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Biomedical subjects

U Holmskov

Publications and source records attributed to U Holmskov.

At least 19 recordsLinked to original sources
PubMed2000

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340.

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

Agglutinins↗
PubMed2000

Cloning of a novel scavenger receptor cysteine-rich type I transmembrane molecule (M160) expressed by human macrophages.

We report the cloning of a novel human type I cell surface Ag mainly expressed by macrophages. The primary structure was established by molecular cloning, which yielded a 4579-bp cDNA sequence encoding a polypeptide chain of 1453 amino acid residues with 16 potential N:-glycosylation sites. We designated this molecule M160. The domain organization features 12 scavenger receptor cysteine-rich domains followed by a transmembrane region and a cytoplasmic domain that occurs in two forms, a predominant form (M160-alpha) of 71 residues and an alternatively spliced form (M160-ss) of 39 residues. M160-alpha contains three possible phosphorylation sites, which are lost in the alternatively spliced form. RT-PCR analyses showed M160 to be expressed by alveolar macrophages and by the monocyte cell lines HL60, U937, and THP1, but not by Jurkat or Raji cells. Stimulation of U937 cells with phorbol ester resulted in an increased expression of M160 from day 5 onward. RT-PCR analysis of 19 different human tissues showed signals for M160-alpha of varying intensity in all tissues, whereas M160-ss was confined to the spleen. We conclude that M160 is a new member of the scavenger receptor cysteine-rich superfamily expressed by the monocyte/macrophage cell lineage.

Alternative Splicing↗
PubMed2000

Structural characterisation of human proteinosis surfactant protein A.

Human surfactant protein-A (SP-A) has been purified from a proteinosis patient and characterised by a combination of automated Edman degradation and mass spectrometry. The complete protein sequence was characterised. The major part of SP-A was shown to consist of SP-A2 gene product, and only a small amount of SP-A1 gene product was shown to be present. A cysteine extension to the N-terminal was indicated by sequence data, but was not definitely proven. All proline residues in the Y position of Gly-X-Y in the collagen-like region were at least partially modified to hydroxy-proline, but no lysine residues were found to be modified. A complex N-linked glycosylation was found on Asn-187 showing great heterogeneity as variants from a mono-antennary to penta-antennary glycosylation with varying amounts of attached pentose were identified. The disulfide bridges in the carbohydrate recognition domain were identified to be in the 1-4, 2-3 pattern common for collectins. Interchain disulfide bridges were discovered between two Cys-48 residues and cysteine residues in the N-terminal region. However, the exact disulfide bridge connections within the bouquet-like ultrastructure could not be established.

Amino Acid Sequence↗
PubMed2000

Localization of lung surfactant protein D on mucosal surfaces in human tissues.

Lung surfactant protein-D (SP-D), a collectin mainly produced by alveolar type II cells, initiates the effector mechanisms of innate immunity on binding to microbial carbohydrates. A panel of mRNAs from human tissues was screened for SP-D mRNA by RT-PCR. The lung was the main site of synthesis, but transcripts were readily amplified from trachea, brain, testis, salivary gland, heart, prostate gland, kidney, and pancreas. Minor sites of synthesis were uterus, small intestine, placenta, mammary gland, and stomach. The sequence of SP-D derived from parotid gland mRNA was identical with that of pulmonary SP-D. mAbs were raised against SP-D, and one was used to locate SP-D in cells and tissues by immunohistochemistry. SP-D immunoreactivity was found in alveolar type II cells, Clara cells, on and within alveolar macrophages, in epithelial cells of large and small ducts of the parotid gland, sweat glands, and lachrymal glands, in epithelial cells of the gall bladder and intrahepatic bile ducts, and in exocrine pancreatic ducts. SP-D was also present in epithelial cells of the skin, esophagus, small intestine, and urinary tract, as well as in the collecting ducts of the kidney. SP-D is generally present on mucosal surfaces and not restricted to a subset of cells in the lung. The localization and functions of SP-D indicate that this collectin is the counterpart in the innate immune system of IgA in the adaptive immune system.

Antibodies, Monoclonal↗
PubMed2000

DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer.

The gene deleted in malignant brain tumors 1 (DMBT1) has been proposed as a candidate tumor suppressor for brain, gastrointestinal, and lung cancer. It codes for a protein of unknown function belonging to the superfamily of scavenger receptor cysteine-rich proteins. We aimed at getting insights into the functions of DMBT1 by expression analyses and studies with a monoclonal antibody against the protein. The DMBT1 mRNA is expressed throughout the immune system, and Western blot studies demonstrated that isoforms of DMBT1 are identical to the collectin-binding protein gp-340, a glycoprotein that is involved in the respiratory immune defense. Immunohistochemical analyses revealed that DMBT1 is produced by both tumor-associated macrophages and tumor cells and that it is deregulated in glioblastoma multiforme in comparison to normal brain tissue. Our data further suggest that the proteins CRP-ductin and hensin, both of which have been implicated in epithelial differentiation, are the DMBT1 orthologs in mice and rabbits, respectively. These findings and the spatial and temporal distribution of DMBT1 in fetal and adult epithelia suggest that DMBT1 further plays a role in epithelial development. Rearrangements of DMBT1 were found in 16 of 18 tumor cell lines, and hemizygous deletions were observed in a subset of normal individuals, indicating that the alterations in tumors may be a result of both pre-existing deletions uncovered by a loss of heterozygosity and secondary changes acquired during tumorigenesis. Thus, DMBT1 is a gene that is highly unstable in cancer and encodes for a protein with at least two different functions, one in the immune defense and a second one in epithelial differentiation.

Agglutinins↗
PubMed2000

Purification and characterization of two mannan-binding lectins from mouse serum.

Mannan-binding lectin (MBL) is a serum protein that activates the complement system after binding to glycoconjugates found on the surface of microorganisms. By molecular cloning two forms of MBL have been identified in the mouse (mMBL-A and mMBL-C), but only mMBL-A has been purified and characterized at the protein level. MBL-C has been termed the liver form of MBL. The present report describes the purification and characterization of mMBL-A and mMBL-C from serum. The two forms of mMBL could be separated both by ion-exchange and carbohydrate-affinity chromatography. The initial identification by immunochemical technique was confirmed by N-terminal amino-acid sequencing. Both proteins give bands corresponding to polypeptide chains of 28 kDa on SDS-PAGE in the reduced state, but mMBL-A migrated more rapidly than mMBL-C in acid/urea-PAGE, in accordance with the calculated pIs. Both forms mediated activation of complement component C4 in mannan-coated microtiter wells. MBL-A showed a higher affinity for d -glucose and alpha-methyl-d -glucose then did MBL-C. Serum concentrations of mMBL-A in laboratory strains and wild mice were found to vary from 5 to 80 microg/ml, with wild mice tending to show higher levels than laboratory strains.

Amino Acid Sequence↗
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↗