Molecular characterization and complement activating functional analysis of a new collectin(TfCol-11) from Trachidermus fasciatus.

The complement system is a crucial component of the innate immune system that links innate and adaptive immunity. CL-11, a protein similar to Mannose-binding lectin (MBL), plays significant role in the innate immune system in mammals and fish, serving as an initiator of the lectin pathway of complement activation. In this study, a CL-11 homolog (TfCol-11) was identified in roughskin sculpin (Trachidermus fasciatus), and its expression and role in immune responses were characterized. The open reading frame of TfCol-11 is 795 bp long, encoding a 264 amino acid polypeptide. The deduced amino acid sequence of this protein is highly homologous to sequences in other teleosts, and is similar to vertebrate CL-11, containing a canonical collagen-like region, a carbohydrate recognition domain, and a neck region. Recombinant TfCol-11 purified from Escherichia coli(E.coli) was able to bind to different microbes in a Ca2+-independent manner. Meanwhile, a 993 bp-long of partial MASP cDNA with a 96 bp 5' untranslated region (UTR) was also cloned from roughskin sculpin, containing 299 amino acids and consisting of three domains (CUB-EGF-CUB). qRT-PCR indicated that TfCol-11 and MASP mRNAs were predominately co-expressed in the liver. The temporal expression of TfCol-11 and MASP were both drastically up-regulated in the liver, skin, and blood by LPS challenge. Recombinant TfCol-11 purified from E.coli BL21(DE3) was able to agglutinate some bacteria in a Ca2+-dependent manner. In addition, an in vitro pull-down experiment demonstrated that TfCol-11 was able to bind to MASP, and in vivo experiments showed that TfCol-11 was associated with increased membrane attack complex (MAC) levels. It is therefore possible that TfCol-11 may plays a role in activating the complement system and in the defense against invading microorganisms in roughskin sculpin.

Surfactant Proteins A and D: Trimerized Innate Immunity Proteins with an Affinity for Viral Fusion Proteins.

Innate recognition of viruses is an essential part of the immune response to viral pathogens. This is integral to the maintenance of healthy lungs, which are free from infection and efficient at gaseous exchange. An important component of innate immunity for identifying viruses is the family of C-type collagen-containing lectins, also known as collectins. These secreted, soluble proteins are pattern recognition receptors (PRRs) which recognise pathogen-associated molecular patterns (PAMPs), including viral glycoproteins. These innate immune proteins are composed of trimerized units which oligomerise into higher-order structures and facilitate the clearance of viral pathogens through multiple mechanisms. Similarly, many viral surface proteins form trimeric configurations, despite not showing primary protein sequence similarities across the virus classes and families to which they belong. In this review, we discuss the role of the lung collectins, i.e., surfactant proteins A and D (SP-A and SP-D) in viral recognition. We focus particularly on the structural similarity and complementarity of these trimeric collectins with the trimeric viral fusion proteins with which, we hypothesise, they have elegantly co-evolved. Recombinant versions of these innate immune proteins may have therapeutic potential in a range of infectious and inflammatory lung diseases including anti-viral therapeutics.

Heteromeric complexes of native collectin kidney 1 and collectin liver 1 are found in the circulation with MASPs and activate the complement system.

The complement system is an important part of the innate immune system. The complement cascade may be initiated downstream of the lectin activation pathway upon binding of mannan-binding lectin, ficolins, or collectin kidney 1 (CL-K1, alias CL-11) to suitable microbial patterns consisting of carbohydrates or acetylated molecules. During purification and characterization of native CL-K1 from plasma, we observed that collectin liver 1 (CL-L1) was copurified. Based on deglycosylation and nonreduced/reduced two-dimensional SDS-PAGE, we detected CL-K1 and CL-L1 in disulfide bridge-stabilized complexes. Heteromeric complex formation in plasma was further shown by ELISA and transient coexpression. Judging from the migration pattern on two-dimensional SDS-PAGE, the majority of plasma CL-K1 was found in complex with CL-L1. The ratio of this complex was in favor of CL-K1, suggesting that a heteromeric subunit is composed of one CL-L1 and two CL-K1 polypeptide chains. We found that the complex bound to mannan-binding lectin-associated serine proteases (MASPs) with affinities in the nM range in vitro and was associated with both MASP-1/-3 and MASP-2 in plasma. Upon binding to mannan or DNA in the presence of MASP-2, the CL-L1-CL-K1 complex mediated deposition of C4b. In favor of large oligomers, the activity of the complex was partly determined by the oligomeric size, which may be influenced by an alternatively spliced variant of CL-K1. The activity of the native heteromeric complexes was superior to that of recombinant CL-K1. We conclude that CL-K1 exists in circulation in the form of heteromeric complexes with CL-L1 that interact with MASPs and can mediate complement activation.

Development of enzyme-linked immunosorbent assay for bovine surfactant protein D in bronchoalveolar lavage fluid.

Surfactant protein D (SP-D) is a pattern recognition molecule that has an important role in pulmonary host defense. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for bovine SP-D and determined the concentration of SP-D in bronchoalveolar lavage fluid (BALF) from calves. Bovine SP-D was purified from BALF using a mannose-Shepharose 6B column. The obtained 44 kDa protein was identified as bovine SP-D by N-terminal amino acid sequence analysis and SDS-PAGE analysis. The peptides corresponding to bovine SP-D amino acid residues SDTRKEGT, which have little homology across bovine serum collectins, were synthesized and used to raise an antibody in rabbits. The obtained antibody was specific for bovine SP-D and did not react with collectins in serum. The anti-bovine SP-D antibody was purified and an ELISA system was developed. The detection range of this assay was 4-125 ng/ml, and the intra-assay and inter-assay coefficients of variation were 5.6 and 9.7%, respectively. The concentrations of SP-D in BALF collected from calves experimentally infected with bovine adenovirus type-3 or Mannheimia haemolytica were determined by the ELISA. Elevation of SP-D was found in BALF from inoculated lobes of infected calves compared with those of non-inoculated lobes and those from control animals. These data suggest that the ELISA developed in this study may be available to investigate the physiological role of bovine SP-D in bovine lung.

Collectrin, a homologue of ACE2, its transcriptional control and functional perspectives.

Collectrin is a type I membrane protein and shares significant homology with C-terminal domain of angiotensin-converting enzyme-2 (ACE2). However, collectrin lacks catalytic domain and it suggests the presence of uncharacterized physiological functions of collectrin. Collectrin is transcriptionally regulated by hepatocyte nuclear factor-alpha and -beta and is highly expressed on renal proximal tubules and collecting ducts as well as pancreatic beta-cells. Recent in vitro and in vivo studies demonstrated interesting physiological roles of collectrin related to insulin secretion, formation of primary cilia, renal cyst formation and amino acid transport. The common underlying molecular mechanism may be suggested by the evidence that collectrin binds to SNARE complex by interacting with snapin. Collectrin is involved in the process of vesicle transport and membrane fusion and thus it delivers insulin for exocytosis or various membrane proteins to apical plasmalemma and primary cilia. Collectrin may be the new therapeutic target for various pathological processes such as diabetes, polycystic kidney disease, hypertension and aminoaciduria.

Collectins: sentinels of innate immunity.

Collectins, present in plasma and on mucosal surfaces, are humoral molecules of the innate immune system. They were discovered a hundred years ago in 1906 as the first association of an animal lectin with the immune system. They are a family of calcium-dependent lectins that recognize pathogen-associated molecular patterns. They share a similar modular domain architecture consisting of four regions; a cysteine-rich N-terminal domain, a collagen-like region, an alpha-helical neck domain and a C-terminal carbohydrate recognition domain. There have been eight collectins members defined so far, of which, MBL, SP-A and SP-D are the most characterized. Collectins represent the first line of host defense. Upon recognition of the infectious agents, collectins put into action effector mechanisms like direct opsonization, neutralization, agglutination, complement activation and phagocytosis to curb the microbial growth. In addition, they also modulate inflammatory and allergic responses and apoptotic cell clearance. These functions limit infection and subsequently modulate the adaptive immune responses. The role of collectins, their structure, function, characteristics and clinical significance are reviewed in this article.

Characterization and expression sites of newly identified chicken collectins.

Collectins are members of the family of vertebrate C-type lectins. They have been found almost exclusively in mammals, with the exception of chicken MBL. Because of their important role in innate immunity, we sought to identify other collectins in chicken. Using the amino acid sequences of known collectins, the EST database was searched and related to the chicken genome. Three chicken collectins were found and designated chicken Collectin 1 (cCL-1), chicken Collectin 2 (cCL-2), and chicken Collectin 3 (cCL-3), which resemble the mammalian proteins Collectin Liver 1, Collectin 11 and Collectin Placenta 1, respectively. Additionally, a lectin was found which resembled Surfactant Protein A, but lacked the collagen domain. Therefore, it was named chicken Lung Lectin (cLL). Tissue distribution analysis showed cCL-1, cCL-2 and cCL-3 are expressed in a wide range of tissues throughout the digestive, the reproductive and the lymphatic system. Similar to SP-A, cLL is mainly localized in lung tissue. Phylogenetic analysis indicates that cCL-1, cCL-2 and cCL-3 represent new subgroups within the collectin family. The newly found collectins may have an important function in avian host defence. Elucidation of the role of these pattern-recognition molecules could lead to strategies that thwart infectious diseases in poultry, which could also be beneficial for public health.

Collectins: players of the innate immune system.

Collectins are a family of collagenous calcium-dependent defense lectins in animals. Their polypeptide chains consist of four regions: a cysteine-rich N-terminal domain, a collagen-like region, an alpha-helical coiled-coil neck domain and a C-terminal lectin or carbohydrate-recognition domain. These polypeptide chains form trimers that may assemble into larger oligomers. The best studied family members are the mannan-binding lectin, which is secreted into the blood by the liver, and the surfactant proteins A and D, which are secreted into the pulmonary alveolar and airway lining fluid. The collectins represent an important group of pattern recognition molecules, which bind to oligosaccharide structures and/or lipid moities on the surface of microorganisms. They bind preferentially to monosaccharide units of the mannose type, which present two vicinal hydroxyl groups in an equatorial position. High-affinity interactions between collectins and microorganisms depend, on the one hand, on the high density of the carbohydrate ligands on the microbial surface, and on the other, on the degree of oligomerization of the collectin. Apart from binding to microorganisms, the collectins can interact with receptors on host cells. Binding of collectins to microorganisms may facilitate microbial clearance through aggregation, complement activation, opsonization and activation of phagocytosis, and inhibition of microbial growth. In addition, the collectins can modulate inflammatory and allergic responses, affect apoptotic cell clearance and modulate the adaptive immune system.

By binding SIRPalpha or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation.

Surfactant proteins A and D (SP-A and SP-D) are lung collectins composed of two regions, a globular head domain that binds PAMPs and a collagenous tail domain that initiates phagocytosis. We provide evidence that SP-A and SP-D act in a dual manner, to enhance or suppress inflammatory mediator production depending on binding orientation. SP-A and SP-D bind SIRPalpha through their globular heads to initiate a signaling pathway that blocks proinflammatory mediator production. In contrast, their collagenous tails stimulate proinflammatory mediator production through binding to calreticulin/CD91. Together a model is implied in which SP-A and SP-D help maintain a non/anti-inflammatory lung environment by stimulating SIRPalpha on resident cells through their globular heads. However, interaction of these heads with PAMPs on foreign organisms or damaged cells and presentation of the collagenous tails in an aggregated state to calreticulin/CD91, stimulates phagocytosis and proinflammatory responses.

SRCL/CL-P1 recognizes GalNAc and a carcinoma-associated antigen, Tn antigen.

SRCL /CL-P1 was recently identified as a scavenger receptor with a C-type lectin domain, which was expressed in vascular endothelial cells and could bind to Gram-positive and Gram-negative bacteria, yeast and oxidized LDL. We found that SRCL was expressed in some but not all nurse-like cells examined. Furthermore, to characterize the C-type lectin domain of SRCL, the secreted form of the C-type lectin domain (LEC-AP) of SRCL, which was fused to the signal sequence of IgG and alkaline phosphatase, was expressed in 293/EBNA-1 cells and the culture medium was used for the in vitro binding assay. LEC-AP specifically bound to GalNAc-conjugated gel in a Ca(2+)-dependent manner, and this binding was inhibited by free GalNAc, L-, D-fucose, D-galactose, lactose, and especially T antigen and Tn antigen. Furthermore, we examined whether or not SRCL could take up saccharide-conjugated particles. 293/EBNA-1 cells stably expressing SRCL were found to take up GalNAc but not mannose-conjugated particles on confocal microscopy. The binding of GalNAc-conjugated particles to these cells was quantitatively measured by comparing the x-means of individual cell populations. An approximately 2.1-fold increase in immunofluorescence intensity was observed for the SRCL transfectants compared to control vector transfectants. Our results provide a basis for understanding the scavenger function of SRCL as to carbohydrate-containing ligands.

Secretion of a collectin-like protein in tunicates is enhanced during inflammatory responses.

The sub-cellular and humoral concentrations of a collectin-like protein from the solitary tunicate, Styela plicata, were measured after in vivo challenge with the inflammatory agent, zymosan. Tunicates were injected with zymosan before hemocytes and serum were harvested, subjected to western blotting and immunostained with an anti-S. plicata collectin antibody to determine the relative titers of collectin-like proteins. Concentrations of the predominant 43kDa collectin polypeptide were found to decrease in hemocytes immediately after zymosan injection, before rising to levels that were six times higher than controls within 96h. Similarly, immunohistochemistry showed that the frequency of collectin-positive hemocytes in the circulating hemolymph increased significantly within 96h of injection. Levels of the 43kDa polypeptide in serum mirrored those of hemocytes. Humoral collectin concentrations decreased immediately after zymosan injection before rising, within 96h post-injection, to levels three times higher than controls. This response to an inflammatory stimulus resembles that of mammalian collectins like mannose-binding lectin. The data suggest that, like its mammalian counterparts, the tunicate collectin acts as an acute phase antigen recognition protein.

CL-46, a novel collectin highly expressed in bovine thymus and liver.

Collectins are oligomeric molecules with C-type lectin domains attached to collagen-like regions via alpha-helical neck regions. They bind nonself glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination, or opsonization. During the characterization of the gene encoding bovine CL-43 (43-kDa collectin), we identified a novel collectin-gene. We report the cloning and partial characterization of the novel collectin CL-46. The mRNA comprises 1188 nucleotides encoding a protein of 371 aa with an included leader peptide of 20 residues. CL-46 has two cysteine residues in the N-terminal segment, a potential N-glycosylation site in the collagen region, and an extended hydrophilic loop close to the binding site of the carbohydrate recognition domain. It is expressed in the thymus, liver, mammary gland, and tissues of the digestive system. Recombinant CL-46 corresponding to the alpha-helical neck region and the C-type lectin domain binds preferential N-acetyl-D-glucoseamine and N-acetyl-D-mannoseamine. The gene encoding CL-46 spans approximately 10 kb and consists of eight exons, with high structural resemblance to the gene encoding human surfactant protein D. It is located on the bovine chromosome 28 at position q1.8 together with the gene encoding conglutinin and CL-43. Several potential thymus-related cis-regulatory elements were identified in the 5'-upstream sequence, indicating that the expression in thymus may be modulated by signals involved in T cell development.