CRP-like Proteins Research Articles

The Global Transcription Regulator XooClp Governs Type IV Pili System-Mediated Bacterial Virulence by Directly Binding to TFP-Chp Promoters to Coordinate Virulence Associated Functions.

Type IV pili (TFP) play a crucial role in the sensing of the external environment for several bacteria. This surface sensing is essential for the lifestyle transitions of several bacteria and involvement in pathogenesis. However, the precise mechanisms underlying TFP's integration of environmental cues, particularly in regulating the TFP-Chp system and its effects on Xanthomonas physiology, social behavior, and virulence, remain poorly understood. In this study, we focused on investigating Clp, a global transcriptional regulator similar to CRP-like proteins, in Xanthomonas oryzae pv. oryzae, a plant pathogen. Our findings reveal that Clp integrates environmental cues detected through diffusible signaling factor (DSF) quorum sensing into the TFP-Chp regulatory system. It accomplishes this by directly binding to TFP-Chp promoters in conjunction with intracellular levels of cyclic-di-GMP, a ubiquitous bacterial second messenger, thereby controlling TFP expression. Moreover, Clp-mediated regulation is involved in regulating several cellular processes, including the production of virulence-associated functions. Collectively, these processes contribute to host colonization and disease initiation. Our study elucidates the intricate regulatory network encompassing Clp, environmental cues, and the TFP-Chp system, providing insights into the molecular mechanisms that drive bacterial virulence in Xanthomonas spp. These findings offer valuable knowledge regarding Xanthomonas pathogenicity and present new avenues for innovative strategies aimed at combating plant diseases caused by these bacteria. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Bactericidal bissulfone B7 targets bacterial pyruvate kinase to impair bacterial biology and pathogenicity in plants.

The prevention and control of rice bacterial leaf blight (BLB) disease has not yet been achieved due to the lack of effective agrochemicals and available targets. Herein, we develop a series of novel bissulfones and a novel target with a unique mechanism to address this challenge. The developed bissulfones can control Xanthomonas oryzae pv. oryzae (Xoo), and 2-(bis(methylsulfonyl)methylene)-N-(4-chlorophenyl) hydrazine-1-carboxamide (B7) is more effective than the commercial drugs thiodiazole copper (TC) and bismerthiazol (BT). Pyruvate kinase (PYK) in Xoo has been identified for the first time as the target protein of our bissulfone B7. PYK modulates bacterial virulence via a CRP-like protein (Clp)/two-component system regulatory protein (regR) axis. The elucidation of this pathway facilitates the use of B7 to reduce PYK expression at the transcriptional level, block PYK activity at the protein level, and impair the interaction within the PYK-Clp-regR complex via competitive inhibition, thereby attenuating bacterial biology and pathogenicity. This study offers insights into the molecular and mechanistic aspects underlying anti-Xoo strategies that target PYK. We believe that these valuable discoveries will be used for bacterial disease control in the future.

Structural Basis of Dual Specificity of Sinorhizobium meliloti Clr, a cAMP and cGMP Receptor Protein.

In bacteria, the most prevalent receptor proteins of 3',5'-cyclic AMP (cAMP) and 3',5'-cyclic GMP (cGMP) are found among transcription factors of the Crp-Fnr superfamily. The prototypic Escherichia coli catabolite activator protein (CAP) represents the main Crp cluster of this superfamily and is known to bind cAMP and cGMP but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by Sinorhizobium meliloti Clr, mapping to cluster G of Crp-like proteins. We present crystal structures of Clr-cAMP and Clr-cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr-cNMP-CBS-DNA complexes (where cNMP is cyclic nucleotide monophosphate) to almost identical active conformations, unlike the situation known for the E. coli CAP-cNMP complex. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in the presence of CBS core motif DNA (equilibrium dissociation constant for cNMP (KDcNMP], ~7 to 11 μM). However, different affinities were determined in the absence of this DNA (KDcGMP, ~24 μM; KDcAMP, ~6 μM). Sequencing of Clr-coimmunoprecipitated DNA as well as electrophoretic mobility shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases that are consistent with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr-cNMP-CBS-DNA crystal structures. IMPORTANCE Cyclic 3',5'-AMP (cAMP) and cyclic 3',5'-GMP (cGMP) are both long known as important nucleotide secondary messengers in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins. Escherichia coli CAP, the prototypic transcription regulator of the main Crp cluster, binds both cyclic mononucleotides, but only the CAP-cAMP complex promotes transcription activation. In contrast, Crp cluster G proteins studied so far are activated by cGMP or by both cAMP and cGMP. Here, we report a structural analysis of the cAMP- and cGMP-activatable cluster G member Clr from Sinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.

Maltose-Enhanced Exopolysaccharide Synthesis of Lactiplantibacillus plantarum through CRP-like Protein

Carbon sources alter the synthesis of exopolysaccharides (EPS) in Lactiplantibacillus plantarum. Maltose increased the EPS production of L. plantarum 163 6.5-fold. Subsequently, EPS production, transcriptome, and proteome were analyzed using glucose or maltose to further clarify the regulatory mechanism. A cAMP receptor protein (UniProtKB: F9UNI5) has been identified to control EPS synthesis in the presence of cAMP by binding to the EPS synthesis promoter Pcps4A-J. Overexpression of the cAMP synthesis gene cyaA increased cAMP content and EPS production 4.5- and 2.2-fold, respectively. Furthermore, yogurt produced with L. plantarum 163-cyaA had a similar viscosity to that of commercial Greek yogurt; it had 20 and 83.7% greater viscosity than that produced with L. plantarum 163 with maltose and glucose, respectively. These findings indicated that L. plantarum 163-cyaA has potential applications in the production of functional fermented dairy products.

Synthesis and Antibacterial Activities of 2-Oxo-N-phenylacetamide Derivatives Containing a Dissulfone Moiety Target on Clp.

Rice bacterial blight and rice bacterial streak are two serious rice diseases and have caused great harm to the production of rice all over the world. To develop an efficient antibacterial agent with a novel target, a series of novel 2-oxo-N-phenylacetamide derivatives containing a dissulfone moiety were synthesized, and their antibacterial activities were evaluated. Among them, compound D14 exhibited the best antibacterial activities, especially against Xoo and Xoc with EC50 values of 0.63 and 0.79 mg/L, respectively, which were much better than the commercial control of bismerthiazol (BT) (76.59 and 83.35 mg/L, respectively) and thiodiazole copper (TC) (91.72 and 114.00 mg/L, respectively). Meanwhile, compound D14 can interact with a CRP-like protein (Clp) of Pxo99A and show strong binding activity with Xoo-Clp with a Kd value of 0.52 μM, which was far superior to the corresponding Kd values of BT (183.94 μM) and TC (222.58 μM). Treatment of D14 and deletion of the clp gene could significantly reduce the expression of the clp gene and attenuate the virulence of pathogenic bacteria. These results indicated that compound D14 could be used as a potential novel agricultural bactericide and Clp can be used as a target protein for the control of plant bacterial diseases. This work provided reliable support for developing novel antibacterial agents based on Clp as a target protein.

Aerobic Respiration and Its Regulation in the Metal Reducer Shewanella oneidensis.

Shewanella oneidensis MR-1 is a facultative anaerobe known for its ability to reduce metal oxides. Anaerobic respiration, especially metal reduction, has been the subject of extensive research. In contrast, S. oneidensis aerobic respiration has received less attention. S. oneidensis expresses cbb3- and aa3-type cytochrome c oxidases and a bd-type quinol oxidase. The aa3-type oxidase, which in other bacteria is the major oxygen reductase under oxygen replete conditions, does not appear to contribute to aerobic respiration and growth in S. oneidensis. Our results indicated that although the aa3-type oxidase does not play a role in aerobic growth on lactate, the preferred carbon source for S. oneidensis, it is involved in growth on pyruvate or acetate. These results highlight the importance of testing multiple carbon and energy sources when attempting to identify enzyme activities and mutant phenotypes. Several regulatory proteins contribute to the regulation of aerobic growth in S. oneidensis including CRP and ArcA. The 3',5'-cAMP phosphodiesterase (CpdA) appears to play a more significant role in aerobic growth than either CRP or ArcA, yet the deficiency does not appear to be the result of reduced oxidase genes expression. Interestingly, the ∆cpdA mutant was more deficient in aerobic respiration with several carbon sources tested compared to ∆crp, which was moderately deficient only in the presence of lactate. To identify the reason for ∆cpdA aerobic growth deficiency, we isolated a suppressor mutant with transposon insertion in SO_3550. Inactivation of this gene, which encodes an anti-sigma factor, restored aerobic growth in the cpdA mutant to wild-type levels. Inactivation of SO_3550 in wild-type cells, however, did not affect aerobic growth. The S. oneidensis genome encodes two additional CRP-like proteins that we designated CrpB and CrpC. Mutants that lack crpB and crpC were deficient in aerobic growth, but this deficiency was not due to the loss of oxidase gene expression.

Modulation of the Tissue Expression Pattern of Zebrafish CRP-Like Molecules Suggests a Relevant Antiviral Role in Fish Skin.

Simple SummaryThe clinical use of the human short pentraxin C-reactive protein as a health biomarker is expanded worldwide. The acute increase of the serum levels of short pentraxins in response to bacterial infections is evolutionarily conserved, as are the main functions of pentraxins. Interestingly, fish orthologs have been found to increase similarly after bacterial and viral stimuli, thus becoming promising candidates for health biomarkers of both types of infection in this group of vertebrates. To preliminarily assess their adequacy for this application, zebrafish and a fish rhabdovirus were chosen as infection model systems for the analysis of the levels of gene expression of all short pentraxins in healthy and infected animals in a wide range of tissues. Because some significant increases were found in skin (a very suitable sampling source for testing purposes), further transcript analyses were carried out in this tissue. Due to the functional similarities between pentraxins and antibodies, it was also checked whether short pentraxins can compensate for the deficiencies in adaptive immunity by using mutant zebrafish lacking this system. In conclusion, the obtained results suggest that short pentraxins are highly reactant against viruses in skin and their overexpression seems to reflect a mechanism to compensate for the loss of adaptive immunity.Recent studies suggest that short pentraxins in fish might serve as biomarkers for not only bacterial infections, as in higher vertebrates including humans, but also for viral ones. These fish orthologs of mammalian short pentraxins are currently attracting interest because of their newly discovered antiviral activity. In the present work, the modulation of the gene expression of all zebrafish short pentraxins (CRP-like proteins, CRP1-7) was extensively analyzed by quantitative polymerase chain reaction. Initially, the tissue distribution of crp1-7 transcripts and how the transcripts varied in response to a bath infection with the spring viremia of carp virus, were determined. The expression of crp1-7 was widely distributed and generally increased after infection (mostly at 5 days post infection), except for crp1 (downregulated). Interestingly, several crp transcription levels significantly increased in skin. Further assays in mutant zebrafish of recombinant activation gene 1 (rag1) showed that all crps (except for crp2, downregulated) were already constitutively highly expressed in skin from rag1 knockouts and only increased moderately after viral infection. Similar results were obtained for most mx isoforms (a reporter gene of the interferon response), suggesting a general overcompensation of the innate immunity in the absence of the adaptive one.

Phosphoglycerate Kinase Is Involved in Carbohydrate Utilization, Extracellular Polysaccharide Biosynthesis, and Cell Motility of Xanthomonas axonopodis pv. glycines Independent of Clp.

Phosphoglycerate kinase (Pgk), catalyzing the reversible conversions between glycerate-1.3-2P and glycerate-3P, plays an important role in carbohydrate metabolism. Here, we show that a Pgk-deficient mutant (NΔpgk) of Xanthomonas axonopodis pv. glycines (Xag) could grow in medium with glucose, galactose, fructose, mannose, or sucrose, as the sole carbon source, suggesting that Xag may employ Entner-Doudoroff (ED) and pentose phosphate pathway (PPP), but not glycolysis, to catabolize glucose. NΔpgk could not utilize pyruvate, suggesting that Pgk might be essential for gluconeogenesis. Mutation in pgk led to a reduction of extracellular polysaccharide (EPS) biosynthesis, cell motility, and intracellular ATP. As a result, the virulence of NΔpgk was significantly compromised in soybean. NΔpgk could be fully complemented by the wild-type pgk, but not by clp (encoding Crp-like protein). qRT-PCR analyses demonstrated that pgk is regulated by the HrpG/HrpX cascade, but not by Clp. These results suggest that Pgk is involved in carbohydrate utilization, EPS biosynthesis, and cell motility of Xag independent of Clp.

C-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

SummaryStreptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

Downregulated CHI3L1 alleviates skeletal muscle stem cell injury in a mouse model of sepsis

Sepsis is an acute systemic inflammatory response of the body to microbial infection and a life-threatening condition associated with multiple organ failure. Recent data suggest that sepsis survivors present with long-term myopathy due to the dysfunction of skeletal muscle stem cells and satellite cells. Accumulating studies have implicated chitinase-3-like-1 protein (CHI3L1) in a variety of infectious diseases, specifically sepsis. Therefore, the aim of the present study is to elucidate the potential mechanism by which CHI3L1 is involved in the injury of skeletal muscle stem cells in mouse models of sepsis. An in vitro cell model was developed by lipopolysaccharide (LPS) and in vivo mouse model of sepsis was induced by CRP-like protein (CLP). To elucidate the biological significance behind the silencing of CHI3L1, modeled skeletal muscle stem cells and mice were treated with siRNA against CHI3L1 or overexpressed CHI3L1. Highly expressed CHI3L1 was found in skeletal muscle tissues of mice with sepsis. Besides, siRNA-mediated silencing of CHI3L1 was revealed to increase Bcl-2 expression along with cell proliferation, while diminishing Bax expression, cell apopstosis as well as serum levels of TNF-α, IL-1β, INF-γ, IL-10, and IL-6. Taken conjointly, this present study provided evidence suggesting that downregulation of CHI3L1 has the potential to prevent the injury of skeletal muscle stem cells in mice with sepsis. Collectively, CHI3L1 may serve as a valuable therapeutic strategy in alleviating sepsis.

Evaluation of C-reactive-like protein in Mytilus galloprovincialis

Abstract C-reactive protein (CRP) is an acute phase protein of the innate immune system with an important role as biomarker in many biological processes in mammals. However, there is a lack of information regarding CRP in molluscs and there is no data about the presence of CRP-like protein in mussels. In the present work, CPR-like protein was identified and purified from the digestive gland of the Mediterranean mussel Mytilus galloprovincialis . In addition, it was demonstrated that CRP-like protein can be measured accurately and precisely by a high-throughput automated method. Finally, higher levels of CRP-like protein in mussels exposed to hypoxia and nanoplastics were detected when compared to controls. The results obtained indicate that CRP-like protein could be a suitable novel biomarker of inflammatory response in mussels for biomonitoring programs being economic, easy and rapid to determine.

Crp-Like Protein Plays Both Positive and Negative Roles in Regulating the Pathogenicity of Bacterial Pustule Pathogen Xanthomonas axonopodis pv. glycines.

The global regulator Crp-like protein (Clp) is positively involved in the production of virulence factors in some of the Xanthomonas spp. However, the functional importance of Clp in X. axonopodis pv. glycines has not been investigated previously. Here, we showed that deletion of clp led to significant reduction in the virulence of X. axonopodis pv. glycines in soybean, which was highly correlated with the drastic reductions in carbohydrates utilization, extracellular polysaccharide (EPS) production, biofilm formation, cell motility, and synthesis of cell wall degrading enzymes (CWDEs). These significantly impaired properties in the clp mutant were completely rescued by a single-copy integration of the wild-type clp into the mutant chromosome via homologous recombination. Interestingly, overexpression of clp in the wild-type strain resulted in significant increases in cell motility and synthesis of the CWDEs. To our surprise, significant reductions in carbohydrates utilization, EPS production, biofilm formation, and the protease activity were observed in the wild-type strain overexpressing clp, suggesting that Clp also plays a negative role in these properties. Furthermore, quantitative reverse transcription polymerase chain reaction analysis suggested that clp was positively regulated by the diffusible signal factor-mediated quorum-sensing system and the HrpG/HrpX cascade. Taken together, our results reveal that Clp functions as both activator and repressor in multiple biological processes in X. axonopodis pv. glycines that are essential for its full virulence.

Structures of inactive CRP species reveal the atomic details of the allosteric transition that discriminates cyclic nucleotide second messengers.

The prokaryotic global transcription factor CRP has been considered to be an ideal model for in-depth study of both the allostery of the protein and the differential utilization of the homologous cyclic nucleotide second messengers cAMP and cGMP. Here, atomic details from the crystal structures of two inactive CRP species, an apo form and a cGMP-bound form, in comparison with a known active conformation, the cAMP-CRP complex, provide macroscopic and microscopic insights into CRP allostery, which is coupled to specific discrimination between the two effectors. The cAMP-induced conformational transition, including dynamic fluctuations, can be driven by the fundamental folding forces that cause water-soluble globular proteins to construct an optimized hydrophobic core, including secondary-structure formation. The observed conformational asymmetries underlie a negative cooperativity in the sequential binding of cyclic nucleotides and a stepwise manner of binding with discrimination between the effector molecules. Additionally, the finding that cGMP, which is specifically recognized in a syn conformation, induces an inhibitory conformational change, rather than a null effect, on CRP supports the intriguing possibility that cGMP signalling could be widely utilized in prokaryotes, including in aggressive inhibition of CRP-like proteins.

Structures of the PelD Cyclic Diguanylate Effector Involved in Pellicle Formation in Pseudomonas aeruginosa PAO1

The second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays a vital role in the global regulation in bacteria. Here, we describe structural and biochemical characterization of a novel c-di-GMP effector PelD that is critical to the formation of pellicles by Pseudomonas aeruginosa. We present high-resolution structures of a cytosolic fragment of PelD in apo form and its complex with c-di-GMP. The structure contains a bi-domain architecture composed of a GAF domain (commonly found in cyclic nucleotide receptors) and a GGDEF domain (found in c-di-GMP synthesizing enzymes), with the latter binding to one molecule of c-di-GMP. The GGDEF domain has a degenerate active site but a conserved allosteric site (I-site), which we show binds c-di-GMP with a K(d) of 0.5 μm. We identified a series of residues that are crucial for c-di-GMP binding, and confirmed the roles of these residues through biochemical characterization of site-specific variants. The structures of PelD represent a novel class of c-di-GMP effector and expand the knowledge of scaffolds that mediate c-di-GMP recognition.

Involvement of a putative cyclic amp receptor protein (CRP)-like binding sequence and a CRP-like protein in glucose-mediated catabolite repression of thn genes in Rhodococcus sp. strain TFB.

Glucose catabolite repression of tetralin catabolic genes in Rhodococcus sp. strain TFB was shown to be exerted by a protein homologous to transcriptional regulators of the cyclic AMP receptor (CRP)-FNR family. The protein was detected bound to putative CRP-like boxes localized at the promoters of the thnA1 and thnS genes.

Mutation of the gene encoding a major outer-membrane protein in Xanthomonas campestris pv. campestris causes pleiotropic effects, including loss of pathogenicity

Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot in crucifers. The xanthan polysaccharide and extracellular enzymes produced by this organism are virulence factors, the expression of which is upregulated by Clp (CRP-like protein) and DSF (diffusible signal factor), which is synthesized by RpfF. It is also known that biofilm formation/dispersal, regulated by the effect of controlled synthesis of DSF on cell-cell signalling, is required for virulence. Furthermore, a deficiency in DSF causes cell aggregation with concomitant production of a gum-like substance that can be dispersed by addition of DSF or digested by exogenous endo-beta-1,4-mannanase expressed by Xcc. In this study, Western blotting of proteins from a mopB mutant (XcMopB) showed Xcc MopB to be the major outer-membrane protein (OMP); Xcc MopB shared over 97 % identity with homologues from other members of Xanthomonas. Similarly to the rpfF mutant, XcMopB formed aggregates with simultaneous production of a gummy substance, but these aggregates could not be dispersed by DSF or endo-beta-1,4-mannanase, indicating that different mechanisms were involved in aggregation. In addition, XcMopB showed surface deformation, altered OMP composition, impaired xanthan production, increased sensitivity to stressful conditions including SDS, elevated temperature and changes in pH, reduced adhesion and motility and defects in pathogenesis. The finding that the major OMP is required for pathogenicity is unprecedented in phytopathogenic bacteria.

Exploring the FL-160-CRP gene family through sequence variability of the complement regulatory protein (CRP) expressed by the trypomastigote stage of Trypanosoma cruzi

The complement regulatory protein (CRP) of Trypanosoma cruzi is a surface glycoprotein which confers to the infectious trypomastigote forms a protection against the lytic activity of the host complement. CRP belongs to the large family of the trans-sialidase-like proteins and its sequence is highly similar to those of the flagellar FL-160 and chronic exoantigen proteins, encoded by a multigene family. To further define the gene family encoding the CRP, we investigated the protein diversity among several strains of T. cruzi through the sequencing of trypomastigote transcripts, and used a phylogenetic analysis based on the multiple alignment of these proteins with the top scoring sequences detected by a database sequence homology search. Intrastrain variations in CRP sequences revealed the existence of several copies per strain. The interstrain variability of CRP was consistent with the genetic subdivisions of T. cruzi into lineages and discrete typing units. The phylogenetic analysis based on a 227 amino acid alignment of CRP sequences with the 200 putative proteins retrieved from the protein databases (including the sequences from the T. cruzi genome project) revealed that the CRP sequences clustered with the FL-160 proteins into a monophyletic group characterized by the presence of the 12 amino acid mimicry epitope that mimics nervous tissues. The phylogeny did not differentiate between the CRP and the FL-160 proteins. The identification of this group of CRP-like proteins and the high sequence similarity observed within it open up new prospects for the exploration of the localization, structure and function of these proteins and a better understanding of their involvement in key aspects of host–parasite interactions, such as the resistance to the complement. This work provides also information for the T. cruzi genome annotation of the trans-sialidase-like putative proteins.

Serum CRP-like protein profile in common carp Cyprinus carpio challenged with Aeromonas hydrophila and Escherichia coli lipopolysaccharide

The potential of C-reactive protein (CRP)-like proteins to be used as a biomarker of health status in cultured carp obtained from various European fish lines has been assessed. Varying CRP-like protein levels in the serum of carp were monitored using an indirect competitive enzyme-linked immunosorbent assay. CRP-like protein basal levels in normal fish varied between carp lines, ranging on average from 2.9+/-0.15 to 12.57+/-1.19 microg ml(-1). Serum levels of CRP-like protein in carp were observed to increase several fold in fish infected with the pathogen Aeromonas hydrophila. However, carp injected with Escherichia coli lipopolysaccharide (LPS) serotype 0111:B4 did not exhibit an increase in CRP-like proteins levels.

Molecular cloning and characterization of cortical rod protein in the giant freshwater prawn Macrobrachium rosenbergii, a species not forming cortical rod structures in the oocytes

The formation of cortical rod structures is a characteristic of fully mature oocytes in penaeid prawns, but such structures are absent from oocytes of giant freshwater prawn Macrobrachium rosenbergii. In the present study, we first demonstrated the presence of a 30-kDa protein, which was immunologically related to kuruma prawn cortical rod protein (CRP), in the ovary of giant freshwater prawn, and subsequently purified this protein. Furthermore, a cDNA encoding the CRP-like protein was isolated. Based on the high homology (98%) in the amino acid sequence with kuruma prawn CRP, the 30-kDa protein has been identified as a CRP homologue of giant freshwater prawn, designated mrCRP. The RT-PCR analysis revealed that mrCRP mRNA was present in the ovary from a prawn with a gonadosomatic index (GSI) of 0.2. Western blot analysis revealed the presence of a CRP-immunoreactive band of 30kDa in the ovary with GSI of 1.6. By immunocytochemistry, CRP-immunopositive signals were detected in the ovary with GSI of 0.9, that had started to accumulate considerable amounts of vitellins and lipids in the peripheral cytoplasm. With progress of vitellogenesis, mrCRP was apparently accumulated in the mature oocytes, although it was not detectable, presumably because a relatively small amount of mrCRP was masked with large amounts of vitellin and lipids. In giant freshwater prawn without forming cortical rod structures, our findings indicate that the oocytes produce mrCRP, a homologue of CRP found in penaeid prawns.

Atypical phosphorylcholine-reactive protein from Atlantic salmon, Salmo salar L.

A phosphorylcholine-reactive protein was isolated from serum of Atlantic salmon ( Salmo salar L.) by affinity chromatography on a phosphorylcholine-conjugated Sepharose column followed by elution with phosphorylcholine. Based on the method used we describe the isolated protein as salmon phosphorylcholine-reactive protein (salmon PRP). Salmon PRP has calcium-independent binding to phosphorylcholine. The protein exists in a monomeric and dimeric form with molecular weight of approximately 80 and 160 kD, respectively. Separation of the protein preparation on SDS-PAGE under reducing conditions resulted in disappearance of the 80 and 160 kD bands and appearance of a major protein band of approximately 100 kD. The N-terminal amino acid sequences of the non-reduced 80 and 160 kD bands and the reduced 100 kD band were identical. Apart from the dimeric form, the molecular weight of salmon PRP and its appearance on SDS-PAGE is similar to human plasminogen. Comparison of the sequence in a protein database resulted in approximately 50% identity with human and bovine plasminogen. In addition, cross-reactivity between antibodies to human plasminogen and salmon PRP was demonstrated. Thus, salmon PRP appears to be different from other phosphorylcholine-reactive proteins which are mostly reported to be CRP-like proteins with calcium-dependent binding to phosphorylcholine, pentameric ring-structure and sequence homology between species. Whether salmon PRP is a new type of phosphorylcholine-binding protein with an unknown function or a plasminogen-like protein with binding specificity for phosphorylcholine calls for further investigation.