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Uffe Holmskov - One of the best experts on this subject based on the ideXlab platform.
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a simple two step purification procedure for the ic3b binding collectin Conglutinin
Journal of Immunological Methods, 2010Co-Authors: Thomas Kroghmeibom, I Tornoe, K L Ingvartsen, Nades Palaniyar, Anthony C Willis, Uffe HolmskovAbstract:Bovine Conglutinin is a serum protein involved in innate immunity. It binds calcium dependently to iC3b, a product of the complement component C3 deposited on cell surfaces, immune complexes or artificial surfaces after complement activation. We here present a simple and efficient two-step procedure for the purification of Conglutinin. In the first step, bovine serum is incubated with non-coupled chromatographic TSK beads at 37°C to allow complement activation and iC3b deposition on the beads and subsequent binding of Conglutinin to iC3b. Conglutinin is then eluted from the beads by EDTA. In the second step, Conglutinin is separated from iC3b and IgM by ion-exchange chromatography. This purification procedure yielded 81 μg of Conglutinin per ml of serum with a recovery of 61.2%. Surface plasmon resonance analysis showed that the purified Conglutinin had a high affinity for mannan (K(d)=2.3-3.2 nM). SDS-PAGE and time-resolved immunofluorometric assays showed that the Conglutinin was not contaminated with other serum collectins such as collectin-43 or mannan-binding lectin.
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the genes encoding bovine sp a sp d mbl a Conglutinin cl 43 and cl 46 form a distinct collectin locus on bos taurus chromosome 28 bta28 at position q 1 8 1 9
Animal Genetics, 2004Co-Authors: Morten F Gjerstorff, Soren Hansen, B Jensen, B Dueholm, P Horn, Christian Bendixen, Uffe HolmskovAbstract:Summary Collectins are a group of C-type lectins involved in the innate immune system, where they mediate and modulate clearance of pathogens. The health status of cattle is of major economical and ethical concern; therefore, the study of bovine collectins is of importance. The collectins Conglutinin, CL-43 and CL-46 are only present in Bovidae and the characterization of their genes indicates that they are structural descendants of another collectin, lung surfactant protein D (SP-D). In this study, we assembled BAC clones into a contig spanning 330–1150 kb, which includes the bovine genes encoding the collectins SP-A (SFTPA), SP-D (SFTPD), mannan-binding lectin A (MBL1), CL-43 (COLEC9), CL-46 (COLEC13) and Conglutinin (COLEC8). In the same contig, we also identified a gene that potentially encodes a novel Conglutinin-like collectin (COLEC14). The arrangement of STFPA, SFTPD and MBL1 is homologous to the organization found in humans and mice, whereas the Bovidae-specific collectin genes, COLEC8, COLEC9 and COLEC13, extend from SFTPD. Proximal to the collectin locus at BTA28q1.8–1.9, and included in the contig, we found the microsatellite IDVGA8, which may be a valuable marker for tracking polymorphisms in the linked collectin genes.
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a time resolved immunofluorometric assay for quantification of the bovine collectin Conglutinin
Journal of Immunological Methods, 2004Co-Authors: Thomas Kroghmeibom, Uffe Holmskov, P Lovendahl, N I Nielsen, K L IngvartsenAbstract:A high capacity time-resolved immunofluorometric assay (TRIFMA) for the bovine collectin Conglutinin was developed. The TRIFMA was constructed as a non-competitive sandwich assay based on polyclonal antibodies as the capture reagent and a novel monoclonal antibody raised against Conglutinin as the detection reagent and was set up to run on an automatic analyzer designed for the TRIFMA detection system. Polyclonal antibodies immobilized on microtiter plate wells were incubated overnight at 4 degrees C with diluted plasma samples, including quality controls (QC) and dilutions of a plasma with known Conglutinin concentration. Conglutinin was sandwiched between the capture antibodies and the monoclonal antibody and the detection optimised with biotin-labelled secondary antibodies and streptavidin-Eu(3+). Plates were washed four times between each step and finally incubated with enhancement solution before measuring the fluorescence. The assay detection limit was 0.34 ng/ml and the working range 0.80 ng/ml-0.20 microg/ml. Intra-plate and inter-plate coefficients of variation (CV) were in the range of 5.0-8.3% and 6.2-7.2%, respectively, at concentrations of 3.4 and 150 ng/ml. Recovery was 90.9+/-2.4% and 98.8+/-2.5% when samples were spiked with 20 ng/ml and 100 ng/ml purified bovine Conglutinin (BK). No circadian rhythm (24-h variation) in Conglutinin plasma levels was observed across animals, indicating that the plasma levels were not influenced by, e.g. feeding. Samples could be stored at -20 degrees Celsius and were not sensitive to repeated freezing and thawing. In conclusion, the developed TRIFMA for bovine Conglutinin is specific and reliable over a measurement range covering most situations.
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Antiviral activity of bovine collectins against rotaviruses
2004Co-Authors: Patrick C. Reading, Uffe Holmskov, Margot E. AndersAbstract:The antiviral activity against rotaviruses of three bovine collectins, Conglutinin, collectin-43 (CL-43) and bovine SP-D, was examined. As shown by ELISA and Western blot, all three collectins bound to the Nebraska calf diarrhoea virus bovine strain of rotavirus, and specifically to the VP7 glycoprotein. Inhibition by mannose or EDTA confirmed that binding was mediated through the lectin domains of the collectins. Binding resulted in haemagglutin-ation inhibition and neutralization of rotavirus in-fectivity, CL-43 displaying the highest activity in both types of assay. In contrast, Conglutinin was the most potent of the three collectins against influenz
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novel characterisation of the gene encoding Conglutinin reveals that previously characterised promoter corresponds to the cl 43 promoter
Molecular Immunology, 2002Co-Authors: Soren Hansen, Christian Bendixen, Vivi Moeller, Dorte Holm, Lars Vitved, Karsten Skjodt, Uffe HolmskovAbstract:Conglutinin, a collectin found in bovine serum, is an opsonin that binds to glycoconjugates on the surface of microorganisms or on deposited iC3b, and acts in concert with phagocytes to establish a first-line of immune defense. We have isolated a genomic Conglutinin phage clone and found that the 5′-flanking region shows 95.8% identity with the sequence previously published, which on the other hand shows 99.7% identity with the CL-43 promoter. We conclude that the previously published promoter corresponds to the CL-43 promoter and that the functional studies performed on it apply to transcription of CL-43. Comparison of potential cis-regulatory elements in relation to the functional studies indicates that the two genes are regulated by different mechanisms.
Kenneth B. M. Reid - One of the best experts on this subject based on the ideXlab platform.
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atomic resolution crystal structures of the immune protein Conglutinin from cow reveal specific interactions of its binding site with n acetylglucosamine
Journal of Biological Chemistry, 2019Co-Authors: Janet M Paterson, Kenneth B. M. Reid, Amy J Shaw, I Burns, Alister W Dodds, Alpana Prasad, Trevor J Greenhough, Annette K ShriveAbstract:Bovine Conglutinin is an immune protein that is involved in host resistance to microbes and parasites and interacts with complement component iC3b, agglutinates erythrocytes, and neutralizes influenza A virus. Here, we determined the high-resolution (0.97–1.46 Å) crystal structures with and without bound ligand of a recombinant fragment of Conglutinin's C-terminal carbohydrate-recognition domain (CRD). The structures disclosed that the high-affinity ligand N-acetyl-d-glucosamine (GlcNAc) binds in the collectin CRD calcium site by interacting with the O3′ and O4′ hydroxyls alongside additional specific interactions of the N-acetyl group oxygen and nitrogen with Lys-343 and Asp-320, respectively. These residues, unique to Conglutinin and differing both in sequence and in location from those in other collectins, result in specific, high-affinity binding for GlcNAc. The binding pocket flanking residue Val-339, unlike the equivalent Arg-343 in the homologous human surfactant protein D, is sufficiently small to allow Conglutinin Lys-343 access to the bound ligand, whereas Asp-320 lies in an extended loop proximal to the ligand-binding site and bounded at both ends by conserved residues that coordinate to both calcium and ligand. This loop becomes ordered on ligand binding. The electron density revealed both α and β anomers of GlcNAc, consistent with the added α/βGlcNAc mixture. Crystals soaked with α1–2 mannobiose, a putative component of iC3b, reported to bind to Conglutinin, failed to reveal bound ligand, suggesting a requirement for presentation of mannobiose as part of an extended physiological ligand. These results reveal a highly specific GlcNAc-binding pocket in Conglutinin and a novel collectin mode of carbohydrate recognition.
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mechanisms of anti influenza activity of surfactant proteins a and d comparison with serum collectins
American Journal of Physiology-lung Cellular and Molecular Physiology, 1997Co-Authors: Kevan L. Hartshorn, Kenneth B. M. Reid, Mitchell R White, Virginia L Shepherd, Jens Christian Jensenius, Edmond C CrouchAbstract:The present study provides the first direct comparison of anti-influenza A virus (IAV) activities of the collectins surfactant protein (SP) A and SP-D, mannose-binding lectin (MBL), and Conglutinin...
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a recombinant polypeptide composed of the α helical neck region and the carbohydrate recogniton domain of Conglutinin self associates to give a functionally intact homotrimer
FEBS Letters, 1995Co-Authors: Jiu Yao Wang, Uday Kishore, Kenneth B. M. ReidAbstract:A recombinant polypeptide composed of the α-helical neck region and carbohydrate recognition domain (CRD) of bovine Conglutinin was expressed in Escherichia coli. The recombinant protein formed inclusion bodies but could be solubilised using a denaturation-renaturation cycle based on urea and then purified by affinity chromatography on a TSK-N-acetylglucosamine column. The purified product behaved as a homotrimer in nondissociating conditions, with three CRDs held together by the α-helical neck regions. The trimer, although lacking the N-terminal and collagen regions of the native Conglutinin, showed the same binding carbohydrate specificities as the native molecule, for the complement fragment C3b and for lipopolysaccharides derived from Gram-negative bacteria.
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comparative study of the structural and functional properties of a bovine plasma c type lectin collectin 43 with other collectins
Biochemical Journal, 1995Co-Authors: Uffe Holmskov, Hanna Wiedemann, G.r. Stuart, I Tornoe, Peder Madsen, Rajneesh Malhotra, Kenneth B. M. Reid, S B Laursen, Rupert Timpl, Jens Chr. JenseniusAbstract:Collectin-43 (CL-43) is a recently described bovine plasma protein containing both collagenous regions and C-type-lectin domains [Holmskov, Teisner, Willis, Reid and Jensenius (1993) J. Biol. Chem. 268, 10120-10125; Lim, Willis, Reid, Lu, Laursen, Jensenius and Holmskov (1994) J. Biol. Chem. 269, 11820-11824]. CL-43 was purified by affinity chromatography on mannan-Sepharose. On SDS/PAGE under reducing conditions the purified lectin showed a double band at about 43 kDa, with the upper band representing the intact molecule and the lower band a truncated form that lacked the N-terminal nine amino acid residues. Under non-reducing conditions, only one band was seen at 120 kDa. Analytical gel chromatography and sucrose-density-gradient centrifugation of the purified molecule, showed a Stokes radius of 9.1 +/- 0.3 nm (91 +/- 3 A) and a sedimentation coefficient (s20,w) of 3.6 +/- 0.1 S. These values correspond to a molecular mass of 119-138 kDa under non-denaturing condition in solution. The frictional coefficient (f/f0) was 2.7, indicating extreme elongation due to the collagenous segment. Only monomer subunits, with 37.4 +/- 1.7-nm-long rods, were seen by electron microscopy. These findings indicate that CL-43, in contrast with the other circulating collectins, is found only as a single subunit composed of three polypeptide chains. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms, with pI values of 4.9 and 5.3, corresponding to the native form and the truncated form of the molecule respectively. CL-43, like Conglutinin, lung surfactant protein A and mannan-binding protein (MBP), was shown to bind to the collectin receptor. Bovine MBP caused the activation of the complement system as revealed by the deposition of complement component C4 upon incubation of diluted serum in wells containing MBP bound to solid-phase mannan. CL-43, lung surfactant protein D (SP-D) and Conglutinin showed no complement-activating properties under the same conditions. Conglutinin binds fluid- and solid-phase iC3b, while CL-43 and MBP do not show such reactivity.
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primary structure of bovine collectin 43 cl 43 comparison with Conglutinin and lung surfactant protein d
Journal of Biological Chemistry, 1994Co-Authors: Boon Leong Lim, Kenneth B. M. Reid, S B Laursen, J C Jensenius, Antony C Willis, Uffe HolmskovAbstract:Abstract Collectin-43 (CL-43) is a bovine serum protein that is composed of subunits of three identical chains, each of which contains a collagen region and a C-type carbohydrate recognition domain; thus, CL-43 belongs to the collectins (group III of the C-type lectins). We have derived the complete primary sequence of CL-43 using partial protein sequencing, cDNA cloning, and reverse transcription-polymerase chain reaction techniques. The primary sequence of CL-43 shows that it contains an N-terminal region of 28 residues, followed by a collagenous domain of 38 repeats of Gly-Xaa-Yaa and then a C-terminal section of 159 residues, containing a short "neck" region and the carbohydrate recognition domain with the conserved residues found in all C-type lectins. The amino acid sequence of CL-43 showed 74% identity to bovine Conglutinin and 70% identity to bovine lung surfactant protein D (SP-D), but the collagen region is considerably shorter than the 57 Gly-Xaa-Yaa triplets found in Conglutinin and SP-D. Northern blot analysis showed that CL-43 was only synthesized in bovine liver, with no detectable signal in a variety of other bovine tissues, including lung. No cross-hybridizing signals were detected in mRNA from sheep, human, rat, or mouse liver. Since CL-43 and Conglutinin have only been detected in members of bovidae, it is probable that an ancestral gene of these two proteins was first derived from a SP-D-like gene, and that this ancestral gene duplicated during evolution.
S B Laursen - One of the best experts on this subject based on the ideXlab platform.
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comparative study of the structural and functional properties of a bovine plasma c type lectin collectin 43 with other collectins
Biochemical Journal, 1995Co-Authors: Uffe Holmskov, Hanna Wiedemann, G.r. Stuart, I Tornoe, Peder Madsen, Rajneesh Malhotra, Kenneth B. M. Reid, S B Laursen, Rupert Timpl, Jens Chr. JenseniusAbstract:Collectin-43 (CL-43) is a recently described bovine plasma protein containing both collagenous regions and C-type-lectin domains [Holmskov, Teisner, Willis, Reid and Jensenius (1993) J. Biol. Chem. 268, 10120-10125; Lim, Willis, Reid, Lu, Laursen, Jensenius and Holmskov (1994) J. Biol. Chem. 269, 11820-11824]. CL-43 was purified by affinity chromatography on mannan-Sepharose. On SDS/PAGE under reducing conditions the purified lectin showed a double band at about 43 kDa, with the upper band representing the intact molecule and the lower band a truncated form that lacked the N-terminal nine amino acid residues. Under non-reducing conditions, only one band was seen at 120 kDa. Analytical gel chromatography and sucrose-density-gradient centrifugation of the purified molecule, showed a Stokes radius of 9.1 +/- 0.3 nm (91 +/- 3 A) and a sedimentation coefficient (s20,w) of 3.6 +/- 0.1 S. These values correspond to a molecular mass of 119-138 kDa under non-denaturing condition in solution. The frictional coefficient (f/f0) was 2.7, indicating extreme elongation due to the collagenous segment. Only monomer subunits, with 37.4 +/- 1.7-nm-long rods, were seen by electron microscopy. These findings indicate that CL-43, in contrast with the other circulating collectins, is found only as a single subunit composed of three polypeptide chains. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms, with pI values of 4.9 and 5.3, corresponding to the native form and the truncated form of the molecule respectively. CL-43, like Conglutinin, lung surfactant protein A and mannan-binding protein (MBP), was shown to bind to the collectin receptor. Bovine MBP caused the activation of the complement system as revealed by the deposition of complement component C4 upon incubation of diluted serum in wells containing MBP bound to solid-phase mannan. CL-43, lung surfactant protein D (SP-D) and Conglutinin showed no complement-activating properties under the same conditions. Conglutinin binds fluid- and solid-phase iC3b, while CL-43 and MBP do not show such reactivity.
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primary structure of bovine collectin 43 cl 43 comparison with Conglutinin and lung surfactant protein d
Journal of Biological Chemistry, 1994Co-Authors: Boon Leong Lim, Kenneth B. M. Reid, S B Laursen, J C Jensenius, Antony C Willis, Uffe HolmskovAbstract:Abstract Collectin-43 (CL-43) is a bovine serum protein that is composed of subunits of three identical chains, each of which contains a collagen region and a C-type carbohydrate recognition domain; thus, CL-43 belongs to the collectins (group III of the C-type lectins). We have derived the complete primary sequence of CL-43 using partial protein sequencing, cDNA cloning, and reverse transcription-polymerase chain reaction techniques. The primary sequence of CL-43 shows that it contains an N-terminal region of 28 residues, followed by a collagenous domain of 38 repeats of Gly-Xaa-Yaa and then a C-terminal section of 159 residues, containing a short "neck" region and the carbohydrate recognition domain with the conserved residues found in all C-type lectins. The amino acid sequence of CL-43 showed 74% identity to bovine Conglutinin and 70% identity to bovine lung surfactant protein D (SP-D), but the collagen region is considerably shorter than the 57 Gly-Xaa-Yaa triplets found in Conglutinin and SP-D. Northern blot analysis showed that CL-43 was only synthesized in bovine liver, with no detectable signal in a variety of other bovine tissues, including lung. No cross-hybridizing signals were detected in mRNA from sheep, human, rat, or mouse liver. Since CL-43 and Conglutinin have only been detected in members of bovidae, it is probable that an ancestral gene of these two proteins was first derived from a SP-D-like gene, and that this ancestral gene duplicated during evolution.
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bovine Conglutinin binds to an oligosaccharide determinant presented by ic3b but not by c3 c3b or c3c
Immunology, 1994Co-Authors: S B Laursen, Uffe Holmskov, Steffen Thiel, B Teisner, Y Wang, Robert B Sim, J C JenseniusAbstract:Bovine Conglutinin is a serum lectin that agglutinates erythrocytes preincubated with antibodies and complement. This agglutination occurs through the binding of Conglutinin to iC3b, a fragment of the complement component C3. It was reported that Conglutinin binds fluid-phase C3b and C3c as well as iC3b. We re-investigated the reactivity of Conglutinin towards fluid-phase C3 degradation products. ELISA wells were coated with Conglutinin and reacted with C3 split products generated in normal human serum, in factor I-deficient serum, or in factor I-depleted serum. Conglutinin-bound C3 fragments were detected with anti-C3c and anti-C3d antibodies. An increased signal was observed during the activation of complement in normal human serum with the peak response after 1-2 hr, following which the signal decreased, reaching background level after 72 hr. The oligosaccharides on C3c, generated in serum, are thus not recognized by Conglutinin. No signal was observed when factor I-deficient serum or factor I-depleted serum was used instead of normal serum. Reconstitution with purified factor I re-established the normal pattern. Examination of the Conglutinin-bound C3 molecules by SDS-PAGE and Western blotting with anti-C3c and anti-C3d antibodies revealed bands characteristic for iC3b, and no bands corresponding to C3b or C3c. Reduction of the disulphide bonds prior to the incubation of the activated serum with the Conglutinin-coated wells revealed a band of 63,000 MW, characteristic of the N-terminal fragment of the alpha-chain of iC3b. We also investigated the binding to the solid-phase Conglutinin of purified C3 and degradation products generated with enzymes. In this case, C3 as well as C3b and C3c were bound, suggesting conformational changes in C3 during purification. In conclusion, when C3 conversion takes place at near physiological conditions, Conglutinin interacts specifically with the oligosaccharide on the alpha-chain of iC3b.
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Primary structure of bovine collectin-43 (CL-43). Comparison with Conglutinin and lung surfactant protein-D
'United States Sports Academy', 1994Co-Authors: J C Jensenius, S B Laursen, Bl Lim, Holmskov U, Lu J, Reid Kbm, A C WillisAbstract:Collectin-43 (CL-43) is a bovine serum protein that is composed of subunits of three identical chains, each of which contains a collagen region and a C-type carbohydrate recognition domain; thus, CL-43 belongs to the collectins (group III of the C-type lectins). We have derived the complete primary sequence of CL-43 using partial protein sequencing, cDNA cloning, and reverse transcription-polymerase chain reaction techniques. The primary sequence of CL-43 shows that it contains an N-terminal region of 28 residues, followed by a collagenous domain of 38 repeats of Gly-Xaa-Yaa and then a C- terminal section of 159 residues, containing a short 'neck' region and the carbohydrate recognition domain with the conserved residues found in all C- type lectins. The amino acid sequence of CL-43 showed 74% identity to bovine Conglutinin and 70% identity to bovine lung surfactant protein D (SP-D), but the collagen region is considerably shorter than the 57 Gly-Xaa-Yaa triplets found in Conglutinin and SP-D. Northern blot analysis showed that CL-43 was only synthesized in bovine liver, with no detectable signal in a variety of other bovine tissues, including lung. No cross-hybridizing signals were detected in mRNA from sheep, human, rat, or mouse liver. Since CL-43 and Conglutinin have only been detected in members of bovidae, it is probable that an ancestral gene of these two proteins was first derived from a SP-D- like gene, and that this ancestral gene duplicated during evolution.published_or_final_versio
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studies on the carbohydrate binding characteristics of human pulmonary surfactant associated protein a and comparison with two other collectins mannan binding protein and Conglutinin
Biochemical Journal, 1993Co-Authors: John S Haurum, S B Laursen, Steffen Thiel, Henk P Haagsman, B Larsen, Jens Chr. JenseniusAbstract:The surfactant-associated protein A (SP-A) belongs to the collectin family, a group of C-type lectins encompassing also surfactant-associated protein D, mannan-binding protein (MBP) and Conglutinin. These proteins all have carbohydrate-recognition domains joined to collagen stalks. It seems likely that SP-A, like MBP and Conglutinin, may mediate anti-microbial activity through binding to carbohydrates on the microorganisms and collectin receptors on phagocytic cells. We have studied the influence of carbohydrates on the binding of SP-A, MBP and Conglutinin to mannan in an enzyme-linked lectin-binding assay. All sugars were of D-configuration, except fucose of which both L- and D-configurations were tested. The order of inhibiting potency on the binding of SP-A was: N-acetylmannosamine > L-fucose, maltose > glucose > mannose. The following sugars were non-inhibitory: galactose, D-fucose, glucosamine, mannosamine, galactosamine, N-acetylglucosamine, and N-acetylgalactosamine. The best inhibitor of MBP was N-acetylglucosamine. Otherwise MBP showed a selectivity similar to that of SP-A. Conglutinin binding was inhibited by all the sugars examined except N-acetylgalactosamine. For Conglutinin, as for MBP, the best inhibitor was N-acetylglucosamine. Normal human SP-A, alveolar-proteinosis SP-A purified by ion-exchange chromatography, and alveolar-proteinosis SP-A purified by n-butanol extraction showed no difference in sugar selectivity. The influence of pH and of the calcium concentration was also examined. Organic solvent-extracted SP-A from patients suffering from alveolar proteinosis and normal SP-A showed different sensitivity profiles.
J C Jensenius - One of the best experts on this subject based on the ideXlab platform.
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mannan binding protein and bovine Conglutinin mediate enhancement of herpes simplex virus type 2 infection in mice
Scandinavian Journal of Immunology, 1994Co-Authors: P B Fischer, Steffen Thiel, J C Jensenius, Svend Ellermanneriksen, Soren C MogensenAbstract:A broad range of plant lectins have recently been shown to inhibit the infectivity of herpes simplex virus type I (HSV-1) in viiro. We decided to investigate the role of mammalian Icctins in infection witb herpes simplex virus. Two lectins, Conglutinin and mannan-binding protein (also called mannose-binding protein. MBP). belonging to the collectin family of lectins, were examined. Four week-old BALB/c mice were injected subcutaneously with 100 μg bovine Conglutinin or 50 μg human MBP 1 day before intravenous infection with 5 × 104 PFU of herpes simplex virus type 2 (HSV-2). A three-fold increase in virus titre of the liver was observed on day 3 of the infection in the mice pretreated with Conglutinin or MBP. whereas no effect was seen on days I and 5. In a standard plaque assay using Vero cells we were not able to demonstrate reproducibly either infection inhibition or infection enhancement, when virus was pre-incubated with differing concentrations ofthe collectins. Tbe concentrations used were similar to tbose used by us in livo, and by others in in vitro experiments showing inhibition of the infectivity of HSV-1 with plant lectins. In an ELISA with HSV-2 antigens captured on anti-HSV-2 antibodies, calcium-dependent and carbohydrate inhibitabte binding of the collectins was observed. Our results indicate that the effect of endogenous mammalian collectins in vivo may not be neutralization as suggested by the data using plant lectins. Instead, the previously described opsonizing activity of the mammalian collectins may provide the virions witb an alternative port of entry into cells leading to infection enhancement.
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primary structure of bovine collectin 43 cl 43 comparison with Conglutinin and lung surfactant protein d
Journal of Biological Chemistry, 1994Co-Authors: Boon Leong Lim, Kenneth B. M. Reid, S B Laursen, J C Jensenius, Antony C Willis, Uffe HolmskovAbstract:Abstract Collectin-43 (CL-43) is a bovine serum protein that is composed of subunits of three identical chains, each of which contains a collagen region and a C-type carbohydrate recognition domain; thus, CL-43 belongs to the collectins (group III of the C-type lectins). We have derived the complete primary sequence of CL-43 using partial protein sequencing, cDNA cloning, and reverse transcription-polymerase chain reaction techniques. The primary sequence of CL-43 shows that it contains an N-terminal region of 28 residues, followed by a collagenous domain of 38 repeats of Gly-Xaa-Yaa and then a C-terminal section of 159 residues, containing a short "neck" region and the carbohydrate recognition domain with the conserved residues found in all C-type lectins. The amino acid sequence of CL-43 showed 74% identity to bovine Conglutinin and 70% identity to bovine lung surfactant protein D (SP-D), but the collagen region is considerably shorter than the 57 Gly-Xaa-Yaa triplets found in Conglutinin and SP-D. Northern blot analysis showed that CL-43 was only synthesized in bovine liver, with no detectable signal in a variety of other bovine tissues, including lung. No cross-hybridizing signals were detected in mRNA from sheep, human, rat, or mouse liver. Since CL-43 and Conglutinin have only been detected in members of bovidae, it is probable that an ancestral gene of these two proteins was first derived from a SP-D-like gene, and that this ancestral gene duplicated during evolution.
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bovine Conglutinin binds to an oligosaccharide determinant presented by ic3b but not by c3 c3b or c3c
Immunology, 1994Co-Authors: S B Laursen, Uffe Holmskov, Steffen Thiel, B Teisner, Y Wang, Robert B Sim, J C JenseniusAbstract:Bovine Conglutinin is a serum lectin that agglutinates erythrocytes preincubated with antibodies and complement. This agglutination occurs through the binding of Conglutinin to iC3b, a fragment of the complement component C3. It was reported that Conglutinin binds fluid-phase C3b and C3c as well as iC3b. We re-investigated the reactivity of Conglutinin towards fluid-phase C3 degradation products. ELISA wells were coated with Conglutinin and reacted with C3 split products generated in normal human serum, in factor I-deficient serum, or in factor I-depleted serum. Conglutinin-bound C3 fragments were detected with anti-C3c and anti-C3d antibodies. An increased signal was observed during the activation of complement in normal human serum with the peak response after 1-2 hr, following which the signal decreased, reaching background level after 72 hr. The oligosaccharides on C3c, generated in serum, are thus not recognized by Conglutinin. No signal was observed when factor I-deficient serum or factor I-depleted serum was used instead of normal serum. Reconstitution with purified factor I re-established the normal pattern. Examination of the Conglutinin-bound C3 molecules by SDS-PAGE and Western blotting with anti-C3c and anti-C3d antibodies revealed bands characteristic for iC3b, and no bands corresponding to C3b or C3c. Reduction of the disulphide bonds prior to the incubation of the activated serum with the Conglutinin-coated wells revealed a band of 63,000 MW, characteristic of the N-terminal fragment of the alpha-chain of iC3b. We also investigated the binding to the solid-phase Conglutinin of purified C3 and degradation products generated with enzymes. In this case, C3 as well as C3b and C3c were bound, suggesting conformational changes in C3 during purification. In conclusion, when C3 conversion takes place at near physiological conditions, Conglutinin interacts specifically with the oligosaccharide on the alpha-chain of iC3b.
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Primary structure of bovine collectin-43 (CL-43). Comparison with Conglutinin and lung surfactant protein-D
'United States Sports Academy', 1994Co-Authors: J C Jensenius, S B Laursen, Bl Lim, Holmskov U, Lu J, Reid Kbm, A C WillisAbstract:Collectin-43 (CL-43) is a bovine serum protein that is composed of subunits of three identical chains, each of which contains a collagen region and a C-type carbohydrate recognition domain; thus, CL-43 belongs to the collectins (group III of the C-type lectins). We have derived the complete primary sequence of CL-43 using partial protein sequencing, cDNA cloning, and reverse transcription-polymerase chain reaction techniques. The primary sequence of CL-43 shows that it contains an N-terminal region of 28 residues, followed by a collagenous domain of 38 repeats of Gly-Xaa-Yaa and then a C- terminal section of 159 residues, containing a short 'neck' region and the carbohydrate recognition domain with the conserved residues found in all C- type lectins. The amino acid sequence of CL-43 showed 74% identity to bovine Conglutinin and 70% identity to bovine lung surfactant protein D (SP-D), but the collagen region is considerably shorter than the 57 Gly-Xaa-Yaa triplets found in Conglutinin and SP-D. Northern blot analysis showed that CL-43 was only synthesized in bovine liver, with no detectable signal in a variety of other bovine tissues, including lung. No cross-hybridizing signals were detected in mRNA from sheep, human, rat, or mouse liver. Since CL-43 and Conglutinin have only been detected in members of bovidae, it is probable that an ancestral gene of these two proteins was first derived from a SP-D- like gene, and that this ancestral gene duplicated during evolution.published_or_final_versio
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the cdna cloning of Conglutinin and identification of liver as a primary site of synthesis of Conglutinin in members of the bovidae
Biochemical Journal, 1993Co-Authors: S B Laursen, Steffen Thiel, J C Jensenius, Kenneth B. M. ReidAbstract:Bovine Conglutinin is a collagen-like, C-type, plasma lectin which belongs to the group of proteins called 'collectins'. Two inosine-containing oligonucleotides were synthesized, based on the published protein sequence for bovine Conglutinin [Lee, Leiby, Allar, Paris, Lerch and Okarma (1991) J. Biol. Chem. 266, 2715-2723], and PCR on target DNA from a bovine liver lambda gt 11 cDNA library yielded a product of the expected size of 210 bp. Screening of the library with this cDNA fragment identified a single positive clone, with an insert of 0.9 kb, coding for bovine Conglutinin from residue 70 to the C-terminus. The 5' cDNA sequence, encompassing 150 bp of the 5' non-translated sequence plus the sequence encoding the leader peptide and the N-terminal residues 1-70, was completed by the use of PCR techniques. The cDNA sequence of bovine Conglutinin showed 86% identity with that of bovine lung surfactant protein D (SP-D), and the derived amino acid sequence of bovine Conglutinin showed 78% identity with that of bovine SP-D, which included complete identity of the leader-peptide sequences. The amino acid sequence derived from the cDNA sequence differs from the published protein sequence at four positions. Northern-blot analysis on total RNA, purified from various tissues from cattle, sheep, humans, rats and mice, showed that a strong signal of approx. 1.8 kb is present in bovine liver RNA. A weak signal of similar size was also observed in sheep liver, but not in human, rat and mouse livers. A weak signal, also of 1.8 kb, is present in the lung RNAs of all the species tested. The signals from the lung tissues are likely to be due to the cross-hybridization of the bovine Conglutinin cDNA to the SP-D mRNAs of the respective species. The finding of significant signals in only the bovine and sheep liver RNA samples is indicative that serum Conglutinin may be present in significant amounts only in members of the Bovidae (the family encompassing cattle, antelopes, sheep and goats) and closely related species.
Jens Chr. Jensenius - One of the best experts on this subject based on the ideXlab platform.
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the plasma levels of Conglutinin are heritable in cattle and low levels predispose to infection
Immunology Letters, 1997Co-Authors: Uffe Holmskov, Jens Chr. Jensenius, I Tornoe, P LovendahlAbstract:Conglutinin, like mannan-binding lectin (MBL) and CL-43, is a serum collection involved in the innate immune defence system. In man, low serum MBL concentrations, resulting from mutations in the collagen region, are associated with a common opsonic defect. Plasma levels of Conglutinin in cattle were assayed by rocket immunoelectrophoresis to examine whether they were genetically determined. Samples were collected from calves (309 bull-calves and 260 heifers with complex pedigree relationships). The number of respiratory infections from the 42nd to 336th day of life was recorded. The number of infections was found to be genetically determined (heritability: h2 = 0.31 +/- 0.07). A wide concentration range of Conglutinin was found in plasma (< 1.25-35 micrograms/ml for females, geometric mean 8.1 micrograms/ml, and < 1.25-47 micrograms/ml for males, geometric mean 15.5 micrograms/ml), and the concentrations was found to be genetically determined (heritability, h2 = 0.52 +/- 0.07). The analysis revealed a negative association between disease frequency and the Conglutinin levels (-0.56 +/- 0.18 for female; -0.50 +/- 0.18 for male). Levels of Conglutinin below the detection limit of the assay (1.25 micrograms/ml) were found in 2% of the animals. If these animals are assumed to be homozygous for a single recessive allele causing low concentrations a gene frequency of 0.15 could be calculated. These findings suggests that selection for resistance against infectious disease is possible in cattle and that the level of plasma Conglutinin may be a helpful trait in such a breeding scheme.
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comparative study of the structural and functional properties of a bovine plasma c type lectin collectin 43 with other collectins
Biochemical Journal, 1995Co-Authors: Uffe Holmskov, Hanna Wiedemann, G.r. Stuart, I Tornoe, Peder Madsen, Rajneesh Malhotra, Kenneth B. M. Reid, S B Laursen, Rupert Timpl, Jens Chr. JenseniusAbstract:Collectin-43 (CL-43) is a recently described bovine plasma protein containing both collagenous regions and C-type-lectin domains [Holmskov, Teisner, Willis, Reid and Jensenius (1993) J. Biol. Chem. 268, 10120-10125; Lim, Willis, Reid, Lu, Laursen, Jensenius and Holmskov (1994) J. Biol. Chem. 269, 11820-11824]. CL-43 was purified by affinity chromatography on mannan-Sepharose. On SDS/PAGE under reducing conditions the purified lectin showed a double band at about 43 kDa, with the upper band representing the intact molecule and the lower band a truncated form that lacked the N-terminal nine amino acid residues. Under non-reducing conditions, only one band was seen at 120 kDa. Analytical gel chromatography and sucrose-density-gradient centrifugation of the purified molecule, showed a Stokes radius of 9.1 +/- 0.3 nm (91 +/- 3 A) and a sedimentation coefficient (s20,w) of 3.6 +/- 0.1 S. These values correspond to a molecular mass of 119-138 kDa under non-denaturing condition in solution. The frictional coefficient (f/f0) was 2.7, indicating extreme elongation due to the collagenous segment. Only monomer subunits, with 37.4 +/- 1.7-nm-long rods, were seen by electron microscopy. These findings indicate that CL-43, in contrast with the other circulating collectins, is found only as a single subunit composed of three polypeptide chains. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms, with pI values of 4.9 and 5.3, corresponding to the native form and the truncated form of the molecule respectively. CL-43, like Conglutinin, lung surfactant protein A and mannan-binding protein (MBP), was shown to bind to the collectin receptor. Bovine MBP caused the activation of the complement system as revealed by the deposition of complement component C4 upon incubation of diluted serum in wells containing MBP bound to solid-phase mannan. CL-43, lung surfactant protein D (SP-D) and Conglutinin showed no complement-activating properties under the same conditions. Conglutinin binds fluid- and solid-phase iC3b, while CL-43 and MBP do not show such reactivity.
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studies on the carbohydrate binding characteristics of human pulmonary surfactant associated protein a and comparison with two other collectins mannan binding protein and Conglutinin
Biochemical Journal, 1993Co-Authors: John S Haurum, S B Laursen, Steffen Thiel, Henk P Haagsman, B Larsen, Jens Chr. JenseniusAbstract:The surfactant-associated protein A (SP-A) belongs to the collectin family, a group of C-type lectins encompassing also surfactant-associated protein D, mannan-binding protein (MBP) and Conglutinin. These proteins all have carbohydrate-recognition domains joined to collagen stalks. It seems likely that SP-A, like MBP and Conglutinin, may mediate anti-microbial activity through binding to carbohydrates on the microorganisms and collectin receptors on phagocytic cells. We have studied the influence of carbohydrates on the binding of SP-A, MBP and Conglutinin to mannan in an enzyme-linked lectin-binding assay. All sugars were of D-configuration, except fucose of which both L- and D-configurations were tested. The order of inhibiting potency on the binding of SP-A was: N-acetylmannosamine > L-fucose, maltose > glucose > mannose. The following sugars were non-inhibitory: galactose, D-fucose, glucosamine, mannosamine, galactosamine, N-acetylglucosamine, and N-acetylgalactosamine. The best inhibitor of MBP was N-acetylglucosamine. Otherwise MBP showed a selectivity similar to that of SP-A. Conglutinin binding was inhibited by all the sugars examined except N-acetylgalactosamine. For Conglutinin, as for MBP, the best inhibitor was N-acetylglucosamine. Normal human SP-A, alveolar-proteinosis SP-A purified by ion-exchange chromatography, and alveolar-proteinosis SP-A purified by n-butanol extraction showed no difference in sugar selectivity. The influence of pH and of the calcium concentration was also examined. Organic solvent-extracted SP-A from patients suffering from alveolar proteinosis and normal SP-A showed different sensitivity profiles.