A novel chicken membrane-associated complement regulatory protein: molecular cloning and functional characterization

A cDNA encoding a membrane-associated complement (C) regulatory protein was identified here for the first time in an oviparous vertebrate, chicken. This protein, named Cremp, possessed five short consensus repeats (SCRs) and one SCR-like domain followed by a transmembrane domain and a cytoplasmic tail. SCR1/SCR2 of Cremp were 43.6% identical with SCR2/SCR3 of human decay-accelerating factor (CD55), and SCR3/SCR4 were 45.3% identical with those of human membrane cofactor protein (CD46). Cremp is likely to be an ancestral hybrid protein of human decay-accelerating factor and membrane cofactor protein rather than a homolog of rodent C receptor 1-related protein y, which structurally resembles human CR1 (CD35). Chinese hamster ovary cells transfected with Cremp were efficiently protected from chicken C but not from human or rabbit C in both classical and alternative pathways. Thus, chicken Cremp is a membrane C regulator for cell protection against homologous C. Cremp mRNA was seen as a doublet comprised of a faint band of 2.2 kb and a thick band of 3.0 kb on RNA blotting analysis. An Ab against chicken Cremp recognized a single band of 46.8 kDa on immunoblotting. mRNA and protein of Cremp were ubiquitously expressed in all chicken organs tested. Minute amounts of dimer were present in some tissues. Surface expression of Cremp was confirmed by flow cytometry and immunofluorescence analysis. These results suggested that even in nonmammals a C regulatory membrane protein with ubiquitous tissue distribution should be a prerequisite for protection of host cells from homologous C attack.     

Molecular cloning and characterization of prokineticin receptors

Recent studies have identified two novel biofunctional proteins, termed prokineticin 1/EG-VEGF and prokineticin 2, which were mammalian homologues of mamba MIT1 and frog Bv8. Prokineticins have been demonstrated to exert their physiological functions through G-protein coupled receptors (GPCRs). In this study, we report the molecular identification of two endogenous prokineticin receptors, designated PK-R1 and PK-R2, through a search of the human genomic DNA database. PK-R1, locating in chromosome 2, and PK-R2, locating in chromosome 20p13, shared 87% homology, which was an extremely high value among known GPCRs. In functional assays, mammalian cells expressing PK-Rs responded to prokineticins in a concentration-dependent manner. Tissue distribution analysis revealed that expression of PK-R1 was observed in the testis, medulla oblongata, skeletal muscle and skin, while that of PK-R2 showed preferential expression in the central nervous system. The tissue distribution of PK-Rs reported in this paper suggests that the prokineticins play multifunctional roles in vivo.     

Identification and molecular cloning of functional chicken IL-12

By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains. These molecules are the first discovered nonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively. The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, while expression was not detected in stimulated primary chicken embryo fibroblast cells. Following transient expression of both molecules in COS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammalian counterparts. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomeric IL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-gamma synthesis and proliferative activity of freshly exposed chicken splenocytes. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates, despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolutionary dichotomy of birds and mammals >300 million years ago. In this article, we describe the first nonmammalian IL-12 molecule and show that this chicken IL-12 molecule is bioactive.     

Molecular cloning and functional characterization of mouse coactosin-like protein.

Coactosin was first isolated from Dictyostelium discoideum and, as reported, human coactosin-like protein (CLP) was identified in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. A mouse CLP (mCLP) cDNA clone was identified among EMBL/GenBank EST sequences. The derived amino acid sequence (142 residues) was 95.1% identical with human CLP. Here, we also show that mCLP interacts with actin and 5LO in the two-hybrid system. High-speed cosedimentation assays and GST-binding assays confirmed these protein interactions. In chemical cross-linking experiments, one molecule of mCLP was covalently linked to either one subunit of actin or one molecule of 5LO. The mCLP-F-actin and mCLP-5LO associations were pH-insensitive and Ca(2+)-independent. However, association with actin was best observed at low salt concentrations, while association with 5LO was favored by salt, indicating different binding characteristics.     

Molecular cloning and functional characterization of mouse chitotriosidase

Mammalian chitinase and chitinase-like proteins are members of a recently discovered gene family. Thus far, neither chitin nor chitin synthase has been found in mammals. The existence of chitinase genes in mammals is intriguing and the physiologic functions of chitinases are not clear. Human chitotriosidase, also called chitinase 1 (chit1), has been cloned. It has been found that high levels of serum chitotriosidase are associated with several diseases, but the physiologic functions of this enzyme are still unclear. To facilitate the studies in animal models we cloned and characterized a cDNA that encodes the mouse chitotriosidase. The open reading frame of this cDNA predicts a protein of 464 amino acids with a typical chitinase structure, including a signal peptide, a highly conserved catalytic domain and a chitin-binding domain. The predicted amino acid sequence is highly homologous to that of human chitotriosidase and to that of mouse acidic mammalian chitinase. Sequence analysis indicates that the mouse chitotriosidase gene has 12 exons, spanning a 40-kb region in mouse chromosome 1. The constitutive expression of mouse chitotriosidase is restricted to brain, skin, bone marrow, kidney, tongue, stomach and testis. Recombinant expression of the cloned cDNA demonstrated that the encoded protein is secreted and has chitinolytic activity that is sensitive to the specific chitinase inhibitor allosamidin and has the ability to bind to chitin particles. Substitution mutations at the conserved catalytic site completely abolished the enzymatic activity of the recombinant protein. These studies illustrate that mouse chitotriosidase is a typical chitinase that belongs to the mammalian chitinase gene family.     

Molecular cloning and functional characterization of zebrafish ATM

Ataxia-telangiectasia mutated (ATM) is the gene product mutated in ataxia-telangiectasia (A-T), which is an autosomal recessive disorder with symptoms including neurodegeneration, cancer predisposition and premature aging. ATM is thought to play a pivotal role in signal transduction in response to genotoxic DNA damage. To study the physiological and developmental functions of ATM using the zebrafish model system, we cloned the zebrafish homolog cDNA of human ATM (hATM), zebrafish ATM (zATM), analyzed the expression pattern of zATM during early development, and further developed the system to study loss of zATM function in zebrafish embryos. Employing information available from the zebrafish genomic database, we utilized a PCR-based approach to isolate zATM cDNA clones. Sequence analysis of zATM showed a high level homology in the functional domains of hATM. The putative FAT, phosphoinositide 3-kinase-like, and FATC domains of zATM, which regulate ATM kinase activity and functions, were the most highly conserved regions, exhibiting 64-94% amino acid identity to the corresponding domains in hATM, while exhibiting approximately 50% amino acid identity outside these domains. The zATM gene is expected to consist of 62 coding exons, and we have identified at least 55 exons encompassing more than 100kb of nucleotide sequence, which encodes about 9 kb of cDNA. By in situ hybridization, zATM mRNA was detected ubiquitously with a dramatic increase at the 18-somite stage, then more specifically in the eye, brain, trunk, and tail at later stages. To inhibit zATM expression and function, we designed and synthesized splice-blocking antisense-morpholino oligonucleotides targeting the phosphoinositide 3-kinase-like domain. We demonstrated that this knockdown of zATM caused abnormal development upon ionizing radiation-induced DNA damage. Our data suggest that the ATM gene is structurally and functionally conserved in vertebrates from zebrafish to human.     

Molecular cloning and functional characterization of a snake venom metalloprotease.

A cDNA clone, MT-d, encoding metalloprotease precursor was isolated from snake (Agkistrodon halys brevicaudus) venom gland cDNA library. MT-d-I protein containing both metalloprotease and disintegrin domains, and MT-d-II protein containing the metalloprotease domain only were expressed in Escherichia coli and refolded successfully into their functional forms. Each of the refolded enzyme species exhibited distinct substrate specificity. Proteolytic activity of the MT-d-1 was able to hydrolyse type I gelatin, type-III and V collagens in contrast with the catalytic function of MT-d-II. MT-d-I protein having metalloprotease activity was also able to inhibit platelet aggregation. Functionally active MT-d-I protein underwent autoproteolytic processing in vitro to produce metalloprotease and disintegrin; this processing was accompanied by significant changes in the substrate specificity of the enzyme activity. Experimental evidence strongly suggests that the disintegrin domain in the metalloprotease precursor modulates the catalytic function of the enzyme in hydrolysing extracellular matrix proteins.     

Molecular cloning and characterization of the chicken pro-opiomelanocortin (POMC) gene.

The gene for pro-opiomelanocortin (POMC), a common precursor of melanocortins, lipotropins and beta-endorphin, was isolated in the chicken first among avian species. The chicken POMC gene was found to be a single copy gene and appeared to show the same structural organization as that of other species of different classes. The predicted POMC displayed the highest identity to Xenopus POMC(A) (60. 1%), and consisted of 251 amino acid residues with nine proteolytic cleavage sites, suggesting that it could be processed to give rise to all members of the melanocortin family, including adrenocorticotropic hormone and alpha-, beta- and gamma-melanocyte-stimulating hormones, as well as the other POMC-derived peptides. RT-PCR analysis detected the POMC mRNA in the brain, adrenal gland, gonads, kidney, uropygial gland and adipose tissues, each of which has been demonstrated to express melanocortin receptors. These results suggest that melanocortins act in a paracrine and/or autocrine manner to control a variety of functions both in the brain and in the peripheral tissues in the chicken.     

Molecular cloning, expression and functional characterization of the chicken cation dependent mannose 6-phosphate receptor protein

Mammalian mannose 6-phosphate (M6P) receptors function in transport of lysosomal enzymes. To understand the structural and functional significance of the chicken cation dependent mannose 6-phosphate receptor (MPR) (Mr 46kDa), a full-length cDNA for the chicken protein was cloned and expressed in mpr((-/-)) MEF cells devoid of both the receptors. The stably transfected cells express the receptor that could be affinity purified by phosphomannan chromatography. The authenticity of the receptor was confirmed by its immuno-reactivity with mammalian MPR 46 antibodies and its ability to sort cathepsin D in transfected cells (92.3%) as compared to mock transfected cells (50.2%), establishing a functional role for the chicken receptor.     

Molecular cloning and functional characterization of chicken brain tau: isoforms with up to five tandem repeats

Tau is a major microtubule-associated protein in mammalian brain, where it exists as multiple isoforms that are produced from a single gene by alternative mRNA splicing. Here we present the first report on the structure and function of tau protein from a nonmammalian vertebrate. In the adult chicken brain, five main tau isoforms are expressed. One isoform has three tandem repeats, two isoforms have four repeats each, and two isoforms have five repeats each. Similar to mammalian tau, some chicken tau isoforms contain an amino-terminal insert of 53 amino acids. Unlike mammalian tau, a 34 amino acid insert in the proline-rich region upstream of the repeats is alternatively spliced in chicken tau. It is preceded by a constitutively expressed sequence of 17 amino acids that is absent in tau from human and rodent brains. The expression of chicken tau isoforms and their phosphorylation are developmentally regulated, similar to what has been described in mammalian brain. Functionally, chicken tau isoforms with five repeats have the greatest ability to promote microtubule assembly, followed by isoforms with four and three repeats, respectively. The 34 amino acid insert positively influences both the rate and the extent of microtubule assembly, whereas the 53 amino acid insert only influences the extent of assembly.     

Molecular cloning and functional characterization of chick lens fiber connexin 45.6.

The avian lens is an ideal system to study gap junctional intercellular communication in development and homeostasis. The lens is experimentally more accessible in the developing chick embryo than in other organisms, and chick lens cells differentiate well in primary cultures. However, only two members of the connexin gene family have been identified in the avian lens, whereas three are known in the mammalian system. We report here the molecular cloning and characterization of the third lens connexin, chick connexin45.6 (ChCx45.6), a protein with a predicted molecular mass of 45.6 kDa. ChCx45.6 was encoded by a single copy gene and was expressed specifically in the lens. There were two mRNA species of 6.4 kilobase (kb) and 9.4 kb in length. ChCx45.6 was a functional connexin protein, because expression in Xenopus oocyte pairs resulted in the development of high levels of conductance with a characteristic voltage sensitivity. Antisera were raised against ChCx45.6 and chick connexin56 (ChCx56), another avian lens-specific connexin, permitting the examination of the distribution of both proteins. Immunofluorescence localization showed that both ChCx45.6 and ChCx56 were abundant in lens fibers. Treatment of lens membranes with alkaline phosphatase resulted in electrophoretic mobility shifts, demonstrating that both ChCx45.6 and ChCx56 were phosphoproteins in vivo.     

Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase

Chicken pancreatic lipase (CPL) was purified from delipidated pancreas. Pure CPL was obtained after ammonium sulphate fractionation, then DEAE-cellulose, Sephacryl S-200 gel filtration, and FPLC Mono-Q Sepharose columns. The pure lipase is a glycosylated monomer having a molecular mass of about 50kDa. The 23 N-terminal amino acid residues of CPL were sequenced. The sequence is similar to those of avian and mammalian pancreatic lipases. CPL presents the interfacial activation phenomenon tested with tripropionin or vinyl ester. When CPL was inhibited by synthetic detergent (TX-100) or amphipathic protein (BSA), simultaneous addition of bile salts and colipase was required to restore the full CPL activity. In the absence of colipase and bile salts, CPL was unable to hydrolyse tributyrin emulsion. This enzyme can tolerate, more efficiently than HPL, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate in the absence of bile salts and colipase. The CPL activity, under these conditions, was linear whereas that of HPL decreased rapidly. Anti-TPL polyclonal antibodies cross-reacted specifically with CPL. The gene encoding the mature CPL was cloned and sequenced. The deduced amino acid sequence of the mature lipase shows a high degree of homology with the mammalian pancreatic lipases. A 3D structure model of CPL was built using the HPL structure as template. We have concluded that a slight increase in the exposed hydrophobic residues on the surface of CPL, as compared to HPL, could be responsible for a higher tolerance to the presence of long-chain free fatty acids at the lipid/water interface.     

Molecular cloning, characterization and localization of chicken type II procollagen gene

Chicken type II procollagen (ccol2a1) has become as an important oral tolerance protein for effective treatment of rheumatoid arthritis. However, its molecular identity remains unclear. Here, we reported the full-length cDNA and nearly complete genomic DNA encoding ccol2a1. We have determined the structural organization, evolutional characters, developmental expression and chromosomal mapping of the gene. The full-length cDNA sequence spans 4837 bp containing all the coding region of the ccol2a1 including 3' and 5' untranslation region. The deduced peptide of ccol2a1, composed of 1420 amino acids, can be divided into signal peptide, N-propeptide, N-telopeptide, triple helix, C-telopeptide and C-propeptide. The ccol2a1 genomic DNA sequence was determined to be 12,523 bp long containing 54 exons interrupted by 53 introns. Comparison of the ccol2a1 with its counterparts in human, mouse, canine, horse, rat, frog and newt revealed highly conserved sequence in the triple helix domain. Chromosomal mapping of ccol2a1 locates it on 4P2. While the ccol2a1 mRNA was expressed in multiple tissues, the protein was only detected in chondrogenic cartilage, vitreous body and cornea. The ccol2a1 was found to contain two isoforms detected by RT-PCR. The distribution of the ccol2a1 lacking exon 2wasfrequently detected in chondrogenic tissues, whereas the exon 2-containing isoform was more abundant in non-chondrogenic tissues. These results provide useful information for preparing recombinant chicken type II collagen and for a better understanding of normal cartilage development.     

Molecular cloning of the chicken avidin cDNA.

A cDNA for chicken avidin was identified in a chicken oviduct cDNA library by screening with antibodies and synthetic oligodeoxyribonucleotides. Four recombinant clones were characterized and each contained the sequence of the oligonucleotide probes used in screening. They were capable also of expressing an antigen recognizable by a polyclonal or a mixture of monoclonal antibodies raised against avidin. The longest clone, lambda cAV4, contained the entire coding sequence of avidin along with a signal peptide of 24 amino acids. An avidin mRNA, approximately 700 nucleotides in length, was induced by a single injection of progesterone over a period of twenty four hours. The avidin mRNA was distributed in a tissue-specific manner, since detectable concentration of the mRNA appeared only in the oviduct after stimulation with progesterone alone or with a combination of progesterone and estrogen. No avidin mRNA was detected in the liver or kidney under these conditions. Preliminary results on the genomic complexity of avidin suggest a single copy gene. Isolation of the natural gene for avidin and studies on its regulation now can be initiated using the cDNA probe.     

Molecular and functional characterization of adrenomedullin receptors in pufferfish

The receptors for the calcitonin gene-related peptide (CGRP)/adrenomedullin (AM) family peptides were characterized in the mefugu Takifugu obscurus, a euryhaline fugu species very close to Takifugu rubripes, which has as many as five adrenomedullin genes (AM1-5). CGRP and AM share a G protein-coupled core receptor called calcitonin receptor-like receptor (CLR), and the specificity of the CLR is determined by the interaction with receptor activity-modifying proteins (RAMPs). Through database mining, three CLRs (CLR1-3) and five RAMPs (RAMP1-5) were identified, and all of them were cloned by RT-PCR and characterized by functional expression in COS7 cells in every possible combination of CLR-RAMP. The following combinations generated cAMP in response to physiological concentrations of CGRP, AM1 (an ortholog of mammalian AM), AM2, and AM5: CLR1-RAMP1/4 (CGRP), CLR1-RAMP2/3/5 (AM1), CLR2-RAMP2 (AM1), CLR1-RAMP3 (AM2), and CLR1-RAMP3 (AM5). Their expressions were found by Northern blot analysis to be tissue specific and salinity dependent. For example, CLR1-RAMP5 and CLR1-RAMP2 are expressed specifically in the gill and kidney, respectively, suggesting their involvement in osmoregulation. Furthermore, relatively high levels of CLRs and RAMPs were found in the spleen and ovary, suggesting roles in the immune and female reproductive systems. Immunohistochemistry revealed that AM receptors of the following types are expressed in the locations, indicated in brackets, of the mefugu gill and kidney: CLR1-RAMP5 (interlamellar vessels), CLR2-RAMP2 (pillar cells), and CLR1-RAMP2 (apical side of renal proximal tubule cells).