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U Holmskov

Publications and source records attributed to U Holmskov.

At least 55 records · Page 3Linked to original sources
PubMed1995

Comparative study of the structural and functional properties of a bovine plasma C-type lectin, collectin-43, with other collectins.

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.

Animals↗
PubMed1995

Difference and ratio plots: simple tools for improved presentation and interpretation of scientific data. Unexpected possibilities for the use of the conglutinin binding assay in inflammatory rheumatic diseases.

11111onglutinin-binding assay (KgBa) has gained widespread use for the detection of circulating immune complexes. A recent paper questioned the interpretation of the results obtained by this method and the validity of the assay (Holmskov et al. (1992) J. Immunol. Methods 148, 225). We now present hitherto unnoted differences between controls and patients with either rheumatoid arthritis or systemic lupus erythematosus. For this we use simple, but unconventional, graphic representations of the data, based on difference plots and ratio plots. Differences between patients with Burkitt's lymphoma and systemic lupus erythematosus from another previously published study (Macanovic, M. and Lachmann, P.J. (1979) Clin. Exp. Immunol. 38, 274) are also represented using ratio plots. Our observations indicate that analysis by regression analysis may often be misleading.

Arthritis, Rheumatoid↗
PubMed1995

Capillary density measurements in skeletal muscle using immunohistochemical staining with anti-collagen type IV antibodies.

An immunohistochemical method, based on an indirect peroxidase method for staining of capillaries containing collagen type IV, is presented. Biopsies from the vastus lateralis muscle of eight endurance trained runners were stained and counted for capillaries with the collagen type IV method and with the widely used amylase-PAS method. The mean number +/- SEM of capillaries per fibre was 2.48 +/- 0.09 with the collagen type IV method and 2.41 +/- 0.09 with the collagen type IV method and 2.41 +/- 0.08 with the amylase method. The 95% confidence intervals for the differences between the two methods were - 0.03 to 0.15. We conclude that the collagen type IV immunohistochemical method for capillary staining is comparable with the amylase method under optimum conditions, but that it is likely to be more dependable. It also has the advantage that it reveals fibre outlines.

Adult↗
PubMed1995

Binding of host collectins to the pathogenic yeast Cryptococcus neoformans: human surfactant protein D acts as an agglutinin for acapsular yeast cells.

Cryptococcus neoformans is an opportunistic pathogen in AIDS patients causing disseminated disease and lethal meningitis after inhalation of acapsular or sparsely encapsulated yeast cells. In this study we have investigated whether a recently described family of primitive opsonins, termed collectins, contribute to innate resistance against C. neoformans. The pulmonary surfactant proteins SP-A and SP-D as well as the serum collectins mannose-binding protein and CL-43 bound in a calcium-dependent manner to acapsular C. neoformans in vitro. Binding was concentration dependent and abolished by competition with defined mono- and oligosaccharides. In contrast, no binding of the collectins was observed with the encapsulated form of the yeast. Furthermore, binding of purified collectin SP-D, but not SP-A, mannose-binding protein, or CL-43, led to a concentration-dependent agglutination of acapsular C. neoformans. These data indicate that collectins recognize carbohydrate structures in the cell wall of an initial infectious form of C. neoformans and may play a role in early antifungal defenses in the lung.

Agglutinins↗
PubMed1995

Collectin-43 is a serum lectin with a distinct pattern of carbohydrate recognition.

Collectin-43 (CL-43) is a C-type serum lectin and a member of the collectin family of soluble proteins that are effector molecules in innate immunity. We have investigated the binding specificity of CL-43 using as model systems a panel of structurally defined oligosaccharides in the form of neoglycolipids, and several glycoproteins derived from the complement glycoprotein C3 during activation of the complement cascade. A specificity is revealed towards fucose as part of the Lea oligosaccharide sequence, and towards mannose as found on high mannose-type chains. These are features shared with other serum collectins, conglutinin and mannan-binding proteins; a major difference is the lack of detectable binding by CL-43 to N-glycosidic oligosaccharides terminating in N-acetylglucosamine. CL-43 has a unique pattern of reactivity towards high mannose-type oligosaccharides on the two glycosylation sites of C3 and derived glycoproteins: it binds to C3c (not bound by conglutinin and mannan-binding protein) but not to hydrolysed C3 [C3(H2O)], C3b or iC3b immobilized on microwells (all bound by conglutinin but not by mannan-binding protein). When these glycoproteins are sodium dodecyl sulphate (SDS)-treated and immobilized on nitrocellulose, CL-43 (but not conglutinin nor mannan-binding protein) binds strongly to C3(H2O), iC3b and C3c. The salient conclusions are, first, that there are remarkable positive or negative effects of carrier protein on oligosaccharide presentation and these differ for the individual collectins. Second, the different though partially overlapping binding patterns among the collectins may be important for their function as circulating effector molecules with broad surveillance capabilities.

Animals↗
PubMed1994

Expression of the carbohydrate recognition domain of lung surfactant protein D and demonstration of its binding to lipopolysaccharides of gram-negative bacteria.

Surfactant protein D is a collagenous C-type lectin (collectin) that is found almost exclusively in the lung. A recombinant protein, composed of the neck-region and the carbohydrate binding domain of bovine lung surfactant protein D, has been overexpressed in E. coli. The recombinant protein showed the same sugar binding specificity as the native protein and was able to bind to the lipopolysaccharides of several strains of Gram-negative bacteria, such as Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli, which are known to cause lung infections. The binding was calcium-dependent and was inhibited by maltose. Native bovine surfactant protein D was also shown to be able to bind to these lipopolysaccharides in the same manner.

Animals↗
PubMed1994

Primary structure of bovine collectin-43 (CL-43). Comparison with conglutinin and lung surfactant protein-D.

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.

Amino Acid Sequence↗
PubMed1994

Collectins: collagenous C-type lectins of the innate immune defense system.

Collectins are humoral lectins found in mammals and birds. They are oligomers whose subunits comprise three polypeptide chains each containing a collagenous section and a C-terminal lectin domain. They are related structurally and functionally to the first component of the classical complement pathway, C1q, and seem to serve important roles in innate immunity through opsonization and complement activation. The lectin domains bind carbohydrates on microorganisms, while the collagenous regions are ligands for the collectin receptor on phagocytes and also mediate C1q-independent activation of the classical complement pathway.

Animals↗
PubMed1994

Collectins, collectin receptors and the lectin pathway of complement activation.

The collectins are a group of soluble multimeric lectins, which contain collagenous segments, and resemble the complement protein C1q in aspects of their structures and functions. This group of proteins, which includes MBP, SP-A, SP-D, conglutinin and CL-43, are known to act as opsonins in various circumstances, and are likely to have roles in innate immunity. The focus of current research is to pursue the hypothesis that the collectins recognize and bind to non-host carbohydrate structures on microorganisms and particles, and participate in the processing or elimination of such material, either by direct interaction with phagocytic cell receptors, or by indirect routes such as complement activation .

Animals↗
PubMed1994

Purification and characterization of mannan-binding protein from mouse serum.

Mouse mannan-binding protein (MBP) was identified in serum by its Ca(2+)-dependent binding to mannan. On gel permeation chromatography, the protein eluted corresponding to a molecular weight of approximately 750 kDa. Analysed on SDS-PAGE under reducing conditions, the polypeptide showed an apparent molecular weight of 28 kDa, while several high molecular weight bands were seen under non-reducing conditions. The presence of collagen-like domains within the molecule was indicated by a high glycine content (14.9%) and substantiated by sensitivity to collagenase. Rabbit anti-mouse MBP antisera were raised. The concentration of MBP in serum from normal mice was measured by rocket immunoelectrophoresis and found to be from below 1 microgram/ml to 100 micrograms/ml (average 50 micrograms/ml, n = 60). The binding of mouse MBP to mannan could be inhibited by mono- and disaccharides in the following order of potency: L-fucose > D-mannose > N-acetyl-D-glucosamine > maltose > D-mannoheptulose > D-glucose > N-acetyl-D-mannosamine >> lactose > D-galactose >> N-acetyl-D-galactosamine. Mouse MBP was shown to activate the classical complement cascade after binding to mannan. The sequence of 14 NH2-terminal amino acid residues of the molecule showed 93% identity to rat MBP-A and complete identity to the translated cDNA sequences for mouse MBP-A and mouse Ra-reactive factor component P28b (RaRF P28b) published previously. The amino acid composition of mouse MBP showed a high degree of homology to MBPs from other species and mouse RaRF P28b.

Amino Acid Sequence↗
PubMed1994

Bovine conglutinin binds to an oligosaccharide determinant presented by iC3b, but not by C3, C3b or C3c.

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.

Animals↗
PubMed1993

Structural similarity between lung surfactant protein D and conglutinin. Two distinct, C-type lectins containing collagen-like sequences.

Preparations of bovine lung surfactant D (SP-D) and conglutinin were examined by electron microscopy, gel-filtration and SDS/PAGE. SP-D is composed of non-covalently linked subunits, of 160 kDa, which each contain three, disulphide-linked, 44-kDa polypeptide chains. In the electron microscope a single 160-kDa subunit of SP-D appears as a 45.8 +/- 3-nm-long rod connected to a small globular 'head'. Particles were also seen which correspond to non-covalently linked dimers, trimers and tetramers of the 160-kDa monomer subunit of SP-D. The tetramer structure contains 12 polypeptide chains and is very similar to the electron microscopy images and model reported by Strang et al. [Strang, C. J., Slayter, US., Lachmann, P. J. and Davis, A. E. (1986) Biochem. J. 236, 3811-389] for bovine conglutinin in which four 160-kDa subunits are disulphide-linked to give a molecule of expected molecular mass of 528 kDa. This study confirmed the findings by Strang et al. in the above paper for intact conglutinin and also emphasised that the rod-like structures, of length 37.6 +/- 3.7 nm, seen in the conglutinin subunits were significantly shorter than those in SP-D despite the close similarity in amino acid sequence (79% identify) and chain length between the two proteins. In addition, a truncated form of conglutinin was found in the conglutinin preparations, due to limited proteolysis of the Arg-Ala bond at position 54 in the 44-kDa chains. These truncated conglutinin chains yield a subunit composed of three shortened, non-disulphide-linked, chains and this subunit appears as a monomer with a rod length of 34.2 +/- 2.8 nm in the electron microscope. On gel-filtration, a proportion of the SP-D preparation behaved, as expected, as a molecule with an apparent molecular mass of 600 kDa. The remainder of the SP-D preparation behaved as aggregated material with a molecular mass greater than 900 kDa which yielded no distinct structures in the electron microscope. Intact conglutinin was eluted at a position greater than 900 kDa but yet provided clear electron microscopy images of the tetramer structure described above.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Purification and characterization of a bovine serum lectin (CL-43) with structural homology to conglutinin and SP-D and carbohydrate specificity similar to mannan-binding protein.

A previously undescribed bovine serum lectin (designated CL-43) was identified by its Ca(2+)-dependent binding to mannan and by its molecular mass of 43 kDa under reducing conditions on SDS-PAGE. The lectin was isolated by polyethylene glycol precipitation, affinity chromatography on mannan-Sepharose (followed by elution with EDTA), and absorption on Sepharose-4B-coupled rabbit anti-bovine Ig (to remove anti-mannan antibodies). Fractions containing the lectin were reapplied to mannan-Sepharose. Bound conglutinin was eluted with GlcNAc, and then the 43-kDa lectin, together with mannan-binding protein (MBP), was eluted with mannose. The 43-kDa lectin was separated from MBP by ion exchange chromatography on Mono-Q. On SDS-PAGE under nonreducing conditions the lectin showed a molecular mass of 120 kDa. On gel chromatography under nondissociating conditions the protein was eluted at a volume corresponding to a molecular mass of approximately 750 kDa. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine and a high content of glycine (24.3%) indicating the presence of a collagen-like structure. This was supported by the susceptibility of the protein to collagenase digestion. The designation CL-43 was chosen since this molecule appears to belong to the collectins, i.e. proteins with collagen structure and lectin activity. The N-terminal sequence (27 amino acids) showed 56% identity with bovine SP-D and 44% identity to bovine conglutinin. An inhibition assay with biotinylated CL-43, using solid-phase mannan as ligand, revealed the following carbohydrate inhibition pattern: mannose and ManNAc > fucose > GlcNAc > glucose and maltose > galactose > lactose >> GalNAc. We conclude that CL-43 is a circulating lectin, with structural similarities to bovine conglutinin and SP-D, and a ligand binding profile resembling that of MBP.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of bovine mannan-binding protein.

Bovine mannan-binding protein (bMBP) was observed in serum by its Ca(2+)-dependent binding to mannan and by an M(r) of 28 kDa under reducing conditions on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The lectin was isolated by precipitation with polyethyleneglycol (PEG), affinity chromatography on mannan-Sepharose eluted with EDTA, and absorption on Sepharose 4B rabbit anti-bovine Ig to remove anti-mannan antibodies. Fractions containing the lectin were reapplied to mannan-Sepharose and eluted first with N-acetyl-D-glucosamine (GlcNAc) to remove conglutinin, and then with mannose to elute the 28 kDa lectin. Further purification was achieved by ion-exchange chromatography on Mono-Q and by mannose-gradient elution from a mannan-Sepharose column. SDS-PAGE of the purified lectin showed three high molecular weight bands under non-reducing conditions. The reduced protein gave a single band of 28 kDa. On gel permeation chromatography under non-dissociating conditions, the protein emerged at a volume corresponding to M(r) approximately 750 kDa. Amino acid analysis showed the presence of hydroxyproline and hydroxylysine, and a high glycine content (17.7%), suggesting the presence of a collagen-like structure. This was supported by the susceptibility of the protein to collagenase digestion. The N-terminal 26 amino acids showed 62% identity with human MBP, when three gaps were allowed in the alignment.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Structure and function of collectins: humoral C-type lectins with collagenous regions.

Collectins are a family of C-type lectins with collagenous regions. Five such lectins have now been described: Lung surfactant protein A and D (SP-A and SP-D), and the plasma proteins, conglutinin, mannan-binding protein (MBP), and CL-43. They are composed of trimeric subunits containing a collagenous section and a C-terminal globular carbohydrate-recognizing domain containing the 14 invariant amino acids characteristic of the C-type lectins. The complete molecules of MBP and SP-A are composed of up to six such subunits, while conglutinin, SP-D and CL-43 contain up to four subunits. The collectins bind to carbohydrates on yeast, bacteria and viruses. The collagenous section reacts with the C1q receptor (collectin receptor) found on many cells including phagocytes. One function of these lectins appears to be the enhancement of phagocytosis, i.e. opsonization. One of the collectins, mannan-binding protein, activates the C1r2C1s2 complex of the classical complement pathway independently of C1q. Collectins have been found in several mammalian species and in the chicken. It seems likely that the biological role of the collectins is to participate in the innate immune defense.

Amino Acid Sequence↗
PubMed1992

Calcium-dependent and calcium-independent signals in the conglutinin-binding assay (KgBa) for immune complexes. Influence of anti-collagen-antibodies.

A solid phase ELISA conglutinin-binding assay (KgBa) was evaluated for the detection of circulating immune complexes. ELISA wells were coated with purified bovine conglutinin and incubated with test sera. Bound IgG was detected with enzyme labelled anti-immunoglobulin. Heat aggregated IgG which had been "solubilized" (i.e., complement treated by incubation with serum) was employed as a reference. The binding of the complement-reacted IgG to solid phase conglutinin was found to be calcium-dependent and inhibitable with N-acetyl-D-glucosamine (GlcNAc). Prolonged incubation (4 days) of aggregated IgG with serum at 37 degrees C abolished the binding to conglutinin, a finding consistent with the complete degradation of deposited C3b to C3c and C3d. The solubilized IgG that bound to solid phase conglutinin was found by gel chromatography to be of high molecular weight (greater than 600 kDa). Binding of IgG to solid phase bovine conglutinin was also observed to a variable degree in normal and pathological sera. However, in this situation the IgG binding was largely calcium-independent, was not inhibited by GlcNAc and did not decrease after prolonged incubation of the serum at 37 degrees C. The reactive IgG eluted on gel chromatography at the position of monomeric IgG suggesting binding via the antigen binding sites. Binding of this IgG was inhibited by both collagen type II and purified conglutinin. These observations suggest that the assay detects cross-reacting autoantibodies against collagen epitopes, or, alternatively, antibodies against the dietary antigen, bovine conglutinin.

Acetylgalactosamine↗
PubMed1992

The concentration of the C-type lectin, mannan-binding protein, in human plasma increases during an acute phase response.

Two ELISAs for estimating mannan-binding protein (MBP) were constructed and the concentration of MBP in plasma was followed in patients undergoing major surgery and in patients having a malarial attack. In both cases increases of MBP in the plasma were observed. The relative increase and the kinetics varied from person to person. The concentration of MBP increased between 1.5- and three-fold following surgery. In some patients an increase was seen at day 1 whereas in others the increase was not observed until days 3-9. In the malaria patients an increased level of MBP was maintained during 30 days of treatment with chloroquine. The relative increase in MBP was independent of the presurgery or premalaria levels.

Acute-Phase Reaction↗