60S Acidic Ribosomal Protein Research Articles

Isolation and characterization of a cDNA clone encoding one IgE-binding fragment of Penicillium brevicompactum

Rationale The abundance of allergenic Penicillium species has been associated with an increased incidence of childhood asthma and pulmonary bleeding. Penicillium brevicompactum has been identified as the most prevalent indoor Penicillium species. However, detailed studies on allergen of the ubiquitous Penicillium species are still lacking. For the characterization of allergens of prevalent Penicillium species, molecular cloning of the allergen genes of P. brevicompactum was performed in the present study. Methods A cDNA library of P. brevicompactum was constructed in Uni-ZAP® XR vector using mRNA isolated from the organism. The cDNA library of P. brevicompactum was screened using pooled atopic sera. Results One positive clone encoding a polypeptide of 11.06 kDa, designated as Pb2 was isolated. This clone was found to be reactive only against the atopic sera. The mRNA sequence of the clone was deduced from the cDNA of 0.5 kb transcript and found to be a 321 nt encoding 107 amino acids. Analysis of the crude extracts of the phage clone by SDS-PAGE immunoblot confirmed its expected molecular size. BLAST analysis showed that clone Pb2 had strong homology to the 60S acidic ribosomal protein P1 family from different eukaryotic sources, including the Alt a 12 allergen of mould Alternaria alternata which is an important source of fungal aeroallergens. Conclusions The results obtained suggest that 60S acidic ribosomal protein P1 is an allergen of P. brevicompactum and clone Pb2 contains the cDNA of this allergen.

Immediate-type hypersensitivity reaction to ingestion of mycoprotein (Quorn) in a patient allergic to molds caused by acidic ribosomal protein P2

Background: Quorn is the brand name for a line of foods made with so-called “mycoprotein,” which springs from the mold Fusarium venenatum . Since the introduction on the food market, there have been complaints from consumers reporting adverse gastrointestinal reactions after ingestion of mycoprotein. To date, it is not clear whether the reported symptoms are IgE-mediated. Objective: The aim of the study was to describe for the first time a case history of an asthmatic patient with severe hypersensitivity reactions to ingested mycoprotein and to identify and characterize the potential allergen that might be responsible for this. Methods: The sensitization pattern of the asthmatic subject was characterized, and food allergy to mycoprotein was assessed by double-blinded placebo-controlled food challenge. Afterward, specific IgE antibodies of the serum of this patient were used to screen a Fusarium culmorum cDNA expression library. The coding sequence of one enriched cDNA-clone was expressed in Escherichia coli to produce a recombinant protein that was further purified and immunologically characterized. Results: The patient showed high sensitization to many known aeroallergens but apart from Quorn not to any other tested food samples. The deduced amino acid sequence of the enriched cDNA-clone (Fus c 1) showed large identity to the 60S acidic ribosomal protein P2 which is highly conserved among several species and also described as minor allergen in other mold species. The frequency of IgE reactivity of sera from F culmorum -sensitized subjects to rFus c 1 was approximately 35%. By enzyme allergosorbent test inhibition, we found 65% inhibition of mycoprotein IgE reactivity by rFus c 1 . On the opposite we found reduced IgE reactivity of rFus c 1 of 68% by using mycoprotein as inhibitor. Conclusions: Sensitization to mold allergens by the respiratory tract and subsequent oral ingestion of cross-reactive proteins may lead to severe food-allergic reactions. Thus, the 60S acidic ribosomal protein P2 of F venenatum probably is the reason for the described severe hypersensitivity reactions of the patient to Quorn-mycoprotein because of its potential cross-reactivity to the F culmorum allergen Fus c 1. (J Allergy Clin Immunol 2003;111:1106-10.)

Molecular cloning and immunological characterisation of potential allergens from the mould Fusarium culmorum

Background: High quality and stability are essential requirements of commercial allergen preparations. Recently we have demonstrated the very low stability of protein allergens in an extract of the ubiquitous mould Fusarium culmorum. Objective: The present study was performed to identify, isolate and characterise allergens of F. culmorum as a basis for a stable allergenic reference material. In addition, the significance of IgE binding to carbohydrate structures in the natural allergen source was investigated. Methods: Sera of 52 subjects with suspected mould allergy were used to determine the IgE binding capacity of a commercial F. culmorum extract and an in-house extract by immunoblotting and enzyme allergo sorbent test (EAST). Binding of IgE-antibodies to putative carbohydrate structures located on glycoproteins was verified by periodate treatment of blot strips prior to immunodetection. A complementary (c)DNA expression library of F. culmorum was prepared and screened for IgE-binding clones using sera from F. culmorum-sensitive individuals. Positive clones were isolated, and the open reading frames were subcloned into expression vectors to produce recombinant proteins in E. coli. The recombinant proteins were tested for their IgE reactivity by immunoblotting and EAST. Results: Using the in-house extract for EAST and immunoblot experiments 44% (23/52) of the sera were found to contain F. culmorum-specific IgE antibodies. Compared to the in-house extract, nearly all IgE-reactivties in the range of 15–30 kD were lacking in the commercial preparation as examined by immunoblot analysis and only 10% (5/52) of the sera were found to contain F. culmorum-specific IgE by EAST. IgE binding to putative carbohydrate structures was observed in the high molecular weight range in approximately 50% (12/23) of the IgE-positive sera by both extracts. Three IgE binding clones were isolated from the cDNA-library. One clone (Fus c 1) is homologous to the highly conserved 60S acidic ribosomal protein P2 described as minor allergen in other moulds. The second (Fus c 2) shows high similarity (64%) to a respiratory allergen from the basidiomycete Coprinus comatus (Cop c 2). The third clone (Fus c 3) was not related to known proteins. With sera from 26 individuals sensitised to F. culmorum the IgE prevalence of recombinant proteins rFus c 1, rFus c 2 and rFus c 3 was found to be 35, 50, and 15%, respectively. Conclusions: F. culmorum may represent an underestimated source of aeroallergens. In contrast to highly labile and poorly standardised F. culmorum extracts, the new recombinant allergens may serve as stable allergenic reference material. A combination of rFus c 1 and rFus c 2 is suitable to diagnose 81% of F. culmorum-sensitised subjects. IgE reactivity to putative carbohydrate structures is relatively frequent, and can not be detected by these allergens.

Characterization of human lactoferricin as a potent protein kinase CK2 activator regulated by A-kinase in vitro.

Lactoferricin (LFcin) hydrolyzed from lactoferrin (LF), a major 80 kDa iron-binding protein in milk and other exocrine secretions, was characterized as a potent activator of protein kinase CK2 (CK2) in vitro. Human LFcin (hLFcin) at 0.5 microg stimulated approx. 5-fold CK2 activity [phosphorylation of 60S acidic ribosomal proteins (P0, P1, P2) and Hsp90 (p98)] in a manner similar to other functional proteins with oligo-Arg clusters, such as salmine A1, sperm histone H2B and HIV-1 Rev. Interestingly, this stimulatory effect of hLFcin was significantly reduced when it was phosphorylated by A-kinase in vitro. These results suggest that (i) hLFcin acts as a potent CK2 activator in vitro; and (ii) the stimulatory effect of hLFcin on CK2 activity is regulated by its phosphorylation by A-kinase in vitro.

Sequence and transcription patterns of 60S ribosomal protein P0, a diapause-regulated AP endonuclease in the flesh fly, Sarcophaga crassipalpis

We have isolated and sequenced a 1308 bp clone from a pupal brain cDNA library of the flesh fly, Sarcophaga crassipalpis, showing 97% amino acid (aa) sequence similarity to Ceratitis capitata 60S acidic ribosomal protein P0 (CcP0) and 93% aa sequence similiarity to Drosophila melanogaster P0 (DmP0). DmP0 is a multifunctional protein necessary for efficient protein translation of the 60S ribosome as well as DNA repair via AP3 endonuclease activity. In this study, we observed that S. crassipalpis P0 ( ScP0) is cyclically regulated throughout the fly's overwintering pupal diapause. Expression of ScP0 cycles out of phase with the 4 day cycles of O 2 consumption: the peak day of O 2 consumption is characterized by low ScP0 expression, while high expression is noted during the trough of the O 2 consumption cycle. The O 2 cycles, which are in turn driven by cycles of juvenile hormone (JH), can be eliminated by application of a JH analog (JHA). Pupae rendered acyclic with a JHA application consume O 2 at a constant high rate and ScP0 is consistently downregulated. Our findings thus suggest that the cyclic nature of ScP0 regulation during pupal diapause is linked to the JH-mediated metabolic cycles characteristic of this species.

Biochemical characterization of 60S acidic ribosomal P proteins from porcine liver and the inhibition of their immunocomplex formation with sera from systemic lupus erythematosus (SLE) patients by glycyrrhizin in vitro.

The three casein kinase II (CK-II) phosphate acceptors (p35, p17 and p15) in the Superdex CK-II fraction prepared from a 1.5 M NaCl extract of porcine liver were selectively purified by glycyrrhizin (GL)-affinity column chromatography (HPLC) as a heterocomplex associated with CK-II. Determination of the N-terminal amino acid sequences and immunological tests confirmed that these three CK-II phosphate acceptors belong to the family of 60S acidic ribosomal proteins (P0, P1 and P2). Three polyphenol-containing anti-oxidant compounds [catechin, epigallocatechin gallate (EGCG) and quercetin] inhibited CK-II activity (phosphorylation of these ribosomal P proteins) in a dose-dependent manner in vitro. Quercetin (ID50 = approx. 50 nM) was found to be an effective CK-II inhibitor. In contrast, CK-II activity was significantly stimulated by lower doses (0.3-3 microl) of GL, but was inhibited at high doses above 30 microM. As expected, GL at high doses above 200 microM inhibited the immunocomplex formation of 60S acidic ribosomal P proteins with their specific antibodies in the sera from patients with systemic lupus erythematosus (SLE). These results suggest that (i) a GL-affinity column is useful for effective purification of 60S acidic ribosomal P proteins from various mammalian cells as a heterocomplex associated with CK-II; and (ii) a relative high dose of GL may prevent the immunocomplex formation of 60S acidic ribosomal P proteins with their specific antibodies in the sera of SLE patients.

Nucleotide Sequence of a cDNA for a P2 60S Acidic Ribosomal Protein from Parthenium argentatum

In screening a stem-bark cDNA library from guayule (Parthenium argentatum) for genes involved in isoprenoid biogenesis, we isolated a full-length gene for a P2 60S acidic ribosomal protein that shows sequence similarity to vertebrate (Rich and Steitz, 1987), invertebrate (Qian et al., 1987), and yeast (Beltrame and Bianchi, 1987, 1990; Remacha et al., 1988) 60S acidic ribosomal proteins (Table I). The guayule P2 sequence is a full-length cDNA; a partial cDNA has been described for Arabidopsis (C. Bardet, M. Axelos, D. Tremousaygue, M. Lebas, T. Lagravere, and B. Lescure, unpublished data; EMBL accession number Z17464) with 65.5% identity at the nucleic acid level and 54.5% identity and 74.5% similarity at the amino acid level. Our interest in this gene extends to its potential use in experimental studies of Parthenium, from which few genes are at present available. Being a ribosomal protein, it should display minimal variations in expression and thus serve as a suitable internal control for gene expression studies in this species. The P2 60S acidic ribosomal protein plays a crucial role in the elongation step of protein synthesis. The P2 proteins exist as heterodimers composed of a and d subunits. Comparison of the P2 Parthenium sequence with both the a (Beltrame and Bianchi, 1987) and p (Beltrame and Bianchi, 1990) subunits of fission yeast shows nearly the same identity, 52.7 versus 50.9%, respectively, making it difficult to classify the guayule protein as P2 a or P2 F. Comparison of the P2 Parthenium sequence to the human LA2 protein (Rich and Steitz, 1987) shows 59.0% identity at the nucleic acid level and 54.5% identity and 79.5% similarity on the amino acid level, with a similar level of identity for the Drosophila gene (Qian et al., 1987).

Expressed Sequence Tags (ESTs) from a Jute (<i>Corchorus olitorius</i>) cDNA Library

Analysis of the genome of jute (Corchorus olitorius) was done by creating a new cDNA library of expressed sequence tags (ESTs) in pBluescript as the previous libraries reported earlier in this journal yielded only small DNA fragments from chloroplast and mitochondrial DNA. This report discusses results from a cDNA library constructed using poly A+ mRNA purified from 7-day old etiolated jute seedlings. Out of 700 recombinant plasmids obtained, 250 were analyzed using WU-BLAST (www.arabidopsis.org) for similar EST sequences in Arabidopsis thaliana and other higher plants. So far the analysis of the library has yielded several significant sequences, including the complete open reading frame of the 60S acidic ribosomal protein P3 and a partial cDNA of Class I chitinase. These results and future EST sequences from this library will be made available in Genbank and the sequence information will be used to clone full length DNA through PCR. Â DOI = 10.3329/ptcb.v16i2.1110Plant Tissue Cult. & Biotech. 16(2): 95-104, 2006 (December)

Novel ESTs from a Jute (Corchorus olitorius L.) cDNA Library

The paper describes the identification of new expressed sequence tags (ESTs) from a cDNA library of Corchorus olitorius L. var. O-4 after the initial description of the library construction reported recently. The sequence homology search in The Arabidopsis Information Source (TAIR) using the WU-BLAST tool revealed four complete and ten partial cDNA sequences. The complete cDNA sequences, based on their similarities to those of Arabidopsis proteins, encode V-ATPase subunit F, mitochondrial NADH-ubiquinone oxidoreductase, RuBisCO small subunit 1A and heat shock protein (HSP 60). Based on similar homology results the partial cDNAs encode proteins, namely, chaperonin, actin 7, RelA-spoT homology, 60s ribosomal protein L 36a, transport protein, chloroplast inner membrane import protein Tic22, formate dehydrogenase, serine hydroxy-methyltransferase, metallothionein 2B and tansmembrane kinase. These cDNA sequences are new additions to the ESTs that encode chitinase-like protein (Class I) and 60S acidic ribosomal protein reported earlier. All the available cDNA sequences including that for the chitinase-like protein have been registered with GenBank, bearing Accession Nos. EU024510 through EU024520 and EU092254, EU057193-95. The cDNA samples are available for any researchers interested in such ESTs. A Clustal-W study of the amino acid sequences of proteins encoded by cDNA isolated in the present investigation with those of cotton, citrus, Arabidopsis, tobacco and other organisms revealed that the homology is maximum between jute and cotton followed by citrus, grapevine, tobacco and Arabidopsis. Key words: Jute cDNA library, Corchorus olitorius, Chitinase, Chaperonin, RSH2, RelA/SpoT, V-ATPase, Small subunit RuBisCO, Metallothionein, Actin 7, and HSPs D.O.I. 10.3329/ptcb.v17i2.3237 Plant Tissue Cult. & Biotech. 17(2): 173-182, 2007 (December)