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U Holmskov

Publications and source records attributed to U Holmskov.

At least 37 records · Page 2Linked to original sources
PubMed2000

The homologue of mannose-binding lectin in the carp family Cyprinidae is expressed at high level in spleen, and the deduced primary structure predicts affinity for galactose.

Mannose-binding lectin (MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. We isolated and characterized cDNA transcripts encoding an MBL homologue from three members of the carp family Cyprinidae, the zebrafish Danio rerio, the goldfish Carassius auratus, and the carp Cyprinus carpio. The carp and zebrafish transcripts contain two polyadenylation sites and RT-PCR on mRNA from carp tissues revealed the carp transcript to be most prominently expressed in the spleen. The deduced mature proteins contain 228 or 233 amino acids with a short N-terminal segment containing a single conserved cysteine expected to form interchain disulfide bridges, a collagen domain interrupted by four amino acids between two glycine residues, a neck region predicted to form an alpha-helical coiled-coil structure, and a C-terminal carbohydrate recognition domain (CRD). Several of the structurally important residues in the CRD are conserved, but the residues known to interact with the calcium ion and hydroxyl groups of the carbohydrate ligand are different. The amino acid motif EPN, important for mannose specificity, was QPD in the Cyprinidae homologue, suggesting specificity for galactose instead. The identity between the deduced amino acid sequences is more than 90% between the carp and the goldfish and 68% and 65% between these two species, respectively, and the zebrafish. The identity with bird and mammalian MBLs ranges from 28 to 33%.

Amino Acid Sequence↗
PubMed1999

Microfibril-associated protein 4 is present in lung washings and binds to the collagen region of lung surfactant protein D.

We have purified a glycoprotein from bovine lung washings using affinity chromatography on a maltose-affinity column. On SDS-polyacrylamide gel electrophoresis the protein showed a molecular mass of 36 kDa in the reduced state and 66 kDa in the unreduced state. On gel permeation chromatography the apparent molecular mass was 250 kDa. N-terminal sequencing showed homology to the human matrix protein microfibril-associated protein (hMFAP4), and the glycoprotein was designated bovine MFAP4 (bMFAP4). Lung surfactant protein D (SP-D) was also purified from lung washings, and calcium-dependent binding was demonstrated between bMFAP4 and SP-D. hMFAP4 was cloned, and recombinant hMFAP4 showed the same binding pattern to SP-D as bMFAP4. No binding was seen to recombinant SP-D composed of the neck region and carbohydrate recognition domain of SP-D, indicating that the interaction between MFAP4 and SP-D is mediated via the collagen region of SP-D. MFAP4 also showed calcium-dependent binding to mannan, which was partially inhibited by maltose. Our findings indicate that MFAP4 has two binding specificities, one for collagen and one for carbohydrate, and we suggest that MFAP4 may fix the collectins in the extracellular compartment during inflammation.

Amino Acid Sequence↗
PubMed1999

The genomic structure of the DMBT1 gene: evidence for a region with susceptibility to genomic instability.

Increasing evidence has accumulated for an involvement of the inactivation of tumour suppressor genes at chromosome 10q in the carcinogenesis of brain tumours, melanomas, and carcinomas of the lung, the prostate, the pancreas, and the endometrium. The gene DMBT1 (Deleted in Malignant Brain Tumours 1) is located at chromosome 10q25.3-q26.1, within one of the putative intervals for tumour suppressor genes. DMBT1 is a member of the scavenger-receptor cysteine-rich (SRCR) superfamily and displays homozygous deletions or lack of expression in glioblastoma multiforme, medulloblastoma, and in gastrointestinal and lung cancers. Based on these properties, DMBT1 has been proposed to be a candidate tumour suppressor gene. We have determined the genomic sequence of DMBT1 to allow analyses of mutations. The gene has at least 54 exons that span a genomic region of about 80 kb. We have identified a putative exon with coding potential for a transmembrane domain. Our data further suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and SRCR interspersed domains. The major part of the gene harbours locus specific repeats. These repeats may point to the DMBT1 locus as a region susceptible to chromosomal instability.

Agglutinins↗
PubMed1999

Structural characterization of human and bovine lung surfactant protein D.

Human and bovine surfactant proteins D (SP-D) were purified from late amniotic fluid and bronchioalveolar lavage on the basis of its Ca(2+)-dependent affinity for maltose. The molecular mass of a trimeric subunit was determined by matrix-assisted laser desorption ionization MS to lie in the range 115-125 kDa for human SP-D and 110-123 kDa for bovine SP-D. A single polypeptide chain was determined at 37-41 and 36-40 kDa for the human and bovine species respectively. The major parts of the primary structures of both SP-D molecules were determined by a combination of MS and Edman degradation. The heterogeneity in SP-D was caused mainly by a high number of post-translational modifications in the collagen-like region. Proline and lysine residues were partly hydroxylated and lysine residues were further O-glycosylated with the disaccharide galactose-glucose. A partly occupied N-linked glycosylation site was characterized in human SP-D. The carbohydrate was determined as a complex type bi-antennary structure, with a small content of mono-antennary and tri-antennary structures. No sialic acid residues were present on the glycan, but some had an attached fucose and/or an N-acetylglucosamine residue linked to the core. Bovine SP-D was determined as having a similar structure.

Amino Acid Sequence↗
PubMed1999

Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D.

Surfactant protein D (SP-D) is an oligomeric C type lectin that promotes phagocytosis by binding to microbial surface carbohydrates. A 340-kDa glycoprotein (gp-340) has been shown to bind SP-D in the presence of calcium but does so independently of carbohydrate recognition. This protein exists both in a soluble form and in association with the membranes of alveolar macrophages. The primary structure of gp-340 has been established by molecular cloning, which yielded a 7,686-bp cDNA sequence encoding a polypeptide chain of 2, 413 amino acids. The domain organization features 13 scavenger receptor cysteine-rich (SRCR) domains, each separated by an SRCR-interspersed domain, except for SRCRs 4 and 5, which are contiguous. The 13 SRCR domains are followed by two C1r/C1s Uegf Bmp1 domains separated by a 14th SRCR domain and a zona pellucida domain. gp-340 seems to be an alternative spliced form of DMBT1. Reverse transcription-PCR analysis showed that the main sites of synthesis of gp-340 are lung, trachea, salivary gland, small intestine, and stomach. Immunohistochemistry revealed strong staining for gp-340 in alveolar and other tissue macrophages. Immunostaining of the macrophage membrane was either uniform or focal in a way that suggested capping, whereas other macrophages showed strong intracellular staining within the phagosome/phagolysosome compartments. In some macrophages, SP-D and gp-340 were located in the same cellular compartment. Immunoreactive gp-340 was also found in epithelial cells of the small intestine and in the ducts of salivary glands. The distribution of gp-340 in macrophages is compatible with a role as an opsonin receptor for SP-D.

Amino Acid Sequence↗
PubMed1999

Lung surfactant proteins (SP-A and SP-D) in non-adaptive host responses to infection.

The lung surfactant proteins A and D (SP-A and SP-D) are collectins composed of C-type lectin domains attached to collagen regions. SP-A and SP-D are mainly found in the surfactant covering the pulmonary epithelial cells, but are also produced by cells lining the gastrointestinal tract. The main role of SP-A and SP-D is to interact directly with carbohydrate on the surface of microbial pathogens, thereby initiating a variety of effector mechanisms. This review focuses on the non-adaptive host responses of SP-A and SP-D to infection. Interaction of SP-A and SP-D with phagocytes is discussed and the structure and function of the putative receptors for SP-A and SP-D is presented. SP-A and SP-D seem to be regulated in a way similar to acute-phase proteins in the course of inflammation and evidence for the involvement of SP-A and SP-D as immunomodulators as well as their role in clearing allergens and modulating effector mechanisms in allergic reactions is discussed.

Adaptation, Physiological↗
PubMed1999

Genomic organization of the mouse gene for lung surfactant protein D.

Lung surfactant protein (SP)-D belongs to the family of soluble collagenous C-type lectins, named collectins. SP-D participates in the local innate immune defense of the lung, eliciting various effector functions by acting as a pattern recognition receptor for the carbohydrate structures on inhaled microorganisms and particulate matter. This work describes the isolation and characterization of the mouse SP-D gene (Sftpd), which spans 8 exons over 14 kb of sequence and shows an overall organization similar to other collectin genes. The complete 5' untranslated region of the messenger RNA, absent from the published complementary DNA for mouse SP-D, was also cloned and is shown to be encoded by a single exon. Analysis of 3.5 kb of 5' flanking nucleotide sequence for Sftpd is described and reveals positional conservation of a number of transcription factor binding sites on comparison of Sftpd with the human SP-D gene and the bovine conglutinin gene. In addition, a single copy SP-D-like gene has been shown to be present in mammals, birds, and amphibians but is absent in fish. An atypical, rodent-specific, long terminal repeat of retroviral origin containing a minisatellite that has become inserted in Sftpd is described. Three new polymorphic microsatellites are also described, one of which is just 160 base pairs upstream of Sftpd. This microsatellite was used to map the gene to the central region of chromosome 14; fine-scale mapping indicates that it lies in a 5. 64-centimorgan area between D14Mit45 and D14Mit60. This will allow the easy identification of the collectin gene cluster and aid in the construction of a physical map over this region.

5' Untranslated Regions↗
PubMed1998

Structural aspects of collectins and receptors for collectins.

The collectins are oligomeric molecules composed of C-type lectin domains attached to collagen regions via alpha-coiled neck regions. Five members of the collectins have been characterized. Mannan-binding lectin (MBL), conglutinin and collectin-43 (CL-43) are serum proteins produced by the liver. Lung surfactant protein A (SP-A) and lung surfactant protein D (SP-D) are mainly found in the lung, where they are synthesized by alveolar type II cells and secreted to the alveolar surface. The collectins are believed to play an important role in innate immunity. They bind oligosaccharides on the surface of a variety of microbial pathogens. After binding of the collectins to the microbial surface effector mechanisms such as agglutination, neutralizing or opsonization of the microorganisms for phagocytosis are initiated. SP-A and SP-D stimulate chemotaxis of phagocytes and once bound to the phagocytes, the production of oxygen radicals can be induced. In the case of MBL the opsonization can be further enhanced by complement activation via the MBLectin pathway while conglutinin interacts with the complement system by binding to the complement degradation product iC3b. A number of receptors and binding molecules interacting with the collectins are found on the membrane or in association with the membrane of various cells responsible for phagocytosis and clearance of microorganisms. This paper focus on the structural aspects of the collectins and the receptors for collectins.

Amino Acid Sequence↗
PubMed1998

The plasma levels of coglutinin are heritable in cattle and low levels predispose to infection.

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.

Animals↗
PubMed1998

Antiviral activity of bovine collectins against rotaviruses.

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 haemagglutination inhibition and neutralization of rotavirus infectivity, CL-43 displaying the highest activity in both types of assay. In contrast, conglutinin was the most potent of the three collectins against influenza virus A/HKx31. Neutralization of rotaviruses by the lectins was dependent on glycosylation of VP7. Furthermore, rotaviruses adapted to growth in Madin-Darby bovine kidney cells, and thus bearing carbohydrate of bovine origin, remained sensitive to neutralization, although slightly less so than virus stocks propagated in the monkey kidney cell line MA104. These findings provide the first description of antiviral activity of collectins against a non-enveloped virus and may indicate a potential role for collectins in host defence against bovine rotavirus infection.

Animals↗
PubMed1997

Fetal antigen 1 (FA1), a circulating member of the epidermal growth factor (EGF) superfamily: ELISA development, physiology and metabolism in relation to renal function.

We describe an ELISA technique for quantification of fetal antigen 1 (FA1), a glycoprotein belonging to the EGF-superfamily. The ELISA is based on immunospecifically purified polyclonal antibodies and has a dynamic range of 0.7-5.3 ng/ml, intra- and inter-assay C.V.s of less than 3.2% and an average recovery of 105% in serum and 98% in urine. Comparison of FA1 in amniotic fluid, serum and urine revealed parallel titration curves, identical elution volumes following size chromatography, immunological identity and similar profiles when analysed by MALDI-MS. The reference interval for serum FA1 was 12.3-46.6 ng/ml and the levels were 10 times higher in patients with renal failure. FA1 showed no diurnal variation, no variation during the menstrual cycle and was not influenced by the acute phase reaction. In humans (n = 10) the renal clearance of FA1 was 11 ml/min and an identical high renal clearance was found in rats when expressed per 100 g body weight. In rats the initial increase in serum FA1 was 10 ng/ml/h following bilateral nephrectomy, explaining the increased serum concentrations of FA1 observed in patients with renal failure.

Acute-Phase Reaction↗
PubMed1997

Isolation and characterization of a new member of the scavenger receptor superfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-D binding molecule.

We have purified a previously unknown glycoprotein (designated gp-340) from human bronchioalveolar lung washings from a patient with alveolar proteinosis. gp-340 was identified by its calcium-dependent binding to lung surfactant protein D (SP-D) and by its molecular mass of 340 kDa in the reduced state on SDS-polyacrylamide gel electrophoresis (PAGE). gp-340 was purified from the 10,000 x g pellet of the lavage fluid by ion-exchange and gel permeation chromatography. On SDS-PAGE, gp-340 showed an apparent molecular mass of 290 kDa in the unreduced state. On gel chromatography under non-dissociating conditions, the apparent molecular mass of gp-340 was >1000 kDa. The presence of N-linked glycosylation was shown by digestion with N-glycosidase F, which reduced the apparent molecular mass of gp-340 under reducing condition to about 300 kDa. Partial amino acid sequence data showed the presence of scavenger-receptor type domains. Monoclonal and polyclonal antibodies were raised against gp-340, and their specificities were confirmed by Western blotting. The antibodies were used for immunohistochemical localization of gp-340 in the lung, where it was found on the surface of and within alveolar macrophages. Direct binding between gp-340 and SP-D took place at physiological ionic strength, required the presence of calcium, and was not inhibited by maltose. The binding between SP-D and mannan also required the presence of calcium, but this interaction was completely inhibited by maltose. The same binding pattern was seen between gp-340 and recombinant human SP-D composed of the trimeric neck region and three carbohydrate recognition domains. These findings indicate that the binding between gp-340 and SP-D is a protein-protein interaction rather than a lectin-carbohydrate interaction and that the binding to gp-340 takes place via the carbohydrate recognition domain of SP-D. We conclude that gp-340 is a new member of the scavenger-receptor superfamily and likely to be a truncated form of a receptor for SP-D.

Amino Acid Sequence↗
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

Structural characterization of bovine collectin-43.

Bovine collectin-43 (CL-43), the most recently disclosed member of the collectin group, has been characterized structurally at the protein level by a combination of mass spectrometry and protein sequencing. The molecular mass of reduced CL-43 was determined by the use of mass spectrometry to be 33.6 +/- 0.1 kDa. Furthermore, the mass spectrum showed the presence of a truncated version of the polypeptide, which has also previously been shown by SDS/PAGE and N-terminal sequencing. N-terminal Edman degradation of peptides from a tryptic digestion of native CL-43 verified the published sequence derived from cDNA studies and partial protein sequencing [Lim, B.-L., Willis, A. C., Reid, K. B. M., Lu, J., Lauersen, S. B., Jensenius, J. C. & Holmskov, U. (1994) J. Biol. Chem. 269, 11820-11824] with two exceptions. Using mass spectrometry and N-terminal sequencing, a large number of post-translational modifications were found in the collagen-like region (repetitive Gly-Xaa-Yaa sequence). All proline residues located in the Yaa-position in the collagen-like region were found to be partially hydroxylated while all lysine residues in the Yaa position were fully hydroxylated and glycosylated. The glycosylation was determined as glycosyl-galactosyl O-linked to a hydroxylated lysine residue. Mass spectrometric analysis of a peptic digest of the N-terminal tryptic peptide revealed that the three polypeptide chains were disulphide linked in a rather surprising pattern. The cysteine residues were inter-chain disulphide linked by Cys15 in polypeptide chain 1 to Cys15 in polypeptide chain 2, Cys20 in chain 2 to Cys20 in chain 3 and Cys20 in chain 1 to Cys15 in chain 3. The four cysteine residues at the C terminus were intra-chain disulphide linked, Cys204 to Cys299 and Cys277 to Cys291, as expected for a C-type lectin.

Amino Acid Sequence↗
PubMed1996

Affinity and kinetic analysis of the bovine plasma C-type lectin collectin-43 (CL-43) interacting with mannan.

Collectins are C-type lectins which have been implied to play an important role in the innate immune defence against microorganisms. The critical discriminatory event in the opsonization of microorganisms by collectins is the interaction of the C-type lectin domain with microbial carbohydrates. Surface plasmon resonance measurements allow for quantitative real-time measurements of binding interaction between immobilized carbohydrate and unlabelled lectin in solution. Binding analysis were carried out with purified collectin-43 (CL-43) which structurally is the simplest collectin consisting of only three polypeptides each terminating in a C-type lectin domain. The target was immobilized yeast mannan. The molecular mass of native CL-43 was determinated by mass spectroscopy to 99.8 kDa. The dissociation rate (kdiss) of the C-type lectin-carbohydrate binding was fast (1.19-1.36 x 10(-2) second-1), and the association rate (kass) was 4.37-5.07 x 10(5) M-1 second-1. The equilibrium constant for dissociation (Kd) was 2.68-2.72 x 10(-8) M.

Animals↗
PubMed1996

Expression of the carbohydrate recognition domain of bovine conglutinin and demonstration of its binding to iC3b and yeast mannan.

Bovine conglutinin is a collagenous C-type lectin (collectin) that is found in bovine serum. A recombinant protein, composed of the neck-region and the carbohydrate binding domain of bovine conglutinin, has been overexpressed in E. coli. The recombinant protein has been successfully renatured and showed the same sugar binding specificity as the native protein and was able to bind to yeast mannan and complement-activated immune complexes. The binding was calcium-dependent and was inhibited by N-acetylglucosamine. The concentration of N-acetylglucosamine required for 50% inhibition of binding to mannan was 1.77 mM for recombinant conglutinin and 0.71 mM for native conglutinin, respectively. The recombinant conglutinin should be useful in the assay and purification of circulating immune complexes and for therapeutic purposes involving the removal of immune complexes from patient's plasma.

Acetylglucosamine↗
PubMed1996

Measurement of pulmonary status and surfactant protein levels during dexamethasone treatment of neonatal respiratory distress syndrome.

BACKGROUND: Early postnatal use of dexamethasone in infants with respiratory distress syndrome (RDS) has been shown effectively to improve pulmonary status and to allow early weaning off mechanical ventilation. However, the mechanisms to explain the beneficial effects of dexamethasone in ventilatory dependent preterm infants remain unclear. METHODS: A double blind, placebo controlled study was performed to determine the change in pulmonary ventilation of premature infants with RDS as a result of dexamethasone treatment, and to evaluate the effect of dexamethasone on the levels of surfactant-associated proteins A (SP-A) and D (SP-D) in the tracheal fluid from 34 premature infants with RDS and 29 control subjects. RESULTS: Dexamethasone treatment decreased fractional inspired oxygen concentration (FIO2), arterial carbon dioxide tension (PCO2), mean airway pressure (MAP), and facilitated successful weaning from mechanical ventilation. SP-A concentrations in the tracheal aspirates were increased at days 7 and 14, and SP-D concentrations were increased during the period from days 3 to 14 in the dexamethasone treated group compared with the control group. However, albumin levels in the tracheal aspirate samples were decreased after dexamethasone treatment over the period from days 3 to 14. There was an inverse correlation between PCO2 values and SP-A concentrations. CONCLUSIONS: These results suggest that early use of dexamethasone can improve pulmonary status and also increase SP-A and SP-D levels in the tracheal fluid in premature infants with RDS.

Albumins↗