Molecular cloning of a mouse scavenger receptor with C-type lectin (SRCL)(1), a novel member of the scavenger receptor family.

The cDNA clone encoding a mouse scavenger receptor with C-type lectin (SRCL), a novel member of the scavenger receptor family, has been isolated from a mouse embryonic cDNA library. The predicted cDNA sequence contains a 2226 bp open reading frame encoding a coiled-coil, collagen-like, C-type lectin/carbohydrate recognition domain with an overall sequence identity of 92% to human SRCL. In contrast to human, mouse SRCL mRNA was expressed ubiquitously in various adult tissues including the liver and spleen, in which human SRCL mRNA was under detection limits. Mouse SRCL mRNA was expressed in the macrophage cell line J774A.1 cells at a high level and in the embryo as early as E9.     

Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus)

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5′ untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3′ UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys⁶¹-Cys¹⁵⁸, Cys¹³⁴-Cys¹⁵⁰) and a Ca²⁺/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca²⁺-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca²⁺-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR.     

Molecular cloning, expression, and functional characterization of a novel member of the CD38 family of ADP-ribosyl cyclases

We report the molecular cloning and functional characterization of a novel member of the CD38 family of cyclic ADP-ribose (cADPr)-generating cyclases. We cloned a cDNA insert that encoded a 298-amino-acid-long protein (M(w) approximately 39 kDa). The predicted protein displayed 69, 61, and 58% similarity, respectively, to mouse, rat, and human CD38. Rabbit CD38 was also 28% homologous to Aplysia ADP-ribosyl cyclase and leukocyte CD157 (another ADP-ribosyl cyclase); the three cyclases shared 10 cysteine and 2 adjacent proline residues. We then transfected CD38-negative NIH3T3 cells with cDNA encoding a CD38-EGFP fusion protein. Epifluorescence microscopy showed intense EGFP fluorescence confirming CD38 expression. We finally confirmed the ADP-ribosyl cyclase activity of the expressed CD38 by measuring its ability to catalyze the cyclization of the nicotinamide adenine dinucleotide (NAD(+)) surrogate, NGD(+), to its fluorescent nonhydrolyzable derivative, cGDPr.     

Molecular cloning and characterization of a novel Dehydrogenase/reductase (SDR family) member 1 genea from human fetal brain

Short-chain dehydrogenases/reductases (SDR) constitute a large protein family of NAD(P)(H)-dependent oxidoreductase. They are defined by distinct, common sequence motifs and show a wide range of substrate specialisms. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human SDR-type dehydrogenase/reductase gene named Dehydrogenase/reductase (SDR family) member 1 (DHRS1). The DHRS1 cDNA is 1411 base pair in length, encoding a 314-amino-acid polypeptide which has a SDR motif. Northern blot reveals two bands, of about 0.9 and 1.4 kb in size. These two forms are expressed in many tissues. The DHRS1 gene is localized on chromosome 14q21.3. It has 9 exons and spans 9.2 kb of the genomic DNA.     

Molecular cloning of a cDNA encoding a novel member of the mouse glutamate receptor channel family.

The primary structure of a novel putative subunit of the mouse glutamate receptor channel, designated as delta 1, has been deduced by cloning and sequencing the cDNA. The delta 1 subunit shows 21-25% amino acid sequence identity with previously characterized rodent glutamate receptor channel subunits and thus may represent a new subfamily of the glutamate receptor channel.     

Molecular cloning and characterization of the human S100A14 gene encoding a novel member of the S100 family

S100 proteins form a growing subfamily of proteins related by Ca2+-binding motifs to the Efhand Ca2+-binding protein superfamily. By analyzing a human lung cancer cell line subtraction cDNA library, we have identified and characterized a new member of the human S100 family that we named S100A14 (GenBank acc. no. NM_020672). It encodes a mRNA present in several normal human tissues of epithelial origin, with the highest level of expression in colon. The full-length cDNA is 1067 nt in length, with a coding region predicting a protein of 104 amino acids that is 68% homologous to the S100A13 protein. The deduced amino acid sequence of the human S100A14 and its mouse homolog (identified as GenBank entry) contains two EF-hand Ca2+-binding domains, a myristoylation motif, a glycosylation site, and several potential protein kinase phosphorylation sites. We have mapped this gene to human chromosome 1q21, within a region where at least 15 other S100 genes are tightly clustered. A 3.2-kb genomic fragment containing the entire S100A14 was cloned and sequenced. The gene is split into four exons and three introns spanning a total of 2165 bp of genomic sequence. We examined the intracellular distribution of the epitope-tagged S100A14 protein in two human lung carcinoma cell lines and one immortalized monkey cell line. Pronounced staining was observed in the cytoplasm, suggesting an association with the plasma membrane and in the perinuclear area. We also provide evidence for heterogenic expression of S100A14 in tumors, demonstrating its overexpression in ovary, breast, and uterus tumors and underexpression in kidney, rectum, and colon tumors, a pattern suggesting distinct regulation with potentially important functions in malignant transformation.     

Molecular cloning and expression of a C-type lectin gene from Venerupis philippinarum

C-type lectins have been demonstrated to play important roles in invertebrate innate immunity by mediating the recognition of pathogens and clearing the micro-invaders. In the present study, a C-type lectin gene (denoted as VpCTL) was identified from Venerupis philippinarum by expressed sequence tag and rapid amplification of cDNA ends approaches. The full-length cDNA of VpCTL consists of 904 nucleotides with an open-reading frame of 456 bp encoding a peptide of 151 amino acids. The deduced amino acid sequence of VpCTL shared high similarity with C-type lectins from other species. The C-type lectin domain and the characteristic EPN and WND motifs were found in VpCTL. The VpCTL mRNA was dominantly expressed in the haemocytes of the V. philippinarum. After Listonella anguillarum challenge, the temporal expression of VpCTL mRNA in haemocytes was increased by 97- and 84-fold at 48 and 96 h, respectively. With high expression level in haemocytes and hepatopancreas, and the up-regulated expression in haemocytes indicted that VpCTL was perhaps involved in the immune responses to L. anguillarum challenge.     

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.     

Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians

C-type lectins are Ca(2+)-dependent carbohydrate-recognition proteins that play crucial roles in innate immunity. The cDNA of C-type lectin (AiCTL1) in the bay scallop Argopecten irradians was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of AiCTL1 was 660 bp, consisting of a 5'-terminal untranslated region (UTR) of 30 bp and a 3' UTR of 132 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The AiCTL1 cDNA encoded a polypeptide of 166 amino acids with a putative signal peptide of 20 amino acid residues and a mature protein of 146 amino acids. The deduced amino acid sequence of AiCTL1 was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 121 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. AiCTL1 mRNA was dominantly expressed in the hemocytes of the bay scallop. The temporal expression of AiCTL1 mRNA in hemocytes was increased by 5.7- and 4.9-fold at 6h after injury and 8h after injection of bacteria, respectively. The structural features, high similarity and expression pattern of AiCTL1 indicate that the gene may be involved in injury healing and the immune response in A. irradians.     

Molecular cloning, heterologous expression and functional characterization of a novel translationally-controlled tumor protein (TCTP) family member from Loxosceles intermedia (brown spider) venom

Envenoming with brown spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of spider venom was identified from a previously obtained cDNA library of the L. intermedia venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.