Abstract
Collectins are a C-lectin family with collagen-like sequences and carbohydrate recognition domains. These proteins can bind to carbohydrate antigens of microorganisms and inhibit their infection by direct neutralization and agglutination, the activation of complement through the lectin pathway, and opsonization by collectin receptors. Here we report the cloning of a cDNA encoding human collectin from liver (CL-L1 (collectin liver 1)) that has typical collectin structural characteristics, consisting of an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain. The cDNA has an insert of 831 base pairs coding for a protein of 277 amino acid residues. The deduced amino acid sequence shows that this collectin has a unique repeat of four lysine residues in its C-terminal area. Northern blot, Western blot, and reverse transcription-polymerase chain reaction analyses showed that CL-L1 is present mainly in liver as a cytosolic protein and at low levels in placenta. More sensitive analyses by reverse transcription-polymerase chain reactions showed that most tissues (except skeletal muscle) have CL-L1 mRNA. Zoo-blot analysis indicated that CL-L1 is limited to mammals and birds. A chromosomal localization study indicated that the CL-L1 gene localizes to chromosome 8q23-q24.1, different from chromosome 10 of other human collectin genes. Expression studies of fusion proteins lacking the collagen and N-terminal domains produced in Escherichia coli affirmed that CL-L1 binds mannose weakly. CL-L1 and recombinant CL-L1 fusion proteins do not bind to mannan columns. Analysis of the phylogenetic tree of CL-L1 and other collectins indicated that CL-L1 belongs to a fourth subfamily of collectins following the mannan-binding protein, surfactant protein A, and surfactant protein D subfamilies including bovine conglutinin and collectin-43 (CL-43). These findings indicate that CL-L1 may be involved in different biological functions.
Highlights
Collectins are a C-lectin family with collagen-like sequences and carbohydrate recognition domains
Identification of a New Human Liver Collectin (CL-L1)—We screened DNA data bases to identify novel members of the collectin family. This resulted in the identification of a cDNA fragment from human expressed sequence tag (EST) data bases that showed carboxylterminal sequence homology to the collectins
Much recent data suggest that collectins play an important role in innate immunity [16]
Summary
Buffers and Media—Escherichia coli lysis buffer for the pMAL-c2 system contained 10 mM phosphate, pH 7.2, 30 mM NaCl, 0.25% (w/v) Tween 20, 10 mM 2-mercaptoethanol, 10 mM EDTA, and 10 mM EGTA. Recombinant CL-L1 CRDs, maltose-binding protein, and recombinant human MBP (1 g of each) were dissolved in SDS sample buffer, separated by SDS-PAGE (10 –20% gradient polyacrylamide gel), and transferred to BioBlot-NC membranes (Coster Co.) by standard procedures. The primary hepatocyte cells (Cell Systems Corp.) were plated and cultured at a density of 3 ϫ 104 cells/0.2 ml in a 14-mm hole of 35-mm plastic culture dishes (Matsunami Glass Industries, Ltd., Kishiwada, Japan) in CS-C complete medium (Cell Systems Corp.) They were fixed in PBS containing 4% paraformaldehyde, pH 7.4, and treated with PBS containing 0.1% Triton X-100, followed by incubation with rabbit antiMBP serum or rabbit anti-CL-L1-CRDhis serum with or without PBS containing 400 g/ml recombinant CL-L1-CRDmal and 25% Block Ace and fluorescein-conjugated anti-rabbit IgG (Chemicon International, Inc.) diluted 1:200. The phylogenetic relationships were analyzed using the computer program PHYLIP Version 3.57c package [25]
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