Jos M. H. Raats

MPP6 is an exosome-associated RNA-binding protein involved in 5.8S rRNA maturation

The exosome is a complex of 3′→5′ exoribonucleases which is involved in many RNA metabolic processes. To regulate these functions distinct proteins are believed to recruit the exosome to specific substrate RNAs. Here, we demonstrate that M-phase phosphoprotein 6 (MPP6), a protein reported previously to co-purify with the TAP-tagged human exosome, accumulates in the nucleoli of HEp-2 cells and associates with a subset of nuclear exosomes as evidenced by co-immunoprecipitation and biochemical fractionation experiments. In agreement with its nucleolar accumulation, siRNA-mediated knock-down experiments revealed that MPP6 is involved in the generation of the 3′ end of the 5.8S rRNA. The accumulation of the same processing intermediates after reducing the levels of either MPP6 or exosome components strongly suggests that MPP6 is required for the recruitment of the exosome to the pre-rRNA. Interestingly, MPP6 appeared to display RNA-binding activity in vitro with a preference for pyrimidine-rich sequences, and to bind to the ITS2 element of pre-rRNAs. Our data indicate that MPP6 is a nucleolus-specific exosome co-factor required for its role in the maturation of 5.8S rRNA.

Experimental autoimmune encephalomyelitis induction in peptidylarginine deiminase 2 knockout mice

During the development of multiple sclerosis the destruction of the myelin sheath surrounding the neurites is accompanied by citrullination of several central nervous system (CNS) proteins, including myelin basic protein and glial fibrillary acidic protein. In experimental autoimmune encephalomyelitis (EAE), a disease induced in animals by immunization with proteins or peptides from the CNS, the animals develop symptoms similar to multiple sclerosis (MS). The increased levels of citrullinated CNS proteins associated with MS are also observed during the development of EAE. To study the role of CNS protein citrullination in EAE development, we induced EAE with a peptide derived from myelin oligodendrocyte glycoprotein (MOG35–55) in mice lacking the peptidylarginine deiminase 2 (PAD2) protein, because this enzyme was the most likely candidate to be involved in catalyzing CNS protein citrullination in the diseased state. Even though the PAD2 knockout mice displayed a dramatic reduction in the amount of citrullination present in the CNS, indicating that PAD2 is indeed responsible for the majority of detectable citrullination observed in EAE, the development of EAE was not impaired by genetic deletion of PAD2, suggesting that PAD2 catalyzed citrullination is not essential to the development of EAE. J. Comp. Neurol. 498:217–226, 2006.

Autoantibodies specific for apoptotic U1-70K are superior serological markers for mixed connective tissue disease.

Modifications occurring on autoantigens during cell death have been proposed to have a role in the initiation of autoimmune diseases. Patients suffering from mixed connective tissue disease (MCTD) produce autoantibodies directed to U1 small nuclear ribonucleoprotein (snRNP), and antibodies against a 70 kDa protein component, the U1-70K (70K) protein, are the most prominent. During apoptosis, 70K is cleaved by caspase-3 to a 40 kDa product, which remains associated with the complex. Autoantibodies preferentially recognizing the apoptotic form of 70K have been described previously, and an apoptosis-specific epitope on 70K has been identified. This study shows that 29 of 53 (54%) MCTD sera preferentially recognize the apoptotic form of 70K over intact 70K. Moreover, we show that antibodies directed to an apoptosis-specific epitope on 70K are more specifically associated with MCTD than other anti-70K antibodies, suggesting that apoptotic 70K is a better antigen for the detection of these antibodies in MCTD patients. Longitudinal analysis of 12 MCTD patients showed in several patients that early sera are relatively enriched with antibodies recognizing an apoptosis-specific epitope, and that the levels of these apoptosis-specific antibodies decrease in time. These findings indicate that the early detection of apoptotic 70K is of considerable interest for anti-U1 snRNP-positive patients.

Structure of the bovine eye lens γs-crystallin gene (formerly βs)

The organization of a number of crystallin genes has already been resolved. One of the remaining genes of which the structure was hitherto unknown is the gamma s gene (formerly beta s). We determined the complete sequence of the bovine gamma s-crystallin-coding gene, apart from the middle region of the first intron. Since it contains three exons and two introns, we conclude that the former beta s, also at the gene level is gamma-crystallin-like. However, it is located on chromosome 3, in contrast to other gamma genes which occur in tandem on the human chromosome 2.

ACPA fine-specificity profiles in early rheumatoid arthritis patients do not correlate with clinical features at baseline or with disease progression.

IntroductionAutoantibodies against citrullinated peptides/proteins (ACPA) are found in approximately 75% of the sera of patients with rheumatoid arthritis (RA). The RA-specific ACPA are frequently present prior to disease onset and their presence associates with a more erosive disease course. ACPA can therefore be used to aid the diagnosis and prognosis of RA. Recently, it became clear that ACPA are very heterogeneous, both in an individual patient and among different patients. The aim of this study was to investigate whether clinically meaningful ACPA profiles exist in early RA patients.MethodsTwenty citrullinated peptides and the corresponding non-citrullinated control peptides were immobilized on microarray sensor chips. Sera from 374 early arthritis patients were analyzed by surface plasmon resonance imaging (i SPR) of biomolecular interactions on the sensor chip.ResultsCluster analysis of the reactivities with the citrullinated peptides, after subtraction of the reactivities with the corresponding control peptides confirmed the heterogeneity of the ACPA response in RA and revealed 12 distinct ACPA profiles. The association of the 5 most frequent profiles with clinical features at diagnosis and during the disease course was examined, showing no statistically significant associations.ConclusionsCompared to the detection of ACPA in RA sera by CCP-based assays, ACPA profiling in early arthritis patients did not reveal associations with disease activity and progression scores.

Recombinant human antibodies specific for the Pfs48/45 protein of the malaria parasite Plasmodium falciparum

We report the first construction of two combinatorial human phage display libraries derived from malaria-immune patients. Specific single-chain Fv fragments (scFv) against Pfs48/45, a gamete surface protein of the sexual stages of Plasmodium falciparum, were selected and analyzed extensively. The selected scFv reacted with the surface of extracellular sexual forms of the parasite and showed Pfs48/45 reactivity on immunoblot. The scFv inhibit binding of human malaria sera to native Pfs48/45 from gametocytes. Moreover, the scFv bind to target epitopes of Pfs48/45 exposed in natural infections. Sequence analysis of eight scFv clones specific for epitope III of Pfs48/45 revealed that these clones could be divided into one VH family-derived germ-line gene (VH1) and two VL family segments (VL2 and VKI).

Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

BackgroundClassical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.ResultsTargeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb)-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA)-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step.ConclusionA novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

Structurally Derived Mutations Define Congenital Heart Block‐Related Epitopes Within the 200–239 Amino Acid Stretch of the Ro52 Protein

Congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with Ro/SSA autoantibodies. During pregnancy, the antibodies are transported across the placenta and affect the fetus. We have previously demonstrated that antibodies directed to the 200–239 amino acid (aa) stretch of the Ro52 component of the Ro/SSA antigen correlate with the development of congenital heart block. In this report, we investigated the antibody–antigen interaction of this target epitope in detail at a molecular and structural level. Peptides representing aa 200–239 (p200) with structurally derived mutations were synthesized to define the epitopes recognized by two Ro52 human monoclonal antibodies, S3A8 and M4H1, isolated from patient‐derived phage display libraries. Analyses by ELISA, circular dichroism and MALDI‐TOF‐MS demonstrate that the antibody recognition is dependent on a partly α‐helical fold within the putative leucine zipper of the 200–239 aa stretch and that the two human anti‐p200 monoclonal antibodies, M4H1 and S3A8, recognize different epitopic structures within the p200 peptide. In addition, we investigated the representation of each fine specificity within the sera of mothers with children born with congenital heart block, and in such sera, antibodies of the S3A8 idiotype were more commonly detected and at higher levels than M4H1‐like antibodies.

Isolation of a monoclonal antibody from a malaria patient-derived phage display library recognising the Block 2 region of Plasmodium falciparum merozoite surface protein-1 ☆

Antigens associated with different life-cycle stages of Plasmodium falciparum (P. falciparum) are being investigated as potential malaria vaccines. However, the difficulties of obtaining immunological reagents have hampered detailed characterisation of these antigens and human antibody responses to different antigenic variants. Phage display antibodies offer an alternative method for the production of high affinity single chain variable fragment (scFv) derivatives of human antibodies of ‘natural host’ origin [1]. These cloned recombinant human B-cell derived antibody fragments, containing a single antigen binding site, are derived from the natural induction of the immune response of the host to the parasite and thus, by-pass the need for animal immunisation. Phage display scFv antibodies are assembled as antibody fragments in the periplasmic space of Escherichia coli in a reducing environment similar to that found in eukaryotic assembly pathways [2,3]. The abundant merozoite surface protein-1 (MSP-1) of P. falciparum is a major blood-stage malaria vaccine candidate. Antibodies to MSP-1 of P. falciparum are involved in the protection of animals against experimental P. falciparum infections [4,5] and MSP-1 has been successfully used as a protective immunogen in animal model vaccination trials [6–8]. Most studies have used the conserved C-terminal end of the protein (MSP-119) [9,10]. Less is known about the function and immunogenicity of the other regions of MSP-1. Block 2 (Bl2), the most variable region of this polymorphic molecule, is at the N-terminus of the protein. All MSP-1 variants can be classified into three types referred to as, K1, MAD20 and RO33 types, based on Block 2 amino acid sequence [11]. K1-type and MAD20-type Block 2 regions are characterised by the presence of internal trior hexa-peptide repeats flanked by type-specific sequences. The Block 2 region of RO33-type variants contains a non-repetitive sequence that varies little between isolates [12]. Recently, a strikingly positive correlation between protection against clinical malaria and the presence of anti-Block 2 (Bl2) antibodies has been demonstrated in a prospective study of disease resistance and susceptibility in the Gambia [13]. MAD 20 Block 2-type specific human or mouse monoclonal antibodies do not exist. To obtain such a reagent, we have isolated phage antibodies that bind a Abbre6iations: E. coli, Escherichia coli ; GST, glutathione-S-transferase; MAD20/Bl2, MAD20 Block 2; MSP-1, merozoite surface protein-1; P. falciparum, Plasmodium falciparum ; scFv, single chain variable fragment. Note : Nucleotide sequence data reported in this paper have been submitted to the DDJB, EMBL and GenBankTM databases under the accession numbers AF240360, AF240361 and AF240362. * Corresponding author. Tel.: +44-131-6508655; fax: +44-1316508655. E-mail address: d.e.arnot@ed.ac.uk (D.E. Arnot).

Citrullination: A Target for Disease Intervention in Multiple Sclerosis and other Inflammatory Diseases?

Citrullinated histone epitopes are involved in the very early stages of inflammatory responses. An important early event is the activation of neutrophils. It has been shown that Peptidyl Arginine Deiminase (PAD) expression levels increase upon pro-inflammatory signalling followed by activation of neutrophils. Subsequently, PAD enzymes cause histone citrullination in the activated neutrophils. Histone citrullination is involved in various processes. One of the most important is NETosis, which results in the release of citrullinated histones to the extracellular space. There, they are involved in Neutrophil Extracellular Trap (NET) formation, which intensifies the inflammatory response. The central role of citrullinated histones in early inflammation makes NETs an attractive target for inflammatory disease intervention. Moreover, the safety profile is expected to be superior to immune-suppressing biologicals, like anti- Tumour Necrosis Factor (TNF) drugs. It is anticipated that shielding citrullinated histone epitopes from the immune system, as well as interfering with their putative roles in the inflammatory response, will have a broad applicability in preventing and treating various inflammatory diseases, including multiple sclerosis.