CLAP: A web-server for automatic classification of proteins with special reference to multi-domain proteins

Background

The function of a protein can be deciphered with higher accuracy from its structure than from its amino acid sequence. Due to the huge gap in the available protein sequence and structural space, tools that can generate functionally homogeneous clusters using only the sequence information, hold great importance. For this, traditional alignment-based tools work well in most cases and clustering is performed on the basis of sequence similarity. But, in the case of multi-domain proteins, the alignment quality might be poor due to varied lengths of the proteins, domain shuffling or circular permutations. Multi-domain proteins are ubiquitous in nature, hence alignment-free tools, which overcome the shortcomings of alignment-based protein comparison methods, are required. Further, existing tools classify proteins using only domain-level information and hence miss out on the information encoded in the tethered regions or accessory domains. Our method, on the other hand, takes into account the full-length sequence of a protein, consolidating the complete sequence information to understand a given protein better.

Results

Our web-server, CLAP (Classification of Proteins), is one such alignment-free software for automatic classification of protein sequences. It utilizes a pattern-matching algorithm that assigns local matching scores (LMS) to residues that are a part of the matched patterns between two sequences being compared. CLAP works on full-length sequences and does not require prior domain definitions.Pilot studies undertaken previously on protein kinases and immunoglobulins have shown that CLAP yields clusters, which have high functional and domain architectural similarity. Moreover, parsing at a statistically determined cut-off resulted in clusters that corroborated with the sub-family level classification of that particular domain family.

Conclusions

CLAP is a useful protein-clustering tool, independent of domain assignment, domain order, sequence length and domain diversity. Our method can be used for any set of protein sequences, yielding functionally relevant clusters with high domain architectural homogeneity. The CLAP web server is freely available for academic use at http://nslab.mbu.iisc.ernet.in/clap/.     

The Arabidopsis Kinome: phylogeny and evolutionary insights into functional diversification

Background

Protein kinases constitute a particularly large protein family in Arabidopsis with important functions in cellular signal transduction networks. At the same time Arabidopsis is a model plant with high frequencies of gene duplications. Here, we have conducted a systematic analysis of the Arabidopsis kinase complement, the kinome, with particular focus on gene duplication events. We matched Arabidopsis proteins to a Hidden-Markov Model of eukaryotic kinases and computed a phylogeny of 942 Arabidopsis protein kinase domains and mapped their origin by gene duplication.

Results

The phylogeny showed two major clades of receptor kinases and soluble kinases, each of which was divided into functional subclades. Based on this phylogeny, association of yet uncharacterized kinases to families was possible which extended functional annotation of unknowns. Classification of gene duplications within these protein kinases revealed that representatives of cytosolic subfamilies showed a tendency to maintain segmentally duplicated genes, while some subfamilies of the receptor kinases were enriched for tandem duplicates. Although functional diversification is observed throughout most subfamilies, some instances of functional conservation among genes transposed from the same ancestor were observed. In general, a significant enrichment of essential genes was found among genes encoding for protein kinases.

Conclusions

The inferred phylogeny allowed classification and annotation of yet uncharacterized kinases. The prediction and analysis of syntenic blocks and duplication events within gene families of interest can be used to link functional biology to insights from an evolutionary viewpoint. The approach undertaken here can be applied to any gene family in any organism with an annotated genome.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-548) contains supplementary material, which is available to authorized users.     

The repertoire of protein kinases encoded in the draft version of the human genome: atypical variations and uncommon domain combinations

Background

Phosphorylation by protein kinases is central to cellular signal transduction. Abnormal functioning of kinases has been implicated in developmental disorders and malignancies. Their activity is regulated by second messengers and by the binding of associated domains, which are also influential in translocating the catalytic component to their substrate sites, in mediating interaction with other proteins and carrying out their biological roles.

Result

Using sensitive profile-search methods and manual analysis, the human genome has been surveyed for protein kinases. A set of 448 sequences, which show significant similarity to protein kinases and contain the critical residues essential for kinase function, have been selected for an analysis of domain combinations after classifying the kinase domains into subfamilies. The unusual domain combinations in particular kinases suggest their involvement in ubiquitination pathways and alternative modes of regulation for mitogen-activated protein kinase kinases (MAPKKs) and cyclin-dependent kinase (CDK)-like kinases. Previously unexplored kinases have been implicated in osteoblast differentiation and embryonic development on the basis of homology with kinases of known functions from other organisms. Kinases potentially unique to vertebrates are involved in highly evolved processes such as apoptosis, protein translation and tyrosine kinase signaling. In addition to coevolution with the kinase domain, duplication and recruitment of non-catalytic domains is apparent in signaling domains such as the PH, DAG-PE, SH2 and SH3 domains.

Conclusions

Expansion of the functional repertoire and possible existence of alternative modes of regulation of certain kinases is suggested by their uncommon domain combinations. Experimental verification of the predicted implications of these kinases could enhance our understanding of their biological roles.

     

Integrin-Linked Kinase Is a Functional Mn2+-Dependent Protein Kinase that Regulates Glycogen Synthase Kinase-3β (GSK-3β) Phosphorylation

Background

Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.

Methodology/Principal Findings

Here, we have expressed full-length recombinant ILK, purified it to >94% homogeneity, and characterized its kinase activity. Recombinant ILK readily phosphorylates glycogen synthase kinase-3 (GSK-3) peptide and the 20-kDa regulatory light chains of myosin (LC₂₀). Phosphorylation kinetics are similar to those of other active kinases, and mutation of the ATP-binding lysine (K220 within subdomain 2) causes marked reduction in enzymatic activity. We show that ILK is a Mn-dependent kinase (the K_m for MnATP is ∼150-fold less than that for MgATP).

Conclusions/Significance

Taken together, our data demonstrate that ILK is a bona fide protein kinase with enzyme kinetic properties similar to other active protein kinases.     

Structure-Function Analysis of Rgs1 in Magnaporthe oryzae: Role of DEP Domains in Subcellular Targeting

Background

Rgs1, a prototypical Regulator of G protein Signaling, negatively modulates the cyclic AMP pathway thereby influencing various aspects of asexual development and pathogenesis in the rice-blast fungus Magnaporthe oryzae. Rgs1 possesses tandem DEP motifs (termed DEP-A and DEP-B; for Dishevelled, Egl-10, Pleckstrin) at the N-terminus, and a Gα-GTP interacting RGS catalytic core domain at the C-terminus. In this study, we focused on gaining further insights into the mechanisms of Rgs1 regulation and subcellular localization by characterizing the role(s) of the individual domains and the full-length protein during asexual development and pathogenesis in Magnaporthe.

Methodology/Principal Findings

Utilizing western blot analysis and specific antisera against the N- and C-terminal halves of Rgs1, we identify and report the in vivo endoproteolytic processing/cleavage of full-length Rgs1 that yields an N-terminal DEP and a RGS core domain. Independent expression of the resultant DEP-DEP half (N-Rgs1) or RGS core (C-Rgs1) fragments, failed to complement the rgs1Δ defects in colony morphology, aerial hyphal growth, surface hydrophobicity, conidiation, appressorium formation and infection. Interestingly, the full-length Rgs1-mCherry, as well as the tagged N-terminal DEP domains (individually or in conjunction) localized to distinct punctate vesicular structures in the cytosol, while the catalytic RGS core motif was predominantly vacuolar.

Conclusions/Significance

Based on our data from sequence alignments, immuno-blot and microscopic analysis, we propose that the post-translational proteolytic processing of Rgs1 and the vacuolar sequestration of the catalytic RGS domain represents an important means of down regulating Rgs1 function and thus forming an additional and alternative means of regulating G protein signaling in Magnaporthe. We further hypothesize the prevalence of analogous mechanisms functioning in other filamentous fungi. Furthermore, we conclusively assign a specific vesicular/membrane targeting function for the N-terminal DEP domains of Rgs1 in the rice-blast fungus.     

A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase

Background

Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored.

Methodology/Principal Findings

We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo.

Conclusions/Significance

This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.     

A computational screen for type I polyketide synthases in metagenomics shotgun data

Background

Polyketides are a diverse group of biotechnologically important secondary metabolites that are produced by multi domain enzymes called polyketide synthases (PKS).

Methodology/Principal Findings

We have estimated frequencies of type I PKS (PKS I) – a PKS subgroup – in natural environments by using Hidden-Markov-Models of eight domains to screen predicted proteins from six metagenomic shotgun data sets. As the complex PKS I have similarities to other multi-domain enzymes (like those for the fatty acid biosynthesis) we increased the reliability and resolution of the dataset by maximum-likelihood trees. The combined information of these trees was then used to discriminate true PKS I domains from evolutionary related but functionally different ones. We were able to identify numerous novel PKS I proteins, the highest density of which was found in Minnesota farm soil with 136 proteins out of 183,536 predicted genes. We also applied the protocol to UniRef database to improve the annotation of proteins with so far unknown function and identified some new instances of horizontal gene transfer.

Conclusions/Significance

The screening approach proved powerful in identifying PKS I sequences in large sequence data sets and is applicable to many other protein families.     

Homodimerization of the death-associated protein kinase catalytic domain: development of a new small molecule fluorescent reporter

Background

Death-Associated Protein Kinase (DAPK) is a member of the Ca²⁺/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases.

Methodology/Principal Findings

In the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution.

Conclusions/Significance

The use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure.     

Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs

Background

Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily.

Results

Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a β-hairpin pointed towards the substrate binding cleft. The invariant β-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism.

Conclusions

The seven families and subfamilies of the lysozyme superfamily all have in common a β-hairpin structure which displays a family-specific sequence motif. These GH β-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily.     

Differential induction of functional IgG using the Plasmodium falciparum placental malaria vaccine candidate VAR2CSA

Background

In Plasmodium falciparum malaria endemic areas placental malaria (PM) is an important complication of malaria. The recurrence of malaria in primigravidae women irrespective of acquired protection during childhood is caused by the interaction between the parasite-expressed VAR2CSA antigen and chondroitin sulfate A (CSA) in the placental intervillous space and lack of protective antibodies. PM impairs fetal development mainly by excessive inflammation processes. After infections during pregnancy women acquire immunity to PM conferred by antibodies against VAR2CSA. Ideally, a vaccine against PM will induce antibody-mediated immune responses that block the adhesion of infected erythrocytes (IE) in the placenta.

Principal Findings

We have previously shown that antibodies raised in rat against individual domains of VAR2CSA can block IE binding to CSA. In this study we have immunized mice, rats and rabbits with each individual domain and the full-length protein corresponding to the FCR3 VAR2CSA variant. We found there is an inherently higher immunogenicity of C-terminal domains compared to N-terminally located domains. This was irrespective of whether antibodies were induced against single domains or the full-length protein. Species-specific antibody responses were also found, these were mainly directed against single domains and not the full-length VAR2CSA protein.

Conclusions/Significance

Binding inhibitory antibodies appeared to be against conformational B-cell epitopes. Non-binding inhibitory antibodies reacted highly against the C-terminal end of the VAR2CSA molecule especially the highly polymorphic DBL6ε domain. Differential species-specific induction of antibody responses may allow for more direct analysis of functional versus non-functional B-cell epitopes.     

Specific syndecan-1 domains regulate mesenchymal tumor cell adhesion, motility and migration

Background

Syndecans are proteoglycans whose core proteins have a short cytoplasmic domain, a transmembrane domain and a large N-terminal extracellular domain possessing glycosaminoglycan chains. Syndecans are involved in many important cellular processes. Our recent publications have demonstrated that syndecan-1 translocates into the nucleus and hampers tumor cell proliferation. In the present study, we aimed to investigate the role of syndecan-1 in tumor cell adhesion and migration, with special focus on the importance of its distinct protein domains, to better understand the structure-function relationship of syndecan-1 in tumor progression.

Methodology/Principal Findings

We utilized two mesenchymal tumor cell lines which were transfected to stably overexpress full-length syndecan-1 or truncated variants: the 78 which lacks the extracellular domain except the DRKE sequence proposed to be essential for oligomerization, the 77 which lacks the whole extracellular domain, and the RMKKK which serves as a nuclear localization signal. The deletion of the RMKKK motif from full-length syndecan-1 abolished the nuclear translocation of this proteoglycan. Various bioassays for cell adhesion, chemotaxis, random movement and wound healing were studied. Furthermore, we performed gene microarray to analyze the global gene expression pattern influenced by syndecan-1. Both full-length and truncated syndecan-1 constructs decrease tumor cell migration and motility, and affect cell adhesion. Distinct protein domains have differential effects, the extracellular domain is more important for promoting cell adhesion, while the transmembrane and cytoplasmic domains are sufficient for inhibition of cell migration. Cell behavior seems to depend also on the nuclear translocation of syndecan-1. Many genes are differentially regulated by syndecan-1 and a number of genes are actually involved in cell adhesion and migration.

Conclusions/Significance

Our results demonstrate that syndecan-1 regulates mesenchymal tumor cell adhesion and migration, and different domains have differential effects. Our study provides new insights into better understanding of the role of syndecans in tumor progression.     

The Evolutionary History of Protein Domains Viewed by Species Phylogeny

Background

Protein structural domains are evolutionary units whose relationships can be detected over long evolutionary distances. The evolutionary history of protein domains, including the origin of protein domains, the identification of domain loss, transfer, duplication and combination with other domains to form new proteins, and the formation of the entire protein domain repertoire, are of great interest.

Methodology/Principal Findings

A methodology is presented for providing a parsimonious domain history based on gain, loss, vertical and horizontal transfer derived from the complete genomic domain assignments of 1015 organisms across the tree of life. When mapped to species trees the evolutionary history of domains and domain combinations is revealed, and the general evolutionary trend of domain and combination is analyzed.

Conclusions/Significance

We show that this approach provides a powerful tool to study how new proteins and functions emerged and to study such processes as horizontal gene transfer among more distant species.     

Evolution of domain combinations in protein kinases and its implications for functional diversity

Protein kinases phosphorylating Ser/Thr/Tyr residues in several cellular proteins exert tight control over their biological functions. They constitute the largest protein family in most eukaryotic species. Protein kinases classified based on sequence similarity in their catalytic domains, cluster into subfamilies, which share gross functional properties. Many protein kinases are associated or tethered covalently to domains that serve as adapter or regulatory modules, aiding substrate recruitment, specificity, and also serve as scaffolds. Hence the modular organisation of the protein kinases serves as guidelines to their functional and molecular properties. Analysis of genomic repertoires of protein kinases in eukaryotes have revealed wide spectrum of domain organisation across various subfamilies of kinases. Occurrence of organism-specific novel domain combinations suggests functional diversity achieved by protein kinases in order to regulate variety of biological processes. In addition, domain architecture of protein kinases revealed existence of hybrid protein kinase subfamilies and their emerging roles in the signaling of eukaryotic organisms. In this review we discuss the repertoire of non-kinase domains tethered to multi-domain kinases in the metazoans. Similarities and differences in the domain architectures of protein kinases in these organisms indicate conserved and unique features that are critical to functional specialization.     

MDAT- Aligning multiple domain arrangements

Background

Proteins are composed of domains, protein segments that fold independently from the rest of the protein and have a specific function. During evolution the arrangement of domains can change: domains are gained, lost or their order is rearranged. To facilitate the analysis of these changes we propose the use of multiple domain alignments.

Results

We developed an alignment program, called MDAT, which aligns multiple domain arrangements. MDAT extends earlier programs which perform pairwise alignments of domain arrangements. MDAT uses a domain similarity matrix to score domain pairs and aligns the domain arrangements using a consistency supported progressive alignment method.

Conclusion

MDAT will be useful for analysing changes in domain arrangements within and between protein families and will thus provide valuable insights into the evolution of proteins and their domains. MDAT is coded in C++, and the source code is freely available for download at http://www.bornberglab.org/pages/mdat.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0442-7) contains supplementary material, which is available to authorized users.     

Protein Synthesis Dependence of Growth Cone Collapse Induced by Different Nogo-A-Domains

Background

The protein Nogo-A regulates axon growth in the developing and mature nervous system, and this is carried out by two distinct domains in the protein, Nogo-A-Δ20 and Nogo-66. The differences in the signalling pathways engaged in axon growth cones by these domains are not well characterized, and have been investigated in this study.

Methodology/Principal Findings

We analyzed growth cone collapse induced by the Nogo-A domains Nogo-A-Δ20 and Nogo-66 using explanted chick dorsal root ganglion neurons growing on laminin/poly-lysine substratum. Collapse induced by purified Nogo-A-Δ20 peptide is dependent on protein synthesis whereas that induced by Nogo-66 peptide is not. Nogo-A-Δ20-induced collapse is accompanied by a protein synthesis-dependent rise in RhoA expression in the growth cone, but is unaffected by proteasomal catalytic site inhibition. Conversely Nogo-66-induced collapse is inhibited ∼50% by proteasomal catalytic site inhibition.

Conclusion/Significance

Growth cone collapse induced by the Nogo-A domains Nogo-A-Δ20 and Nogo-66 is mediated by signalling pathways with distinguishable characteristics concerning their dependence on protein synthesis and proteasomal function.     

A computational study of elongation factor G (EFG) duplicated genes: diverged nature underlying the innovation on the same structural template

Background

Elongation factor G (EFG) is a core translational protein that catalyzes the elongation and recycling phases of translation. A more complex picture of EFG''s evolution and function than previously accepted is emerging from analyzes of heterogeneous EFG family members. Whereas the gene duplication is postulated to be a prominent factor creating functional novelty, the striking divergence between EFG paralogs can be interpreted in terms of innovation in gene function.

Methodology/Principal Findings

We present a computational study of the EFG protein family to cover the role of gene duplication in the evolution of protein function. Using phylogenetic methods, genome context conservation and insertion/deletion (indel) analysis we demonstrate that the EFG gene copies form four subfamilies: EFG I, spdEFG1, spdEFG2, and EFG II. These ancient gene families differ by their indispensability, degree of divergence and number of indels. We show the distribution of EFG subfamilies and describe evidences for lateral gene transfer and recent duplications. Extended studies of the EFG II subfamily concern its diverged nature. Remarkably, EFG II appears to be a widely distributed and a much-diversified subfamily whose subdivisions correlate with phylum or class borders. The EFG II subfamily specific characteristics are low conservation of the GTPase domain, domains II and III; absence of the trGTPase specific G2 consensus motif “RGITI”; and twelve conserved positions common to the whole subfamily. The EFG II specific functional changes could be related to changes in the properties of nucleotide binding and hydrolysis and strengthened ionic interactions between EFG II and the ribosome, particularly between parts of the decoding site and loop I of domain IV.

Conclusions/Significance

Our work, for the first time, comprehensively identifies and describes EFG subfamilies and improves our understanding of the function and evolution of EFG duplicated genes.     

Correlated Mutation Analysis on the Catalytic Domains of Serine/Threonine Protein Kinases

Background

Protein kinases (PKs) have emerged as the largest family of signaling proteins in eukaryotic cells and are involved in every aspect of cellular regulation. Great progresses have been made in understanding the mechanisms of PKs phosphorylating their substrates, but the detailed mechanisms, by which PKs ensure their substrate specificity with their structurally conserved catalytic domains, still have not been adequately understood. Correlated mutation analysis based on large sets of diverse sequence data may provide new insights into this question.

Methodology/Principal Findings

Statistical coupling, residue correlation and mutual information analyses along with clustering were applied to analyze the structure-based multiple sequence alignment of the catalytic domains of the Ser/Thr PK family. Two clusters of highly coupled sites were identified. Mapping these positions onto the 3D structure of PK catalytic domain showed that these two groups of positions form two physically close networks. We named these two networks as θ-shaped and γ-shaped networks, respectively.

Conclusions/Significance

The θ-shaped network links the active site cleft and the substrate binding regions, and might participate in PKs recognizing and interacting with their substrates. The γ-shaped network is mainly situated in one side of substrate binding regions, linking the activation loop and the substrate binding regions. It might play a role in supporting the activation loop and substrate binding regions before catalysis, and participate in product releasing after phosphoryl transfer. Our results exhibit significant correlations with experimental observations, and can be used as a guide to further experimental and theoretical studies on the mechanisms of PKs interacting with their substrates.     

Cartilage oligomeric matrix protein specific antibodies are pathogenic

Introduction

Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP-specific monoclonal antibodies (mAbs).

Methods

B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated and the pathogenicity of mAbs was investigated by passive transfer experiments.

Results

B cell immunodominant epitopes were localized within 4 antigenic domains of the COMP but with preferential response to the epidermal growth factor (EGF)-like domain. Some of our anti-COMP mAbs showed interactions with the native form of COMP, which is present in cartilage and synovium. Passive transfer of COMP-specific mAbs enhanced arthritis when co-administrated with a sub-arthritogenic dose of a mAb specific to collagen type II. Interestingly, we found that a combination of 5 COMP mAbs was capable of inducing arthritis in naive mice.

Conclusions

We have identified the specificities of mAbs to COMP and their contribution to the development of arthritis. These findings will further improve our understanding of the autoantibody mediated immunopathologies occurring widely in rheumatoid arthritis (RA), as well as in other autoimmune disorders.     

Evaluation of the Quality of Guidelines for Myasthenia Gravis with the AGREE II Instrument

Background

Clinical practice guidelines (CPGs) are systematically developed statements to assist practitioners in making decisions about appropriate healthcare in specific clinical circumstances. The methodological quality of CPGs for myasthenia gravis (MG) are unclear.

Objective

To critically evaluate the methodological quality of CPGs for MG using AGREE II instrument.

Method

A systematical search strategy on PubMed, EMBASE, DynaMed, the National Guideline Clearinghouse (NGC) and the Chinese Biomedical Literature database (CBM) was performed on September 20th 2013. All guidelines related to MG were evaluated with AGREE II. The software used for analysis was SPSS 17.0.

Results

A total of 15 CPGs for MG met the inclusion criteria (12 CPGs in English, 3 CPGs in Chinese). The overall agreement among reviews was moderate or high (ICC >0.70). The mean scores (mean ± SD) for al six domains were presented as follows: scope and purpose (60.93% ±16.62%), stakeholder involvement (40.93% ±20.04%), rigor of development (37.22% ±30.46%), clarity of presentation (64.26% ±16.36%), applicability (28.19% ±20.56%) and editorial independence (27.78% ±28.28%). Compared with non-evidence-based CPGs, evidence-based CPGs had statistically significant higher quality scores for all AGREE II domains (P<0.05). All domain scores appear slightly higher for CPGs published after AGREE II instrument development and validation (P>0.05). The quality scores of CPGs developed by NGC/AAN were higher than the quality scores of CPGs developed by other organizations for all domains. The difference was statistically significant for all domains with the exception of clarity of presentation (P = 0.07).

Conclusions

The qualities of CPGs on MG were generally acceptable with several flaws. The AGREE II instrument should be adopted by guideline developers, particularly in China.