Polymorphisms in the promoter region and at codon 54 of the MBL2 gene are not associated with IgA nephropathy.
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BACKGROUND: IgA nephropathy (IgAN) occurs sporadically in unrelated individuals. Several different polymorphic genes have been investigated in recent years in order to demonstrate their possible association with IgAN. Three recent, different studies with conflicting conclusions have discussed the role of the mannose binding lectin (MBL), a serum lectin involved in natural immunity, in the IgAN pathogenesis by examination of MBL deposits in biopsies. In the present study we investigated several polymorphisms of the MBL gene located in the promoter region and in the first exon. METHODS: MBL polymorphism detection was performed in 22 Italian patients with familial IgA nephropathy and in 138 Italian patients with the sporadic form of the disease. The polymorphisms in the MBL2 promoter region and in the exon 1 were investigated, respectively, by direct sequencing and by amplification refractory mutation system-polymerase chain reaction on genomic DNA collected from peripheral blood. Seventy-four unrelated healthy subjects matched for ethnic origin were used as controls. RESULTS: Allelic and genotypic frequencies of the polymorphisms at position -550, -328, -221 and at codon 54 did not show any differences between patients and controls. Similar frequency distributions of these polymorphisms were also found in the subgroups of IgAN patients subdivided according to the clinical manifestations and the progression of the disease. CONCLUSIONS: This study indicates that the analysed polymorphisms of the MBL gene do not appear to be primarily involved in the susceptibility and severity of IgAN. (+info)
An extended conformation of the macrophage mannose receptor.
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The macrophage mannose receptor mediates phagocytosis of pathogenic microorganisms and endocytosis of potentially harmful soluble glycoproteins by recognition of their defining carbohydrate structures. The mannose receptor is the prototype for a family of receptors each having an extracellular region consisting of 8-10 domains related to C-type carbohydrate recognition domains (CRDs), a fibronectin type II repeat and an N-terminal cysteine-rich domain. Hydrodynamic analysis and proteolysis experiments performed on fragments of the extracellular region of the receptor have been used to investigate its conformation. Size and shape parameters derived from sedimentation and diffusion coefficients indicate that the receptor is a monomeric, elongated and asymmetric molecule. Proteolysis experiments indicate the presence of close contacts between several pairs of domains and exposed linker regions separating CRDs 3 and 6 from their neighboring domains. Hydrodynamic coefficients predicted for modeled receptor conformations are consistent with an extended conformation with close contacts between three pairs of CRDs. The N-terminal cysteine-rich domain and the fibronectin type II repeat appear to increase the rigidity of the molecule. The rigid, extended conformation of the receptor places domains with different functions at distinct positions with respect to the membrane. (+info)
The mannose/N-acetylgalactosamine-4-SO4 receptor displays greater specificity for multivalent than monovalent ligands.
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Recognition of carbohydrates on glycosylated molecules typically requires multivalent interactions with receptors. Monovalent forms of terminal saccharides engaged by the receptor binding sites typically display weak affinities in the mm range and poor specificity. In contrast, multivalent forms of the same saccharides are bound with strong affinity (10(-7)-10(-9) m) and significantly greater specificity. Although multivalency can readily account for increased affinity, the molecular basis for enhanced specificity is not well understood. We have examined the specificity of the cysteine-rich domain of the mannose/GalNAc-4-SO4 receptor using monovalent and multivalent forms of the trisaccharide GalNAcbeta1,4GlcNAcbeta1,2Manalpha (GGnM) sulfated at either the C4 (S4GGnM) or C3 (S3GGnM) hydroxyl of the terminal GalNAc. Monovalent S4GGnM and S3GGnM have K(i) values of 25.8 and 16.2 microm, respectively. Multivalent conjugates of the same GalNAc-4-SO4- and GalNAc-3-SO4-bearing trisaccharides (6.7 mol of trisaccharide/mol of bovine serum albumin) have K(i) values of 0.013 and 0.170 microm, respectively. The 2000-fold versus 95-fold change in affinity seen for the multivalent forms of these 4-sulfated and 3-sulfated trisaccharides reflects a difference in the impact of conformational entropy. A large fraction of the SO4-3-GalNAc structures exists in a form that is not favorable for binding to the Cys-rich domain. This reduces the effective concentration of SO4-3-GalNAc as compared with SO4-4-GalNAc under the same conditions and results in a markedly lower association rate. This difference in association rate accounts for the 12-fold difference in the rate of clearance from the blood seen with S4GGnM-BSA and S3GGnM-BSA in vivo. (+info)
In vivo recognition of mannosylated proteins by hepatic mannose receptors and mannan-binding protein.
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In vivo recognition of mannosylated proteins by hepatic mannose receptors and serum mannan-binding protein (MBP) was investigated in mice. After intravenous administration, all three different (111)In-mannosylated proteins were taken up mainly by liver, and uptake was saturated with increasing doses. (111)In-Man-superoxide dismutases and (111)In-Man(12)- and (111)In-Man(16)-BSA had simple dose-dependent pharmacokinetic profiles, whereas other derivatives ((111)In-Man(25)-, -Man(35)-, and -Man(46)-BSA and (111)In-Man-IgGs) showed slow hepatic uptake at <1 mg/kg. Purified MBP experiments in vitro indicated that these derivatives bind to MBP in serum after injection, which interferes with their hepatic uptake. To quantitatively evaluate these recognition properties in vivo, a pharmacokinetic model-based analysis was performed for (111)In-Man-BSAs, estimating some parameters, including the Michaelis-Menten constant of the hepatic uptake and the dissociation constant of MBP, which correlate to the affinity of Man-BSAs for mannose receptors and MBP, respectively. The dissociation constant of Man-BSA and MBP decreased dramatically with increasing density of mannose, but the Michaelis-Menten constant of hepatic uptake of Man-BSA was not so sensitive to the change in density. This suggests that the in vivo recognition of MBP has a stronger cluster effect than that of mannose receptors. Differences obtained here are due to the unique arrangement of carbohydrate recognition domains on each mannose-specific lectin available for mannosylated ligand recognition. (+info)
Polymorphisms of the mannose binding lectin gene in patients with Sjogren's syndrome.
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OBJECTIVE: To investigate polymorphisms of both codon 54 allele and promoter variants of the mannose binding lectin (MBL) gene in patients with primary Sjogren's syndrome (SS). METHODS: Polymorphisms of codon 54 allele and promoter variants of the MBL gene in 104 patients with SS and 143 healthy controls were determined by polymerase chain reaction-restriction fragment length polymorphism and allele specific polymerase chain reaction respectively. RESULTS: The allele frequency of the wild type of MBL codon 54 was significantly higher in patients with SS than in controls (0.836 v 0.741; p=0.011), and the frequency of the homozygous wild type of MBL codon 54 was significantly higher in patients with SS than in controls (0.692 v 0.539; p=0.024). On the other hand, the allele frequencies of the MBL promoter gene did not differ between patients and controls (chi(2)=4.01, df=2, p=0.135). CONCLUSION: The polymorphism of the MBL gene may be one of the genetic factors that determines susceptibility to SS. (+info)
Quantitative ER <--> Golgi transport kinetics and protein separation upon Golgi exit revealed by vesicular integral membrane protein 36 dynamics in live cells.
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To quantitatively investigate the trafficking of the transmembrane lectin VIP36 and its relation to cargo-containing transport carriers (TCs), we analyzed a C-terminal fluorescent-protein (FP) fusion, VIP36-SP-FP. When expressed at moderate levels, VIP36-SP-FP localized to the endoplasmic reticulum, Golgi apparatus, and intermediate transport structures, and colocalized with epitope-tagged VIP36. Temperature shift and pharmacological experiments indicated VIP36-SP-FP recycled in the early secretory pathway, exhibiting trafficking representative of a class of transmembrane cargo receptors, including the closely related lectin ERGIC53. VIP36-SP-FP trafficking structures comprised tubules and globular elements, which translocated in a saltatory manner. Simultaneous visualization of anterograde secretory cargo and VIP36-SP-FP indicated that the globular structures were pre-Golgi carriers, and that VIP36-SP-FP segregated from cargo within the Golgi and was not included in post-Golgi TCs. Organelle-specific bleach experiments directly measured the exchange of VIP36-SP-FP between the Golgi and endoplasmic reticulum (ER). Fitting a two-compartment model to the recovery data predicted first order rate constants of 1.22 +/- 0.44%/min for ER --> Golgi, and 7.68 +/- 1.94%/min for Golgi --> ER transport, revealing a half-time of 113 +/- 70 min for leaving the ER and 1.67 +/- 0.45 min for leaving the Golgi, and accounting for the measured steady-state distribution of VIP36-SP-FP (13% Golgi/87% ER). Perturbing transport with AlF(4)(-) treatment altered VIP36-SP-GFP distribution and changed the rate constants. The parameters of the model suggest that relatively small differences in the first order rate constants, perhaps manifested in subtle differences in the tendency to enter distinct TCs, result in large differences in the steady-state localization of secretory components. (+info)
In vivo-matured Langerhans cells continue to take up and process native proteins unlike in vitro-matured counterparts.
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We have been able to identify the cell subset derived from Langerhans cells in the total dendritic cell population of the peripheral lymph node and hence to follow their trafficking under normal physiological conditions as well as upon skin irritation. As expected, the rapid mobilization of Langerhans cells triggered by inflammatory signals into the draining lymph node correlated with an up-regulation of costimulatory molecules and with an enhanced immunostimulatory capacity. Surprisingly, however, these cells, instead of shutting down, maintain the capacity to capture and process protein Ags during the couple of days they stay alive in stark contrast to in vitro-matured dendritic cells. (+info)
Mannosylated lipoarabinomannans inhibit IL-12 production by human dendritic cells: evidence for a negative signal delivered through the mannose receptor.
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IL-12 is a key cytokine in directing the development of type 1 Th cells, which are critical to eradicate intracellular pathogens such as Mycobacterium tuberculosis. Here, we report that mannose-capped lipoarabinomannans (ManLAMs) from Mycobacterium bovis bacillus Calmette-Guerin and Mycobacterium tuberculosis inhibited, in a dose-dependent manner, the LPS-induced IL-12 production by human dendritic cells. The inhibitory activity was abolished by the loss of the mannose caps or the GPI acyl residues. Mannan, which is a ligand for the mannose receptor (MR) as well as an mAb specific for the MR, also inhibited the LPS-induced IL-12 production by dendritic cells. Our results indicate that ManLAMs may act as virulence factors that contribute to the persistence of M. bovis bacillus Calmette-Guerin and M. tuberculosis within phagocytic cells by suppressing IL-12 responses. Our data also suggest that engagement of the MR by ManLAMs delivers a negative signal that interferes with the LPS-induced positive signals delivered by the Toll-like receptors. (+info)