Fibrinogen-like Proteins Research Articles

The role of Fibrinogen-like proteins in Cancer.

Fibrinogen-associated protein (FREP) family is a family of proteins with a fibrin domain at the carboxyl terminus. Recent investigations illustrated that two members of FREP family, fibrinogen-like protein-1 (FGL1) and fibrinogen-like protein-2 (FGL2), play crucial roles in cancer by regulating the proliferation, invasion, and migration of tumor cells, or regulating the functions of immune cells in tumor microenvironment. Meanwhile, they are potential targets for medical intervention of tumor development. In this review, we discussed the structure, and the roles of FGL1 and FGL2 in tumors, especially the roles in regulating immune cell functions.

Characterization and functional analysis of fibrinogen-related protein (FreP) in the black tiger shrimp, Penaeus monodon

Ficolin is classified as an immune related protein containing collagen-like and fibrinogen-related domain (FreD). In invertebrates, the functions of fibrinogen-related proteins (FrePs) are of importance to innate immunity. In this study, a FreP in the black tiger shrimp Penaeus monodon was identified and characterized. The PmFreP cDNA is 1,007 bp long with a 921 bp-open reading frame that encodes for 306 amino acids. The deduced PmFreP sequence consists of a signal peptide, an unknown region and the FreD. Phylogenetic analysis showed that PmFreP was clustered with fibrinogen-like proteins in crustaceans which was separated from vertebrate ficolin-like proteins. The deduced fibrinogen-like domain contains four conserved cysteine residues (Cys96, Cys127, Cys249, and Cys262) that are responsible for the formation of disulfide bridges. Gene expression analysis shows that Pmfrep is mainly expressed in the intestine and the expression is significantly upregulated after Vibrio harveyi and white spot syndrome virus (WSSV) challenge. Recombinant PmFreP (rPmFreP) were successfully expressed and purified, and forms a trimeric structure as judged by native-PAGE. Bacterial binding assay showed that the rPmFreD can bind and agglutinate Gram-negative and Gram-positive bacteria in the presence of calcium (Ca2+) ions. Moreover, the rPmFreP facilitates the clearance of V. harveyi in vivo. Overall, our results suggested that the PmFreP may serve as pattern recognition receptors implicated in shrimp innate immunity.

Two fibrinogen-like proteins, FGL1 and FGL2 are disulfide-linked subunits of oligomers that specifically bind nonviable spermatozoa

Nevertheless, a nonviable sperm population is present in the cauda epididymidis of many species. Degenerating spermatozoa release enzymes that could have detrimental effects on the viability of neighboring cells, and they are source of autoantigens that induce an autoimmune response if they escape the blood-epididymis barrier. Does the epididymis have specialized protective mechanism(s) to segregate the viable sperm population from defective spermatozoa? Previously, we identified a fibrinogen-like protein-2 (fgl2) that specifically binds to and polymerizes into a cocoon-like complex coating defective spermatozoa and sperm fragments. The objective of the present study is to identify the subunit composition of the fgl2-containing oligomers both in the soluble and cocoon-like complex. Our proteomic studies indicate that the 260/280kDa oligomers (termed eFGL) contain two distinct disulfide-linked subunits; 64kDa fgl2 and 33kDa fgl1. Utilizing a PCR-based cloning strategy, the 33kDa polypeptide has been identified as fibrinogen-like protein-1 (fgl1). Immunocytochemical studies revealed that fgl1 selectively binds to defective spermatozoa in the cauda epididymidis. Northern blot analysis and in situ hybridization demonstrated the high expression of fgl1 in the principal cells of the proximal cauda epididymidis. Co-immunoprecipitation analyses of cauda epididymal fluid, using anti-fgl2, demonstrate that both fgl1 and fgl2 are present in the soluble eFGL. Our study is the first to show an association of fgl1 and fgl2 both in the soluble and in the sperm-associated eFGL. We conclude that our results provide new insights into the mechanisms by which the potentially unique epididymal protein functions in the recognition and elimination of defective spermatozoa.

Human ficolin-2 recognition versatility extended: An update on the binding of ficolin-2 to sulfated/phosphated carbohydrates

Ficolin-2 has been reported to bind to DNA and heparin, but the mechanism involved has not been thoroughly investigated. X-ray studies of the ficolin-2 fibrinogen-like domain in complex with several new ligands now show that sulfate and phosphate groups are prone to bind to the S3 binding site of the protein. Composed of Arg132, Asp133, Thr136 and Lys221, the S3 site was previously shown to mainly bind N-acetyl groups. Furthermore, DNA and heparin compete for binding to ficolin-2. Mutagenesis studies reveal that Arg132, and to a lesser extent Asp133, are important for this binding property. The versatility of the S3 site in binding N-acetyl, sulfate and phosphate groups is discussed through comparisons with homologous fibrinogen-like recognition proteins.

Comparative genomic and phylogenetic analyses of the intelectin gene family: Implications for their origin and evolution

Intelectin is a newly characterized gene family involved in early embryogenesis, host-pathogen interactions and iron metabolism. In this study, we searched the genomes of metazoans by extensive BLAST survey and found no intelectin homologs in invertebrate metazoans but 12 in amphioxus Branchiostoma floridae and 21 in ascidians Ciona intestinalis. Some ascidians oocyte cortical granule lectins (CGLs) have unknown insertion sequences between fibrinogen-related domain (FReD) and Intelectin Domain, the boundaries of which are equivalent to exon structures. In addition to ascidians intelectins/CGLs located in the base, phylogenetic tree comprises four main clades representing mammal, frog, fish, and amphioxus, indicating that intelectin genes undergo extensive lineage-specific duplication or gene conversion. However, genomic neighborhood surrounding analysis shows that clear proto-orthologies are difficult to be established among these counterparts. In addition, sequence comparison and phylogenetic analysis of FReDs from intelectins and other fibrinogen-like proteins from choanoflagellate, anemone, frog and human indicate FReDs of intelectins are unique. Likewise, these choanoflagellate and anemone genes may be close to intelectin gene.

Comprehensive Egg Coat Proteome of the Ascidian Ciona intestinalis Reveals Gamete Recognition Molecules Involved in Self-sterility

Despite central roles of egg coat proteins in gamete recognition, their functions and composition are poorly understood. Here, we report that the proteome of the egg coat in the solitary ascidian Ciona intestinalis, called vitelline coat (VC) fraction, contains more than 800 proteins identified by mass spectrometry-based analyses. Over 100 proteins were enriched in the VC fraction compared with the VC-free egg proteome. The most abundant component in the VC was an apolipoprotein-like protein. The VC contained multiple homologs of mammalian zona pellucida (ZP) proteins, the number of which was unexpectedly large and most of which possessed epidermal growth factor-like repeats. Furthermore, the present study revealed that two fibrinogen-like proteins, v-Themis-A and -B, both of which are expressed in the VC, are the molecules responsible for the two self-sterility loci that were identified by our previous genetic study in this species.

Expression profiling and binding properties of fibrinogen-related proteins (FREPs), plasma proteins from the schistosome snail host Biomphalaria glabrata

A growing body of evidence suggests an important role for fibrinogen-like proteins in innate immunity in both vertebrates and invertebrates. It has been shown that fibrinogen-related proteins (FREPs), plasma proteins present in the freshwater snail Biomphalaria glabrata, the intermediate host for the human blood fluke Schistosoma mansoni, are diverse and involved in snail innate defense responses. To gain further insight into the functions of FREPs, recombinant FREP proteins (rFREPs) were produced in Escherichia coli and antibodies (Abs) were raised against the corresponding rFREPs. We first show that most FREP proteins exist in their native conformation in snail hemolymph as multimeric proteins. Western blot analyses reveal that expression of multiple FREPs including FREP4 in plasma from M line and BS-90 snails, which are susceptible and resistant to S. mansoni infection, respectively, is up-regulated significantly after infection with the trematode Echinostoma paraensei. Moreover, our assays demonstrate that FREPs are able to bind E. paraensei sporocysts and their secretory/excretory products (SEPs), and a variety of microbes (Gram-positive and Gram-negative bacteria and yeast). Furthermore, this binding capability shows evidence of specificity with respect to pathogen type; for example, 65-75-kDa FREPs (mainly FREP4) bind to E. paraensei sporocysts and their SEPs whereas 95-kDa and 125-kDa FREPs bind the microbes assayed. Our results suggest that FREPs can recognize a wide range of pathogens, from prokaryotes to eukaryotes, and different categories of FREPs seem to exhibit functional specialization with respect to the pathogen encountered.

Identification of novel genes related to tetrodotoxin intoxication in pufferfish

To investigate the genes related to the biosynthesis or accumulation of tetrodotoxin (TTX) in pufferfish, mRNA expression patterns in the liver from pufferfish, akamefugu Takifugu chrysops and kusafugu Takifugu niphobles, were compared by mRNA arbitrarily primed reverse transcription-polymerase chain reaction (RAP RT-PCR) with fish bearing different concentrations of TTX and its derivatives. RAP RT-PCR provided a 383 bp cDNA fragment and its transcripts were higher in toxic than non-toxic pufferfish liver. Its deduced amino acid sequence was similar to those of fibrinogen-like proteins reported for other vertebrates. Northern blot analysis and rapid amplification of cDNA ends (RACE) revealed that the cDNA fragment of 383 bp was composed of at least three fibrinogen-like protein (flp) genes, flp-1, flp-2 and flp-3. Relative mRNA levels of flp-1, flp-2 and flp-3 showed a linear correlation with toxicity of the liver for two pufferfish species.

Structure of the Angiopoietin-2 Receptor Binding Domain and Identification of Surfaces Involved in Tie2 Recognition

The angiopoietins comprise a small class of secreted glycoproteins that play crucial roles in the maturation and maintenance of the mammalian vascular and lymphatic systems. They exert their effects through a member of the tyrosine kinase receptor family, Tie2. Angiopoietin/Tie2 signaling is unique among tyrosine kinase receptor-ligand systems in that distinct angiopoietin ligands, although highly homologous, can function as agonists or antagonists in a context-dependent manner. In an effort to understand this molecular dichotomy, we have crystallized and determined the 2.4 A crystal structure of the Angiopoietin-2 (Ang2) receptor binding region. The structure reveals a fibrinogen fold with a unique C-terminal P domain. Conservation analysis and structure-based mutagenesis identify a groove on the Ang2 molecular surface that mediates receptor recognition.

Bovine babesiosis: The immunization of cattle with fractions of erythrocytes infected with [formula omitted] (syn [formula omitted

Soluble antigen which protected susceptible cattle against challenge with Babesia bovis was extracted from B. bovis -infected erythrocytes by sonic disintegration and separation of the soluble from the insoluble matter by ultracentrifugation. The material was then fractionated by the precipitation of fibrinogen-like proteins. The precipitate contained the babesial antigens that were located on the stroma of the infected erythrocytes. Antigen originally located on the parasite remained in solution. Both fractions conferred protection on splenectomized calves against challenge with B. bovis . However, the fraction containing the parasite antigens appeared to have more potential for development as a killed vaccine because it was not heavily contaminated with antigenic material from bovine erythrocytes.

The irradiation of Babesia bovis. 1. The difference in pathogenicity between irradiated and non-irradiated populations.

Babesia bovis parasites attenuated by 35 krads gamma irradiation and parasites not exposed to irradiation, were injected into intact 2-year-old Hereford steers. All five animals receiving non-irradiated blood died but the five animals which received irradiated blood were only mildly affected. Highly significant differences were observed in changes to plasma fibrinogen, serum fibrinogen-like proteins, packed cell volume, partial thromboplastin time, prothrombin time, blood kinins, and plasma kininogen levels in the control animals but non-significant changes in these parameters occurred in the group receiving iradiated blood. Significant changes in the antiplasmin alpha 2M, and the antithrombin levels occurred in control cattle but not in the group receiving irradiated blood. Parasite multiplications rates and maximum parasitaemias were similar in both groups. Irradiation reduced the dose of living parasites from 1 x 10(8) to 2.5 x 10(3), but this was not the reason for the mild reactions. It was concluded that irradiation had selected an avirulent parasite population.

Babesia bovis (argentina): analysis of fibrinogen-like proteins during infection.

Fibrinogen and fibrinogen-like proteins (FLP) were isolated from plasma and serum of cattle acutely infected with Babesia bovis. The sizes and chain structures of these proteins were examined and clotting assays performed. The results indicated that the blood was in a hypercoagulable state due mainly to enhanced production of hydrogen bonded fibrin and offset partly by slight inhibition of chain cross-linking. The latter appeared due to a Factor XIII inhibitor. Reduction of A alpha chains of plasma FLP was not evident, nor could lower molecular weight remnants be regularly detected strongly suggesting that fibrin(ogen) lysis rarely occurred. Similarly the size and chain structure of the majority of noncoagulable FLP of serum was consistent with their being the product of coagulation and not fibrinolysis. Only in heavily infected splenectomized cattle were products from lysed cross-linked fibrin detected and these constituted only about 3% of total serum FLP.

Babesia bovis (argentina): Components of the cryofibrinogen complex and their contribution to pathophysiology of infection in splenectomized calves

A cryofibrinogen complex, found in the plasma of cattle acutely infected with Babesia bovis (argentina), was characterized. The fibrinogen-like proteins of the complex were isolated and the structure of their polypeptide chains analysed. In general, the chain structure was similar to that of soluble non-crosslinked fibrin (fibrinogen) although chains indicating some degree of fibrin crosslinking were often detected. Only rarely did the chain structure suggest that fibrinolysis occurred. It was concluded that the complex was produced by activation of the coagulation system but that fibrinolysis did not occur to any marked degree. The complex was implicated in assistance to the sludging of erythrocytes in the internal organs which is a feature of the pathogenesis of the disease.

Babesia bovis (argentina): observations of coagulation parameters, fibrinogen catabolism and fibrinolysis in intact and splenectomized cattle.

The results of this paper indicate that cattle infected with B. bovis (argentina) have a markedly altered and activated coagulation system. A degree of thrombin activation occurs due partly to release of thromboplastin-like substances from lysed erythrocytes but due primarily to activation of kallikrein by babesial proteases. This produces a hyperfibrinogenaemia, particularly in intact cattle, with soluble fibrin complexes constituting up to one-third of the total fibrinogen concentration. High molecular weight non-coagulable fibrinogen-like proteins are detected terminally but more so in splenectomized cattle. Plasminogen concentration decreases in splenectomized but not intact cattle while low molecular weight fibrinogen degradation products are not easily detected. It is suggested that a hypercoagulable intermediate state with little or no fibrin deposition occurs rather than terminal disseminated intravascular coagulation.

Fibrinogen-like proteins and fibrinolytic proteins in a saline eluate from sucrose washed bovine erythrocytes.

Plasminogen and a spectrum of fibrinogen-like proteins are found in a saline eluate of sucrose washed bovine erythrocytes. It seems probable that these proteins are membrane bound in vivo, and, as such, their physiological significance is discussed.

A cold precipitable fibrinogen complex in the plasma of cattle dying from infection with Babesia argentina.

Plasma from cattle dying from infection with Babesia argentina formed a cold precipitable gel when stored at 4 degrees C. Immunodiffusion and electrophoresis showed the soluble phase of the gel to consist mainly of a fibrinogen-like protein with small amounts of other plasma proteins. Gel filtration and specific clotting assays demonstrated a wide molecular weight spectrum of fibrinogen-like proteins, most of which appeared to be soluble fibrin in either monomer or complex forms. The possibility that proteolytic enzymes might be responsible for the formation of the gel is discussed.