Biomedical subjects

S Thiel

Publications and source records attributed to S Thiel.

At least 73 records · Page 4Linked to original sources

PubMed1998

Constitutive internalization and association with adaptor protein-2 of the interleukin-6 signal transducer gp130.

The transmembrane protein gp130 is the common signalling receptor subunit for the interleukin-6 (IL-6)-type cytokines. It has recently been shown that the cytoplasmic domain of gp130 contains a dileucine internalization motif and that endocytosis of gp130 occurs signal-independent. Here, we have studied whether gp130 itself undergoes constitutive internalization or whether its endocytosis is stimulated by formation of the IL-6/IL-6R/gp130 complex. Using two different assays, we found that gp130 is internalized independent from IL-6/IL-6R stimulation. In addition, we show that gp130 is constitutively associated with the cell surface adaptor complex AP-2. Our findings strongly suggest endocytosis of gp130 to be constitutive.

Adaptor Protein Complex alpha Subunits↗

PubMed1998

Mannan-binding lectin in human serum, cerebrospinal fluid and brain tissue and its role in Alzheimer's disease.

Mannan-Binding lectin (MBL) is a serum lectin which can activate the classical complement pathway. Complement proteins of the classical pathway have been found in the brains of patients with Alzheimer's disease (AD) in association with AD brain pathology. To investigate the role for MBL in AD we have looked for its presence in the brain by immunohistochemistry and determined the levels of MBL in paired samples of cerebrospinal fluid and serum from AD patients and controls. MBL was detected in association with blood vessels in the brain tissue of both AD patients and control subjects. There was no apparent difference in the distribution of MBL in the brain tissue between the two groups. The mean concentration of MBL in the CSF was 44% lower in AD patients than in controls (AD 154 +/- 35 pg/ml, n = 19; non-AD 276 +/- 50 pg/ml, n = 15, p < 0.05). The levels of MBL in serum were not significantly different in the two groups. Thus, this study shows that MBL is associated with blood vessels in the brains of both AD and control subjects. Moreover, CSF levels of MBL appear to be lower in AD patients than in control subjects which may indicate a higher degree of MBL consumption connected with complement activation in the AD patients.

Aged↗

PubMed1998

Internalization of the interleukin 6 signal transducer gp130 does not require activation of the Jak/STAT pathway.

Signalling receptors often undergo receptor-mediated endocytosis. In many cases this internalization is stimulated by ligand binding and activation of intrinsic receptor tyrosine kinases, resulting in a receptor down-regulation. We have analysed whether internalization of the interleukin 6 signal transducer gp130 is dependent on the activation of receptor-associated Jak kinases. By using a chimaeric receptor system we found that receptor mutants that lack box1 and therefore are not capable of activating Jak and signal transducer and activator of transcription (STAT) proteins are still endocytosed efficiently. A chimaeric receptor with the recently identified dileucine internalization motif being replaced by two alanine residues was not efficiently internalized but still capable of recruiting STATs. Furthermore an antagonistic antibody that inhibits the signalling of all interleukin-6-type cytokines via gp130 was internalized as efficiently as an agonistic one that activates the Jak/STAT pathway. Our findings suggest that the endocytosis of gp130 is signal-independent.

Animals↗

PubMed1998

MASP-2, the C3 convertase generating protease of the MBLectin complement activating pathway.

Mannan-binding lectin (MBL) activates the complement system through cleavage of C4 and C2. Until recently it was thought that only one serine protease in complex with MBL (MBL-associated serine protease, MASP) mediates complement activation, but with the finding of a second MBL-associated serine protease, MASP-2, the activation process appears more elaborate, possibly resembling that of the C1 complex. The two MASPs share the domain organisation of C1r and C1s and it may be speculated that interaction between the two MASPs is required for complement activation in the same manner as with the C1 proteases. We have demonstrated that MASP-2 is a C4 cleaving component of the MBL/MASP complex. By analogy, one may thus speculate that, upon binding of MBL to carbohydrate, MASP-1 autoactivates and then activates MASP-2, but there is as yet no evidence for this. The components of C1 are present in serum in approximately equimolar amounts, whereas MASP-1 is in large excess over MBL. Pairwise comparison of the four proteases shows the primary structures to be approximately 40% identical. Phylogenetic analysis indicates that MASP-2 is closer to C1r and C1s than is MASP-1, but no particular association between MASP-2 and the C4 cleaving enzyme, C1s, can be deduced from sequence comparison.

Animals↗

PubMed1998

Cloning and sequencing of a cDNA encoding chicken mannan-binding lectin (MBL) and comparison with mammalian analogues.

The serum lectin, mannan-binding lectin (MBL) (also denoted mannan-binding protein or mannose-binding protein, MBP) has been identified in mammals (humans, monkey, cow, rabbit, mouse and rat). Upon binding to carbohydrates on the surface of microorganisms, MBL mediates activation of the complement system, leading to killing of the microorganism. MBL thus exerts a role in the innate immune defence. We have described the isolation and partial characterization of an analogous protein in chicken serum. Oligonucleotides based on the N-terminal sequence of this protein were used in a reverse transcription-polymerase chain reaction (RT-PCR) with chicken liver RNA as template. The PCR product was sequenced and found to encode part of the NH2 terminus of chicken MBL. A perfect match probe was synthesized and used to screen a chicken liver cDNA library. The isolated clones carried a cDNA insert of 1692 bp with an open reading frame of 714 bp encoding a mature protein of 238 amino acids including a signal peptide of five amino acids. The deduced amino acid sequence agrees with those determined by conventional amino acid sequence analysis of the peptides except for four residues. We have compared the deduced primary structure of chicken MBL with the mammalian analogues. The phylogenetic analysis indicates that the gene duplication leading to two different MBL forms in mammals occurred after the split from birds and reptiles. This concurs with the finding of only one form of MBL in chickens.

Amino Acid Sequence↗

PubMed1998

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.

Animals↗

PubMed1998

Reconstitution of opsonizing activity by infusion of mannan-binding lectin (MBL) to MBL-deficient humans.

Mannan-binding lectin (MBL, previously named mannan-binding protein, MBP) is a serum collectin, which activates complement upon binding to microbial carbohydrates. This results in opsonization of the microorganisms as well as direct complement-mediated killing. Clinical evidence indicates that MBL has an important role in the innate immune defence against various pathogens. Genetically determined MBL deficiency is associated with increased susceptibility to infections. We have infused two MBL-deficient individuals with clinical grade MBL, purified from pooled donor plasma, in doses sufficient to attain normal concentration of MBL in serum. This resulted in normalization of complement-mediated opsonization. An initial rapid decrease in the serum concentration of MBL was followed by a slower decline with an estimated half-life of about 6 days. No adverse effects were observed and anti-MBL antibodies could not be detected following several MBL infusions. One of the two MBL recipient, a two-year-old girl, who had been suffering from repeated infections from the age of 4 months, was given a total of six MBL infusions. She has subsequently remained healthy for more than three years.

Adult↗

PubMed1997

A second serine protease associated with mannan-binding lectin that activates complement.

The complement system comprises a complex array of enzymes and non-enzymatic proteins that is essential for the operation of the innate as well as the adaptive immune defence. The complement system can be activated in three ways: by the classical pathway which is initiated by antibody-antigen complexes, by the alternative pathway initiated by certain structures on microbial surfaces, and by an antibody-independent pathway that is initiated by the binding of mannan-binding lectin (MBL; first described as mannan-binding protein) to carbohydrates. MBL is structurally related to the complement C1 subcomponent, C1q, and seems to activate the complement system through an associated serine protease known as MASP (ref. 4) or p100 (ref. 5), which is similar to C1r and C1s of the classical pathway. MBL binds to specific carbohydrate structures found on the surface of a range of microorganisms, including bacteria, yeasts, parasitic protozoa and viruses, and exhibits antibacterial activity through killing mediated by the terminal, lytic complement components or by promoting phagocytosis. The level of MBL in plasma is genetically determined, and deficiency is associated with frequent infections in childhood, and possibly also in adults (for review, see ref. 6). We have now identified a new MBL-associated serine protease (MASP-2) which shows a striking homology with the previously reported MASP (MASP-1) and the two C1q-associated serine proteases C1r and C1s. Thus complement activation through MBL, like the classical pathway, involves two serine proteases and may antedate the development of the specific immune system of vertebrates.

Amino Acid Sequence↗

PubMed1997

The effect of mannan-binding lectin on opsonophagocytosis of Neisseria meningitidis.

Mannan-binding lectin (MBL), an acute phase protein with a structure and a function very similar to that of C1q, is known to act as an opsonin binding to a number of microorganisms. In order to investigate the effect of MBL on the phagocytic killing of meningococci, a serogroup B meningococcal strain (H44/76) and its unencapsulated variant v24, as well as a serogroup A meningococcal strain were opsonized with MBL (purified from normal human plasma at the State Serum Institute, Denmark) and used in a phagocytic killing assay at a density of 7 x 10(3) CFU/ml. Polymorphonuclear cells (PMNs) from one healthy donor were isolated by density gradient centrifugation over Percoll and added to the system (7 x 10(6) cells/ml). In a first set of experiments without addition of serum or complement, no influence of MBL was observed on the killing of any of these strains. Addition of MBL to non-opsonized bacteria of the serogroup A strain did not result in enhanced killing either; on the contrary, the growth of this strain increased significantly when a high MBL concentration (40 micrograms/ml) was used in the presence of PMNs. Further investigations were performed using sera of five individuals with late complement component deficiency (LCCD) and a concomitant MBL deficiency, vaccinated with a tetra-valent (ACYW135) meningococcal capsular polysaccharide vaccine. Pre- and post-vaccination sera (50% final concentration) were tested against a group A strain opsonized or not with MBL. In only one patient was there a moderate increase of killing of the opsonized bacteria after vaccination compared to pre-vaccination serum. Our results suggest that MBL may not play a significant role in the opsonophagocytosis of meningococci, irrespective of its binding to unencapsulated and serogroup A strains.

Bacterial Vaccines↗

PubMed1997

Mannan-binding lectin serum concentrations in HIV-infected patients are influenced by the stage of disease.

Serum concentrations of mannan-binding lectin (MBL) were determined in the sera of 67 HIV-seropositive patients in different stages of HIV disease and in the sera of 75 HIV-seronegative healthy individuals. In the asymptomatic (AS) HIV-infected persons MBL concentrations were found to be significantly (P < 0.05) lower than in the HIV-seronegative controls, whereas in the AIDS patients they were not. Very low (< or = 25 ng/ml) MBL serum concentrations were detected in 5/19 (26.3%) and 7/75 (9.3%) of the AS HIV-seropositive and HIV-seronegative individuals, respectively (P = 0.06). In the sera of the HIV-infected patients, MBL levels positively correlated to the neopterin concentrations (Spearman correlation coefficient, 0.401, P = 0.0009) while they negatively correlated to the percentage (-0.447, P = 0.0011) and absolute number (-0.453, P = 0.0012) of the CD4+ lymphocytes. These observations indicate that MBL level, which is under strict genetic control, may influence the susceptibility to HIV infection and the progression of HIV disease.

Blood Proteins↗

PubMed1997

A rainbow trout lectin with multimeric structure.

A novel lectin has been identified in rainbow trout serum and plasma. The lectin binds to Sepharose (an agarose polymer) in a calcium-dependent manner. Glucose, N-acetyl-glucosamine, mannose, N-acetyl-mannosamine, L-fucose, maltose and alpha-methyl-mannoside are good inhibitors of this binding, whereas glucosamine and D-fucose inhibits to a lesser degree and mannosamine and galactose do not inhibit the binding to Sepharose. When analysed by SDS-PAGE under non-reducing conditions, the lectin appears as a characteristic ladder of bands with approximately 16 kDa between consecutive bands. Upon reduction, the lectin appears as a 16-kDa band. On size-exclusion chromatography of trout serum and plasma, the protein emerges over a broad range corresponding to sizes from about 2000 kDa to less than 200 kDa. The NH2-terminal sequence (AAENRNQXPPG) shows no significant homology with known proteins. Because of the characteristic appearance in non-reducing SDS-PAGE and the lectin activity, we propose to name the protein "ladderlectin."

Animals↗

PubMed1997

Modification of global erythemally effective irradiance by clouds.

The role clouds play in the modification of global radiation is still a major uncertainty in the risk assessment of UV effects on ecological systems and human health. This study presents cloud transmission data obtained from measurements with Robertson-Berger meters and simultaneous cloud observations. The global transmission of erythemally weighted irradiance depends strongly on cloud amount and can be described by a cubic function. The comparison with results derived from long-term records of total global irradiance indicates no statistically significant difference between the attenuation of erythemal and total global radiation. The large variance of data results from lumping together data from different cloud types. Classification of data according to cloud forms yields a more satisfactory fit. The coefficient of the cubic term characterizes the ability of various cloud forms to attenuate UV radiation. It varies between 0.4 for high clouds and approximately 1.0 for cumulonimbus. This attenuation parameter allows a quantitative description of the cloud influence on irradiance and therefore a more accurate risk assessment.

Algorithms↗

PubMed1996

Improvements on the purification of mannan-binding lectin and demonstration of its Ca(2+)-independent association with a C1s-like serine protease.

Mannan-binding lectin (MBL), previously called 'mannan-binding protein' or MBP, is a plasma C-type lectin which, upon binding to carbohydrate structures on micro-organisms, activates the classical pathway of complement. Purification of MBL relies on its Ca(2+)-dependent affinity for carbohydrate, but existing methods are susceptible to contamination by anti-carbohydrate antibodies. In the present study a sequential-sugar-elution method has been developed which can achieve a preparation of virtually pure MBL and its associated serine protease (MBL-associated serine protease, MASP) by two steps of affinity chromatography. In further separation of MASP from MBL, it was found that activated MASP was associated with MBL independent of Ca2+. Since MBL was found to bind to underivatized Sepharose 4B, the MBL-MASP complex was purified using Sepharose 4B and protease inhibitors were included to purify the complex with MASP in its proenzyme form. Analysis of thus-purified MBL-MASP complex by gel filtration on a Sephacryl S-300 column at pH 7.8 showed that the proenzyme MASP was also associated with MBL independently of Ca2+, but that the complex could be disrupted at a low pH (5.0). Therefore the mechanism of MBL-MASP-mediated complement activation appears to be significantly different from the C1-mediated classical pathway.

Calcium↗

PubMed1996

Glycopeptide mimics of mammalian Man9GlcNAc2. Ligand binding to mannan-binding proteins (MBPs).

A novel and simple approach for rational design of oligosaccharide mimics has been developed. Mammalian high-mannose triantennary structure Man9GlcNac2 has been subjected to molecular modelling using the NMR data available on structural fragments of the oligosaccharide. The analysis indicated four different low energy conformations, and the spatial arrangement of terminal disaccharides of the oligosaccharide antennae were stimulated with glycopeptides carrying disaccharides by applying weak constraints between the saccharide parts in MD-simulations on a large array of tri- to octaglycopeptides. The five glycopeptides exhibiting the best fit with the four minimum energy confirmations of the oligosaccharide were synthesized by solid phase glycopeptide assembly using glycosylated fluoren-9-ylmethyloxycarbonyl-amino acid-O-pentafluorophenyl esters as building blocks. The glycan was acyl protected alpha-D-Man-(1-->2)-alpha-D-Man and Ser, Thr and Hyp were the glycosylated amino acids. The deprotected and purified glycopeptides were subjected to NMR analysis for characterization, and in order to investigate the cis-trans isomerism of the carbimide bonds to Hyp. The glycopeptides were tested for their ability to inhibit binding of mannan-binding protein to mannan from Saccharomyces cerevisiae. They were found to be weak inhibitors showing no indication of multivalent interaction with the mannan-binding protein.

Carbohydrate Conformation↗

PubMed1996

Human autoantibodies against Clq: lack of cross reactivity with the collectins mannan-binding protein, lung surfactant protein A and bovine conglutinin.

The collectins, a group of humoral C-type lectins, have globular and collagen-like regions and share structural features with the complement protein C1q. The question was asked if autoantibodies to the collagen-like region of C1q (anti-C1qCLR) might cross-react with collectins, such as mannan-binding protein (MBP), lung surfactant protein A (SP-A) and bovine conglutinin (BK). Anti-C1qCLR antibodies of the systemic lupus erythematosus (SLE) type and anti-C1qCLR antibodies of the hypocomplementemic urticarial vasculitis syndrome (HUVS) type were investigated. Cross-absorption and elution experiments combined with antibody detection by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis gave no evidence of cross-reactive anti-C1qCLR antibodies. However, one serum with HUVS type anti-C1qCLR antibodies contained anti-MBP antibodies that were cross-reactive with SP-A. Judging from results of ELISA inhibition experiments and immunoblot analysis, four SLE sera contained antibodies to native BK, while two sera with HUVS type anti-C1qCLR antibodies contained antibodies to epitopes of denatured BK. This might imply that autoimmunity to collagen-like structures is not restricted to C1qCLR in HUVS and HUVS/SLE overlap syndromes.

Animals↗

PubMed1995

Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein.

Mannan-binding protein (MBP) is a serum lectin participating in the innate immune defense by opsonizing various microorganisms for phagocytosis. Opsonization defect due to MBP deficiency and low levels of the protein can partially be explained by the dominant effect of three different mutations in the structural part of the MBP gene. Large interracial differences in the frequencies of these variants have previously been described, but they cannot explain the large interindividual variation in MBP serum concentration. We describe the existence of additional polymorphisms at positions -550 (H/L variants) and -221 (X/Y variants) in the promoter region of the gene. The promoter haplotypes, HY, LY, and LX, show associations with high, medium, and low levels of MBP serum concentrations, respectively. Moreover, this represents a genetic system with additive effect of haplotypes in which a low producing LX haplotype in the homozygous state down-regulates the basal expression of MBP as effectively as a single structural variant. Populations of pure Eskimos, Caucasoids, and black Africans show marked interethnic differences in the frequencies of promoter haplotypes regulating the expression of the normal peptide, with the HY haplotype frequency varying from 0.83 in Eskimos via 0.33 in Caucasoids to 0.08 in Africans. The LY haplotype frequency varies from 0.04 in Eskimos via 0.39 in Caucasoids to 0.23 in Africans. The LX haplotype frequency varies from 0.03 in Eskimos via 0.24 in Caucasoids to 0.23 in Africans. The effect of the promoter variants can explain almost all of the ethnic differences not explainable by the structural variants alone.

Base Sequence↗

PubMed1995

Use of factorial experimental design to delineate the strong calcium- and pH-dependent changes in binding of human surfactant protein-A to neutral glycosphingolipids--a model for studies of protein-carbohydrate interactions.

Human surfactant protein-A (SP-A) is a C-type lectin belonging to the collection supergroup of mammalian lectins. It is produced by alveolar type II cells and has been shown to bind to lactosylceramide (R. A. Childs, J. R. Wright, G. F. Ross, C-T Yuen, A. M. Lawson, C. Chai, K. Drickamer, and T. Feizi (1992) J. Biol. Chem. 267, 9972-9979), galactosylceramide, and gangliotriaosylceramide (Y. Kuroki, S. Gasa, Y. Ogasawara, A. Makita, and T. Akino (1992) Arch. Biochem. Biophys. 299, 261-267). To evaluate the pH- and calcium-dependent binding of SP-A to neutral glycosphingolipids we employed a microtiter plate technique and performed a series of full factorial design experiments using lactosylceramide, galactosylceramide, and gangliotetraosylceramide and native nonderivatized SP-A. The optimal binding conditions were drastically different for these three receptors. At pH 7.4 and at 5 mM Ca concentration the binding affinity of SP-A followed the order galactosylceramide > lactosylceramide > gangliotetraosylceramide. However, this was close to optimal conditions for binding of SP-A to lactosylceramide and at minimum for binding to gangliotetraosylceramide. Binding of SP-A to galactosylceramide was relatively insensitive to pH but increased significantly with increasing Ca concentrations. These experiments using factorial experimental design emphasize the importance of critical interpretation of all earlier studies on protein-carbohydrate interactions especially when transferring experimental data into complex biological systems.

Animals↗

PubMed1995

Collectin in a non-mammalian species: isolation and characterization of mannan-binding protein (MBP) from chicken serum.

A chicken serum lectin was isolated by affinity chromatography on TSK-75 beads derivatized with the monosaccharide N-acetyl-D-mannosamine (ManNAc). Serum was applied to the column in a Ca(2+)-containing buffer and proteins were eluted with EDTA. After recalcification, the eluate was passed through a new ManNAc-derivatized column. Bound proteins were eluted with 50 mM ManNAc. Anti-carbohydrate antibodies present in the eluate were removed by passage through a rabbit anti-chicken immunoglobulin derivatized column, and the lectin was further purified by ion-exchange chromatography and gel-permeation chromatography. The purified chicken lectin shows an overall structure similar to mammalian mannan-binding protein (MBP). SDS-PAGE revealed two polypeptides of M(r) 33 and 34 kDa (reduced) with identical sequence for the first 30 NH2-terminal residues. The NH2-terminal sequence shows 43% identity with the human MBP. Like mammalian MBP, the polypeptides of the chicken lectin are degraded by treatment with collagenase. Residues 26-30 (G-L-P(OH)-G-D) are likely to represent the beginning of the collagenous region. Mobilities on SDS-PAGE of the COOH-terminal collagenase-resistant fragment under reduced and non-reduced conditions indicate the presence of intrachain disulphide bonds, as are also found in mammalian MBP. Gel chromatography showed an intact mol. wt of 750 kDa. Binding of the chicken MBP to mannan was inhibited by monosaccharides in the following order of potency: ManNAc > L-fucose > mannose > N-acetylglucosamine. Other monosaccharides inhibited poorly or not at all. Chicken MBP, bound to mannan, activated the classical complement pathway in human serum. Electron micrographs show structures and dimensions resembling human MBP. Overall, the results show that the purified lectin is the chicken homologue to mammalian MBP and indicate the presence of a MBP-like clearance system outside mammals.

Amino Acid Sequence↗