Collectins and collectin receptors in innate immunity.
This thesis is based on nine papers and a review on the collectins and collectin receptors in innate immunity. The collectins are a family of proteins in which the individual chains consist of a C-type lectin domain attached to a collagen domain via an alpha-coiled neck region. The chains are organized into a triple collagen helix and oligomerized through N-terminally located cysteines. The collectins have a dual function: one is to bind specifically to carbohydrate structures on the surface of a pathogen; the other is subsequently to recruit other cells and molecules to destroy the pathogen. The C-type lectin domains contain 110-130 amino-acid residues arranged in a conserved sequence pattern which allows the domain to fold into a well-defined tertiary structure. Five collectins have been described. Lung surfactant proteins A and D (SP-A and SP-D) are mainly found in the surfactant coating the luminal surface of the pulmonary epithelial cells, but are also produced by cells lining the gastrointestinal tract. Mannan-binding lectin (MBL), conglutinin and collectin-43 (CL-43) are serum proteins produced by the liver. Conglutinin and CL-43 have so far only been found in Bovidae. The collectins are involved in innate, nonadaptive immune defense. They bind to microbial surface carbohydrates, inducing aggregation and thereby impeding infectivity or mediating phagocytosis through specific receptors on the phagocytes. After binding microbial carbohydrate, MBL can activate the complement system through a newly discovered pathway which makes use of two serine proteases (MASP-1 and MASP-2) to activate the complement factors C4 and C2. In man, low serum MBL concentrations resulting from mutations in the collagen region are associated with a common opsonic defect. CL-43 was identified as a new collectin by its calcium-dependent binding to mannan and by its M(r) of 43 kDa in the reduced state on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lectin was isolated by affinity chromatography on mannan-Sepharose, absorption with rabbit anti-bovine Ig coupled to Sepharose-4B and ion-exchange chromatography. CL-43 shows an apparent molecular mass of 120 kDa in the unreduced state on SDS-PAGE and elutes with an apparent molecular mass of 750 kDa on gel chromatography under nondissociating conditions. Amino-acid analysis and susceptibility to collagenase digestion indicated that CL-43 was a collectin. Electron microscopy of purified CL-43 revealed only rod-like monomer subunits 37.4 nm long. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms of pI 4.9 and 5.3 respectively, corresponding to the native form of CL-43 and a truncated form which lacks the first 9 amino-acid residues. The N-terminal amino-acid sequence of CL-43 was used to design primers for PCR with a bovine liver cDNA as template. The cDNA of CL-43 was cloned and the open reading frame was found to encode a protein of 301 amino-acid residues, including an N-terminal region of 28 residues, a collagen region of 114 residues and a neck-CRD region of 159 residues. The amino-acid sequence of CL-43 shows 74% identity with bovine conglutinin and 70% identity with bovine SP-D, but the collagen region is considerably shorter than those of conglutinin and SP-D. Northern blot analysis showed that CL-43 was only synthesized in bovine liver, no signal being detected in a variety of other bovine tissues, including lung. No cross-hybridizing signals were detected in mRNA from ovine, human, rat or mouse liver. Since CL-43 and conglutinin have only been detected in members of the 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 underwent duplication during evolution. The carbohydrate binding profile of CL-43 was analyzed by an inhibition assay with biotinylated CL-43, using solid-phase mannan as the ligand. (ABSTRACT TRUNCATED)
Holmskov
U L
UL
Institute of Medical Biology, University of Southern Denmark, Denmark.
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000276:immunology*; D037282:Calreticulin; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002448:Cell Adhesion; D037222:Collectins; D003141:Communicable Diseases / Q000276:immunology / Q000378:metabolism / Q000382:microbiology / Q000469:parasitology; D006262:Health; D006801:Humans; D007154:Immune System Diseases / Q000276:immunology / Q000378:metabolism; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D012261:Ribonucleoproteins / Q000276:immunology*
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D002241:Carbohydrates; D002352:Carrier Proteins; D037222:Collectins; D037102:Lectins; D012261:Ribonucleoproteins