV infection, non-HCV liver disease and healthy controls. Intrapatient consistency of MBL/MASP-1 complex activity levels was assessed in sequential samples from a subgroup of patients. Median values of MBL/MASP-1 complex activity were higher in sera from patients with liver disease compared with healthy controls. MBL/MASP-1 complex activity levels correlate with severity of fibrosis after adjusting for confounding factors (P = 0.003). MBL/MASP-1 complex activity was associated more significantly with fibrosis than was MBL concentration. The potential role of MBL/MASP-1 complex activity in disease progression is worthy of further study to investigate possible mechanistic links.     

Mannose-binding lectin (MBL2) gene polymorphism in sickle cell anemia: an Egyptian study

Sickle cell disease (SCD) is an inherited disorder of sickle hemoglobin affecting millions of people worldwide. The current study aimed at detecting the prevalence of MBL2 exon-1(codons 52, 54, and 57) and promoter region (-221, X/Y) genetic polymorphisms in Egyptian children with SCD to clear out its possible role as a genetic risk factor for susceptibility to vaso-occlusive crisis (VOC) and/or infections. Genotyping of exon-1 and the promoter region was done by polymerase chain reaction for 50 SCD patients and 50 healthy controls. The frequency of MBL2 promoter polymorphism was 32% for the heteromutant genotype, Y/X and 8% for the homomutant genotype, and X/X with no statistical difference in the distribution of the mutant genotypes between SCD patients and controls. MBL2 exon-1 gene mutation in SCD patients was 18% for the heteromutant genotype A/O and 32% for the homomutant genotype O/O. The O/O genotype was significantly higher in SCD patients. Mutation at codon 57 of exon-1 (C allele) was significantly higher in SCD patients. The frequency of intermediate MBL2 expressers was significantly higher in the control group, while the frequency of low MBL2 expressers was higher among the patients. The distribution of MBL2 expressers did not differ between SCD patients with or without recurrent attacks of VOC. There was no association between MBL2 exon-1 or promoter region (-221 Y/X) genetic polymorphisms and the susceptibility to neither VOC nor infections in Egyptian children with SCD.     

Mannose-binding lectin (MBL) as prognostic factor in paediatric oncology patients

Deficiency of mannose-binding lectin (MBL) has been suggested to influence duration of febrile neutropenia and prognosis in paediatric oncology patients. However, there is no consensus on the definition of MBL deficiency. In a cohort of children with cancer, we investigated (i) how to determine MBL deficiency and (ii) whether MBL is a prognostic factor for disease severity. In 222 paediatric oncology patients, 92 healthy children and 194 healthy adults, MBL plasma levels and MBL2 genotype (wild-type: A, variant: O) were determined. Event-free survival (EFS), overall survival (OS) and paediatric intensive care unit (PICU) admissions were recorded prospectively. In febrile neutropenic patients admitted to the PICU, disease severity was assessed by clinical, microbiological and laboratory parameters. An optimal cut-off value for MBL deficiency was determined to be < 0·20 μg/ml. Wild-type MBL2 genotype patients, including the XA/XA haplotype, had increased MBL levels compared to healthy individuals. MBL deficiency was associated with decreased EFS (P = 0·03), but not with need for PICU admission. A trend for a twice increased frequency of septic shock (80% versus 38%, P = 0·14), multiple organ failure (40% versus 17%, P = 0·27) and death (40% versus 21%, P = 0·27) was observed in the absence of microbiological findings. MBL deficiency was associated with decreased EFS and possibly with an increased severity of disease during PICU admission after febrile neutropenia in the absence of any association with microbiological findings. These findings suggest prognosis to be worse in MBL-deficient compared to MBL-sufficient paediatric oncology patients.     

Relationship between gene polymorphisms of mannose-binding lectin (MBL) and two molecular forms of MBL

Mannose-binding lectin (MBL) activates complement through MBL-associated serine proteases (MASP). A deficiency in MBL due to mutations at exon 1 of the human MBL gene is reported to cause vulnerability to infection. We examined sera of known MBL genotype by gel filtration and assessed their elution patterns using an ELISA for MBL and identified two MBL forms, a high-molecular-mass form and a lower-molecular-mass form. By the identification of either or both forms in individual sera, three types of patterns emerged: type 1 consisted of a high-molecular form; type 2, of a low-molecular form; and type 3, of both forms. Types 1, 2 and 3 corresponded, respectively, to a wild type (A/A), a homozygous mutation at codon 54 (B/B) and their heterozygote (A/B). One exception was a heterozygous LXPA/LYPB phenotype that exhibited the type-2 pattern. Binding to mannan and MASP-1/3 occurred exclusively with the high-molecular form. An apparent MBL deficiency does not in fact representa deficiency in MBL molecules but rather the presence of circulating oligomeric mutant MBL with impaired function.     

Mannose-binding lectin (MBL2) polymorphisms and inflammation in hypertensive patients

We investigated the possible role of Mannose binding lectin 2 (MBL2) functional polymorphisms in the prevalence of hypertension and hypertensive end-organ damage in 300 hypertensive patients and 313 normotensive individuals from Southern Brazil. Hypertensive subjects with MBL2 AO/OO genotypes presented lower C-reactive protein levels than AA individuals and consequently lower inflammatory status.     

High prevalence of mannose-binding lectin (MBL) deficiency in premature neonates

Mannose-binding lectin (MBL) is a component of innate immunity and thus particularly important in neonates in whom adaptive immunity is not yet completely developed. Promoter polymorphisms and structural exon-1 mutations in the MBL2 gene cause reduced or deficient MBL plasma concentrations. The aim of our study was to determine the prevalence of MBL deficiency in neonates admitted to the neonatal intensive care unit (NICU). Eighty-five NICU patients (69 premature) were included in the study. We measured MBL concentrations in umbilical cord and neonatal blood within 24 h after birth by ELISA technique. MBL2 genotypes (n = 67) were determined by Taqman analysis. MBL concentrations were measured longitudinally during three weeks in 26 premature neonates. The association between pre- and intra-partum clinical data and MBL concentrations was investigated. At birth, 29 (42%) premature and six (38%) term neonates had MBL plasma concentrations < or = 0.7 microg/ml which was regarded as deficient. Twenty-one (38%) premature and four (36%) term neonates had variant MBL2 haplotypes, corresponding to exon-1 mutations and the LXPA haplotype. MBL concentrations increased over time in neonates with wild-type MBL2 haplotypes, but not in neonates with variant haplotypes. Low MBL plasma concentrations were related to lower gestational age and variant MBL2 haplotypes. Umbilical cord and neonatal MBL plasma concentrations appeared to be similar. In conclusion, almost half of our NICU patients, especially the premature ones, were MBL-deficient at birth. These infants may be at increased risk of neonatal infections. MBL concentration can reliably be measured in umbilical cord blood and it is positively correlated with gestational and postnatal age.     

Mannan-binding lectin (MBL) in chickens: molecular and functional aspects

Mannan-binding lectin (MBL) is a serum collectin (i.e. mosaic protein with collagenous and lectin domains) involved in the innate immune defence against various microbes. In vitro studies indicate that MBL exerts its function by binding to the microbial surface through its carbohydrate recognition domains followed by direct opsonization or complement activation via the MBL associated serine proteases MASP-1 and MASP-2. In Aves (i.e. chickens), as in man, only one MBL form has been found, while traditional laboratory animals (i.e. mouse and rat) have two MBL forms in serum. MBL has been extensively studied in mammals but recently also in Aves. This review summarizes the present knowledge of MBL in chickens and compares it to the situation in mammals.