Separate and overlapping specificities in rheumatoid arthritis antibodies binding to citrulline- and homocitrulline-containing peptides related to type I and II collagen telopeptides

IntroductionOur objective was to find out if there are antibodies binding to homocitrulline-containing type I and II collagen carboxyterminal telopeptides in sera of patients with rheumatoid arthritis (RA), and if these antibodies cross-react with citrulline and homocitrulline in the same peptide sequence.MethodsA total of 72 RA and 72 control sera were analyzed for binding using enzyme-linked immunosorbent assay to citrulline- or homocitrulline-containing type I and II collagen carboxyterminal telopeptides, as well as to cyclic citrullinated peptide (CCP) and to mutated citrullinated vimentin (MCV). Specificities of the antibodies were tested using inhibition-ELISA.ResultsOf the RA sera, 39 (54%) and 41 (57%) were positive for binding to CCP and MCV, respectively. Further, 34 (47%) and 30 (42%) of the patients had specific antibodies binding to and being inhibited by citrulline-containing type I collagen telopeptides and by citrulline-containing type II collagen carboxyterminal telopeptides, respectively. The corresponding figures regarding homocitrulline-containing type I and homocitrulline-containing type II collagen telopeptides were 16 (22%) and 14 (19%). Most of the patients, who were seropositive for citrullinated peptides, showed binding in multiple assays. A total of 10 (14%) RA patients were positive for all the tested peptide pairs, while 28 (39%) of them had antibodies that contained overlapping specifities between citrulline and homocitrulline in the same peptide sequence.ConclusionsAntibodies to both citrulline and homocitrulline containing type I and II collagen telopeptides can be found in sera of RA patients. These antibodies are not constant from one RA patient to another, but contain separate or overlapping specificities within the same peptide sequence varying between individuals. Our results suggest some relationship between citrulline and homocitrulline-recognizing antibodies, since homocitrulline antibodies exist mainly in individuals seropositive to anti-CCP and anti-MCV.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0515-z) contains supplementary material, which is available to authorized users.     

Temperament clusters in a normal population: implications for health and disease

Background

The object of this study was to identify temperament patterns in the Finnish population, and to determine the relationship between these profiles and life habits, socioeconomic status, and health.

Methods/Principal Findings

A cluster analysis of the Temperament and Character Inventory subscales was performed on 3,761 individuals from the Northern Finland Birth Cohort 1966 and replicated on 2,097 individuals from the Cardiovascular Risk in Young Finns study. Clusters were formed using the k-means method and their relationship with 115 variables from the areas of life habits, socioeconomic status and health was examined.

Results

Four clusters were identified for both genders. Individuals from Cluster I are characterized by high persistence, low extravagance and disorderliness. They have healthy life habits, and lowest scores in most of the measures for psychiatric disorders. Cluster II individuals are characterized by low harm avoidance and high novelty seeking. They report the best physical capacity and highest level of income, but also high rate of divorce, smoking, and alcohol consumption. Individuals from Cluster III are not characterized by any extreme characteristic. Individuals from Cluster IV are characterized by high levels of harm avoidance, low levels of exploratory excitability and attachment, and score the lowest in most measures of health and well-being.

Conclusions

This study shows that the temperament subscales do not distribute randomly but have an endogenous structure, and that these patterns have strong associations to health, life events, and well-being.     

The development of a high-content screening binding assay for the smoothened receptor

In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-μM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.     

Effect of Glycosylation and Additional Domains on the Thermostability of a Family 10 Xylanase Produced by Thermopolyspora flexuosa

The effects of different structural features on the thermostability of Thermopolyspora flexuosa xylanase XYN10A were investigated. A C-terminal carbohydrate binding module had only a slight effect, whereas a polyhistidine tag increased the thermostability of XYN10A xylanase. In contrast, glycosylation at Asn26, located in an exposed loop, decreased the thermostability of the xylanase. The presence of a substrate increased stability mainly at low pH.The thermophilic actinomycete Thermopolyspora flexuosa, previously named Nonomuraea flexuosa and before that Actinomadura flexuosa or Microtetraspora flexuosa (15), produces family 11 and family 10 xylanases, which show high thermostability (16, 17, 22). T. flexuosa xylanase XYN10A has a C-terminal family 13 carbohydrate binding module (CBM) (22). Many xylanases have an additional CBM, which can be a cellulose binding domain (CBD) or a xylan binding domain (XBD) (1, 5, 7, 22, 25, 28). XBD typically increases activity against insoluble xylan (1, 5, 24), although some XBDs also bind soluble xylans (21, 25).We studied the thermostability of T. flexuosa xylanase XYN10A and how CBM and other additional groups affect its thermostability. In addition to confirming the previously described importance of terminal regions, our study identified a loop that is important for the thermostability of T. flexuosa XYN10A. In general, identification of sites important for protein stability is necessary for targeted mutagenesis attempts to increase thermostability.The T. flexuosa xyn10A gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AJ508953","term_id":"57897980","term_text":"AJ508953"}}AJ508953) (22), which encodes the full-length XYN10A xylanase (1-AAST… SYNA-448) containing the catalytic domain and CBM, and a truncated gene, which encodes the catalytic domain only (1-AAST… DALN-301) were expressed in Trichoderma reesei as 3′ fusions to a sequence that encodes the Cel6A CBD (A+B) carrier polypeptide and a Kex2 cleavage site (RDKR) (27). In this article, the catalytic domain and the full-length enzyme are referred to as XYN10A and XYN10A-CBM, respectively. The catalytic domain was also produced in Escherichia coli. For production in E. coli, the sequence encoding the catalytic domain was cloned into a pKKtac vector (33) with and without an additional 3′ sequence encoding a 6×His tag at the protein C terminus (… DALNHHHHHH).The proteins were purified by hydrophobic interaction chromatography using a Phenyl Sepharose column and by ion-exchange chromatography using a DEAE Sepharose FF column (Amersham Pharmacia Biotech). The 6×His-tagged XYN10A xylanase produced in E. coli was purified by affinity chromatography using Ni-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen).Mass spectrometric (MS) analyses were performed on a high-resolution 4.7-T hybrid quadrupole-Fourier transform ion cyclotron resonance (FT-ICR) instrument (APEX-Qe; Bruker Daltonics), which employs electrospray ionization (ESI) (see supplemental material for details).Xylanase activity was measured with a 3,5-dinitrosalicylic acid assay by using 1% solubilized birchwood xylan as a substrate (33). The optimum temperature, residual activity, and half-life assays were performed as described earlier (36). SWISS-MODEL (4) was used to automatically model T. flexuosa XYN10A and XYN10A-CBM (PDB codes for the modeling templates are 1v6w and 1e0w, respectively [12, 14]).The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the masses of XYN10A xylanase and XYN10A-CBM produced in Trichoderma reesei were ∼37 kDa and ∼50 kDa, respectively (Fig. ​(Fig.1A).1A). MS analysis of the 6×His-tagged XYN10A produced in E. coli (SDS-PAGE not shown) indicated the presence of a single protein form (Fig. ​(Fig.1B),1B), with a measured mass of 34,943.25 Da. This is consistent with the theoretical mass of 6×His-tagged XYN10A (34,942.93 Da). In contrast, XYN10A produced in T. reesei was heterogeneously modified, and six protein forms (numbered 1 to 6) were detected (Fig. ​(Fig.1B).1B). The mass of form 1 (34,120.76 Da) is in excellent agreement with the calculated mass of XYN10A (34120.73 Da). The masses of forms 2 and 3, with mass increments of ∼203 and ∼162 Da, respectively, suggested protein glycosylation (+203 Da = GlcNAc; +162 Da = Man). There are two potential sites for N-glycosylation in XYN10A, Asn26 and Asn95. These six protein forms were resolved only by the high-resolution FT-ICR MS technique, not by SDS-PAGE (eluted as a single band [Fig. ​[Fig.1A1A]).Open in a separate windowFIG. 1.(A) SDS-PAGE of purified XYN10A and XYN10A-CBM produced in Trichoderma reesei. Lane 1, molecular weight markers; lane 2, catalytic domain (XYN10A); lane 3, full-length enzyme (XYN10A-CBM). (B) ESI FT-ICR mass spectra of XYN10A with a 6×His tag produced in E. coli (bottom) and XYN10A produced in T. reesei (top). Only the expanded view at m/z 1260 to 1300, with the signals representing the most abundant protein ion charge state z = 27+, is presented. For the measured and calculated masses of the protein forms identified, see the supplemental material.In order to locate the glycosylation site or sites, XYN10A proteins produced in E. coli and T. reesei were subjected to on-line pepsin digestion (see supplemental material for details). The sequence coverage for XYN10A xylanase produced in E. coli was 62%. For XYN10A produced in T. reesei, a lower sequence coverage was obtained, but three glycopeptides (residues 20 to 44, 20 to 46, and 20 to 59), carrying one GlcNAc residue, were detected (glycopeptides A to C in Fig. S1B in the supplemental material). A triply charged glycopeptide A was further analyzed by collision-induced dissociation (CID) measurement (see inset in Fig. S1B in the supplemental material). A ladder of b-type fragment ions further identified this peptide and verified Asn26 as the N-glycosylation site in XYN10A, carrying GlcNAc(Man) as a glycan core structure.The additional sequences attached to the catalytic domain affected the thermostability of XYN10A xylanase. The deletion of the native C-terminal CBM domain (XYN10A produced in T. reesei) slightly decreased (∼2°C) the apparent temperature optimum in the region of 70 to 75°C (Table ​(Table11 and Fig. ​Fig.2A).2A). However, at 80°C, the deletion of the CBM domain increased the activity (Fig. ​(Fig.2A).2A). Furthermore, the half-life in the presence of the substrate at 80°C was lower when the CBM was present (Table ​(Table22).Open in a separate windowFIG. 2.Enzyme activity and stability profiles. (A) Enzyme activity as a function of temperature. The enzymes were incubated for 30 min at each temperature at pH 7. (B) Enzyme inactivation as a function of temperature. The enzyme samples were incubated without the substrate for 30 min at each temperature (pH 7), and the residual activity was measured at 70°C. Values are means ± standard deviations (error bars) for three experiments. Symbols: ⧫, XYN10A xylanase produced in T. reesei; ⋄, XYN10A-CBM produced in T. reesei; ▪, XYN10A produced in E. coli; □, XYN10A-6×His produced in E. coli.

TABLE 1.

Peaks of the optimum temperatures (30-min assay)^a

Identification of antigenic components of Toxoplasma gondii by an immunoblotting technique

Proteins of Toxoplasma gondii were separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to a nitrocellulose sheet by electrophoretic blotting. Immunologically reactive polypeptides were detected by human sera with previously known toxoplasma antibody levels. Heavy chain-specific, peroxidase-conjugated anti-human immunoglobulins were used as the indicator antibodies for the separate identification of IgG and IgM reactive polypeptides. IgG toxoplasma antibodies reacted with several antigens of M_r ≈27 000–67 000, while toxoplasma-specific IgM seemed to detect only a few polypeptides. The M_r of 35 000 for the dominating IgM reactive polypeptide was observed.     

Rôle of labelled dietary fatty acids and acetate in phospholipids during the metamorphosis of Pieris brassicae

The incorporation of labelled dietary palmitic, linoleic, and linolenic acids into neutral (NL) and phospholipids (PL) during the metamorphosis of Pieris brassicae was studied, and the ability of the fat body to incorporate acetate into PL determined. Thirty-three per cent of total lipid in early fifth instar larvae (minus haemolymph) is PL, while the corresponding value in female 4-day pupae is 13·0 per cent and in the fat body of 4-day pupae 6·3 per cent. Incorporation of label into PL was studied more closely and in all cases the label was recovered from phosphatidylcholine (PTC) and phosphatidylethanolamine (PTE). The label from palmitate was also found in sphingomyelin and possibly phosphatidylserine. Specific activity of PL in the case of palmitic and linolenic acids was greatest in late fifth instar larvae. In early fifth instar larvae on palmitic acid-1-¹⁴C 39·0 per cent of label was in PTC, 52·8 per cent in PTE, and 2·0 per cent in sphingomyelin. In late fifth instar 45·0 per cent was in PTC, 45·5 per cent in PTE, and 6·5 per cent in sphingomyelin, while in 4-day female pupae 45·2 per cent was in PTC, 41·3 per cent in PTE, and 13·5 per cent in sphingomyelin. The label from linolenic acid only varied a little from early fifth instar to 4-day pupae, 51·8 per cent being in PTC and 48·2 per cent in PTE in early fifth instar larvae. The label from linoleic acid is incorporated in fat body PL almost exclusively in PTC and PTE, 55·8 and 43·2 per cent respectively in 4-day female pupae. Injected acetate is distributed after 1 hr between PTC (58·6 per cent), PTE (24·4 per cent), and sphingomyelin (17·0 per cent). It was concluded that the polyunsaturated acids are proportionately more common in PTE than in other PL types, and that the fatty acids of sphingomyelin are mainly those that the insect is capable of synthesizing from acetate. Palmitic acid is desaturated by Pieris to palmitoleic acid and the latter possibly utilized in PTE to compensate for a deficiency of linolenic acid in the artificial diet. No saturation of linoleic or linolenic acid was found. The rates of PL and NL synthesis during development and the rôle of the investigated fatty acids in the biosynthesis of PL are discussed.     

Crocidolite asbestos causes an induction of p53 and apoptosis in cultured A-549 lung carcinoma cells

A number of genotoxic chemicals and agents, such as benzo(a)pyrene and ultraviolet light, are able to induce nuclear accumulation of p53 protein. Usually, this response is transient and a consequence of stabilization of the wild-type p53 protein. After withdrawal of the exposure, the amount of p53 protein returns to a normal level within hours or a few days. We have studied the p53 response to the exposure of crocidolite asbestos in A-549 lung carcinoma cells using three different methods, i.e., p53 immunohistochemistry, Western blotting and metabolic labelling followed by p53 immunoprecipitation. With these techniques we demonstrate a dose-dependent p53 nuclear response to crocidolite exposure. The half-life of p53 protein in A-549 lung carcinoma cells cultured in serum-free media increased from 30 up to 80 min, and the protein reacted with a wild-type specific antibody suggesting that it was in a wild-type conformation. In situ 3′-end labelling of A-549 cells demonstrated a dose-dependent increase in apoptotic activity. Our data support the idea that increased apoptotic activity, induced by crocidolite, is mediated by p53. This revised version was published online in June 2006 with corrections to the Cover Date.