Autoantibodies against protein disulfide isomerase ER-60 are a diagnostic marker for low-grade testicular inflammation.

STUDY QUESTION: Is there a non-invasive biomarker for the diagnosis of testicular inflammatory lesions? SUMMARY ANSWER: In sera from infertile azoospermic patients with histologically confirmed low-grade testicular inflammation, significantly elevated titers of autoantibodies against disulfide isomerase family A, member 3 (ER-60) were found. WHAT IS KNOWN ALREADY: Infection and inflammation of the genital tract are supposed to be responsible for up to 15% of cases among infertile males. However, specific seminal or serological markers are not available to assess subacute or chronic inflammatory conditions in the testis. STUDY DESIGN, SIZE, DURATION: This study consisted of the identification of autoantibodies for testicular antigens in sera of patients with low-grade testicular inflammation, validation of candidates, development of an ELISA for the most promising target antigen and measurement of autoantibodies titers in healthy normozoospermic men (n = 20); male blood donors (n = 14); men with impaired semen quality without (n = 14) or with (n = 26) symptoms of genital tract infection/inflammation; azoospermic men with histologically confirmed testicular inflammatory lesions (n = 16); men after pharmacotherapy of genital tract infection/inflammation (n = 15) and men with acute epididymo-orchitis (n = 30). PARTICIPANTS/MATERIALS, SETTING, METHODS: Proteins in lysates of normal testicular tissue were separated by high-resolution 2D gel electrophoresis and probed with sera of 13 patients with histologically confirmed chronic testicular inflammation. There were 14 proteins that immunoreacted with a majority of these sera and could be identified by mass spectrometry. Of these 14 proteins, disulfide isomerase family A, member 3 (ER-60), transferrin and chaperonin containing TCP1 complex, subunit 5 (epsilon) (CCT5) were considered as specific. Since ER-60 reacted with 92% of patient sera, an ER-60-autoantibody ELISA was developed. MAIN RESULTS AND THE ROLE OF CHANCE: The newly established ELISA detected significantly elevated titers of autoantibodies against ER-60 in the sera from infertile men with histologically confirmed chronic testicular inflammation (median 8.6; P < 0.01) compared with the control groups. Moreover, elevated levels of anti-ER-60 titers were detected in patients suffering from acute epididymo-orchitis (median 3.3; P < 0.05) as compared with healthy normozoospermic men (median 2.13; P < 0.001), male blood donors with unknown fertility status (median 2.72; P < 0.01), patients with impaired semen quality but no infection/inflammation (median 2.59; P < 0.001) and patients with symptoms of genital tract infections and/or inflammation (median 2.18; P < 0.001). Significantly lower levels of anti-ER-60 antibodies were measured in sera from patients after application of anti-inflammatory pharmacotherapy (median 1.9; P < 0.01) compared with those with histologically confirmed chronic testicular inflammation. The cut-off value of the assay was set to 6.6 U/ml based on a calculated sensitivity of 100% and a specificity of 81.2%. LIMITATIONS, REASONS FOR CAUTION: The results obtained in this study showed statistically significant elevated titers of ER-60 antibodies in sera from patients with histologically confirmed testicular inflammatory lesions and from a few patients with acute epididymo-orchitis. However, the number of serum samples tested was limited. Severe testicular damage seen in azoospermic patients could represent a bias towards ER-60 reactivity, while the assay does not allow for different etiologies of the lesions to be distinguished. Due to ethical reasons, the prevalence of testicular inflammatory lesions among controls and non-azoospermic men cannot be studied at the histological level. WIDER IMPLICATIONS OF THE FINDINGS: Measurement of ER-60 autoantibody titers in serum could be a novel non-invasive marker for the diagnosis of asymptomatic testicular inflammation causing male fertility disturbances. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a grant of the Deutsche Forschungsgemeinschaft (ME 1323/4-4) and the Translational Science Fund (Wirtschafts-und Strukturbank Hessen-WI Bank). M.F., A.P., W.W., H.-C.S. and A.M. are supported by the LOEWE focus group 'MIBIE' (Male infertility during infection and inflammation). The ER-60 ELISA is protected by a patent to the Justus-Liebig-University of Giessen with A.M. and M.F. as inventors (patent no. DE 10 2008 053 503). T.Z. as employee of the DRG Company was responsible for the ELISA development.

Eimeria bovis-induced modulation of the host cell proteome at the meront I stage.

The proteome of Eimeria bovis meront I-carrying host cells was analyzed by two-dimensional gel electrophoresis (2DE) at 14 days p.i. and compared to non-infected control cells. A total of 221 protein spots were modulated in their abundance in E. bovis-infected host cells and were subsequently analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectometry (MALDI-TOF-MS). These analyses identified 104 proteins in total with 25 host cell proteins being up-regulated and 79 proteins being down-regulated in E. bovis-infected host cells. Moreover, 20 newly expressed proteins were identified exclusively in E. bovis-infected host cells and were most likely of parasite origin. Parasite-induced differences in protein abundance concerned distinct functional categories, with most proteins being involved in host cell metabolism, cell structure, protein fate and gene transcription. Some of the modulated molecules also indicated regulatory processes on the level of host cell stress response (HSP70, HSP90), host cell apoptosis (caspase 8) and actin elongation/depolymerization (α-actinin-1, gelsonin, tropomodulin-3, transgelin). Since merozoites I were already released shortly after cell sampling, the current data reflect the situation at the end of first merogony. This is the first proteomic approach on E. bovis-infected host cells that was undertaken to gain a rather broad insight into Eimeria-induced host cell modulation. The data processed in this investigation should provide a useful basis for more detailed analyses concerning Eimeria-host cell interactions.

A 5'-fluorosulfonylbenzoyladenosine-based method to identify physiological substrates of a Drosophila p21-activated kinase.

Nearly all processes in cells are regulated by the coordinated interplay between reversible protein phosphorylation and dephosphorylation. Therefore, it is a great challenge to identify all phosphorylation substrates of a single protein kinase to understand its integration into intracellular signaling networks. In this work, we developed an assay that holds promise as being useful for the identification of phosphorylation substrates of a given protein kinase of interest. The method relies on irreversible inhibition of endogenous kinase activities with the ATP analogue 5'-fluorosulfonylbenzoyladenosine (5'FSBA). 5'FSBA-treated cell extracts are then combined with a purified activated kinase to allow phosphorylation of putative substrate proteins, followed by a two-step purification protocol and identification by fingerprint mass spectrometry. Specifically, we applied this method to identify new phosphorylation substrates of the Drosophila p21-activated kinase (PAK) protein Mbt. Among candidate proteins identified by mass spectrometry, the dynactin complex subunit dynamitin was verified as a bona fide Mbt phosphorylation substrate and interaction partner, suggesting an involvement of this PAK protein in the regulation of dynactin-dependent cellular processes.

Singlet oxygen affects the activity of the thylakoid ATP synthase and has a strong impact on its gamma subunit.

Singlet oxygen is reported to have the most potent damaging effect upon the photosynthetic machinery. Usually this reactive oxygen molecule acts in concert with other ROS types under stressful conditions. To understand the specific role of singlet oxygen we took advantage of the conditional flu mutant of Arabidopsis thaliana. In flu, the negative feedback loop is abolished, which blocks chlorophyll biosynthesis in the dark. Therefore high amounts of free protochlorophyllide accumulate during darkness. If flu gets subsequently illuminated, free protochlorophyllide acts as a photosensitiser leading almost exclusively to high amounts of (1)O2. Analysing the thylakoid protein pattern by using 2D PAGE and subsequent MALDI-TOF analysis, we could show, in addition to previous described effects on photosystem II, that singlet oxygen has a massive impact on the thylakoid ATP synthase, especially on its gamma subunit. Additionally, it could be shown that the activity of the ATP synthase is reduced upon singlet oxygen exposure and that the rate of non-photochemical quenching is affected in flu mutants exposed to (1)O2.

Microbial metalloproteinases mediate sensing of invading pathogens and activate innate immune responses in the lepidopteran model host Galleria mellonella.

Thermolysin-like metalloproteinases such as aureolysin, pseudolysin, and bacillolysin represent virulence factors of diverse bacterial pathogens. Recently, we discovered that injection of thermolysin into larvae of the greater wax moth, Galleria mellonella, mediated strong immune responses. Thermolysin-mediated proteolysis of hemolymph proteins yielded a variety of small-sized (<3 kDa) protein fragments (protfrags) that are potent elicitors of innate immune responses. In this study, we report the activation of a serine proteinase cascade by thermolysin, as described for bacterial lipopolysaccharides (LPS), that results in subsequent prophenoloxidase activation leading to melanization, an elementary immune defense reaction of insects. Quantitative real-time reverse transcription-PCR analyses of the expression of immune-related genes encoding the inducible metalloproteinase inhibitor, gallerimycin, and lysozyme demonstrated increased transcriptional rates after challenge with purified protfrags similar to rates after challenge with LPS. Additionally, we determined the induction of a similar spectrum of immune-responsive proteins that were secreted into the hemolymph by using comparative proteomic analyses of hemolymph proteins from untreated larvae and from larvae that were challenged with either protfrags or LPS. Since G. mellonella was recently established as a valuable pathogenicity model for Cryptococcus neoformans infection, the present results add to our understanding of the mechanisms of immune responses in G. mellonella. The obtained results support the proposed danger model, which suggests that the immune system senses endogenous alarm signals during infection besides recognition of microbial pattern molecules.

Evidence for an RNA chaperone function of polypyrimidine tract-binding protein in picornavirus translation.

The cellular polypyrimidine tract-binding protein (PTB) is recruited by the genomic RNAs of picornaviruses to stimulate translation initiation at their internal ribosome entry site (IRES) elements. We investigated the contribution of the individual RNA recognition motif (RRM) domains of PTB to its interaction with the IRES of foot-and-mouth disease virus (FMDV). Using a native gel system, we found that PTB is a monomer, confirming recent reports that challenged the previous view that PTB is a dimer. Mapping the spatial orientation of PTB relative to the bound IRES RNA, we found that the two C-terminal RRM domains III and IV of PTB bind in an oriented way to the IRES. Domain III contacts the IRES stem-loop 2, while domain IV contacts the separate IRES 3' region. PTB domain I appears not to be involved directly in RNA binding, but domain II stabilizes the RNA binding conferred by domains III and IV. A PTB protein containing only these two C-terminal PTB domains is sufficient to enhance the entry of initiation factor eIF4G to the IRES and stimulate IRES activity, and the long-lived PTB-IRES interaction stabilized by domain II is not a prerequisite for this function. Thus, PTB most likely acts as an RNA chaperone to stabilize IRES structure and, in that way, augment IRES activity.

Molecular comparison of apocrine released and cytoplasmic resident carbonic anhydrase II.

We have previously shown that carbonic anhydrase II usually described as a cytoplasmic resident isoform (cCAH II) is secreted by the rat coagulating gland (sCAH II) via the apocrine secretion mode. To get more detailed information why CAH II is cytoplasmic resident in some organs and secreted in others we cloned and sequenced the cDNA of rat coagulating gland sCAH II. The sequence of the secretory form was found to be completely identical with the cCAH II. Therefore, a signal peptide targeting sCAH II for apocrine secretion can be excluded. Considering the fact that other apocrine secreted proteins are glycosylated, cCAH II and sCAH II were analyzed for carbohydrate substitutions. As expected for a cytoplasmic protein, no glycan modification could be identified in cCAH II. In contrast, sCAH II carried exclusively Gal, GlcNAc and Fuc residues in a molar ratio of 1:0.8:0.5. Carbohydrate linkage analyses demonstrated the presence of terminal Fuc, terminal, 3-substituted and 3,6-disubstituted Gal as well as 4-substituted and 3,4-disubstituted GlcNAc. The composition of the glycan constituents as well as deglycosylation experiments clearly proved that sCAH II carries neither conventional mammalian-type N-glycans nor mucin-type O-linked sugar chains. Lacking a signal peptide for ER translocation, glycosylation of sCAH II must occur within the cytoplasmic compartment. Further studies have to elucidate whether or not glycosylation of sCAH II is essential for the apocrine release of the protein.

Binding of urokinase plasminogen activator to gp130 via a putative urokinase-binding consensus sequence.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are instrumental in cellular activities during inflammation, angiogenesis and tumor metastasis. Recent studies suggest that uPA might exert its function on cell proliferation and migration in a uPAR-independent manner or through an adaptor to the uPA-uPAR system. By applying phage display technology, we have identified a putative uPA-binding consensus sequence BXXSSXXB (where B represents a basic amino acid and X represents any amino acid), which has no apparent sequence correlation to uPAR. This uPA-binding motif apparently recognizes the kringle domain of the protease and has an agonistic effect on uPA binding to immobilized uPAR, thereby possibly serving as part of an adaptor component for uPAR signaling. As a result of protein database searches, this motif was found in the extracellular domain of several cell surface proteins, some of which were proposed to be associated with the uPA-uPAR system. Among these, gp130, a common signal transducer for cytokines, was identified as a uPA-binding protein. The specificity of this interaction was demonstrated by inhibition of uPA binding to immobilized gp130 with soluble gp130. Furthermore, the binding could be partially inhibited by a uPA-binding consensus sequence-containing fusion protein in a dose-dependent manner, with an IC50 of approximately 1 microM, indicating that the uPA-binding motif is apparently involved in the uPA-gp130 interaction. The association of gp130 with uPA may link the uPA-uPAR system to various signal transduction pathways.

The junctional adhesion molecule 3 (JAM-3) on human platelets is a counterreceptor for the leukocyte integrin Mac-1.

The recently described junctional adhesion molecules (JAMs) in man and mice are involved in homotypic and heterotypic intercellular interactions. Here, a third member of this family, human JAM-3, was identified and described as a novel counterreceptor on platelets for the leukocyte beta2-integrin Mac-1 (alphaMbeta2, CD11b/CD18). With the help of two monoclonal antibodies, Gi11 and Gi13, against a 43-kD surface glycoprotein on human platelets, a full-length cDNA encoding JAM-3 was identified. JAM-3 is a type I transmembrane glycoprotein containing two Ig-like domains. Although JAM-3 did not undergo homophilic interactions, myelo-monocytic cells adhered to immobilized JAM-3 or to JAM-3-transfected cells. This heterophilic interaction was specifically attributed to a direct interaction of JAM-3 with the beta2-integrin Mac-1 and to a lower extent with p150.95 (alphaXbeta2, CD11c/CD18) but not with LFA-1 (alphaLbeta2, CD11a/CD18) or with beta1-integrins. These results were corroborated by analysis of K562 erythroleukemic cells transfected with different heterodimeric beta2-integrins and by using purified proteins. Moreover, purified JAM-3 or antibodies against JAM-3 blocked the platelet-neutrophil interaction, indicating that platelet JAM-3 serves as a counterreceptor for Mac-1 mediating leukocyte-platelet interactions. JAM-3 thereby provides a novel molecular target for antagonizing interactions between vascular cells that promote inflammatory vascular pathologies such as in atherothrombosis.