Abnormal collagen fibrils in cartilage of matrilin-1/matrilin-3-deficient mice

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1(-/-)/Matn3(-/-) and Matn3(-/-) mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.

A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin

In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin-Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin-Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unknown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively.

Pseudovacuoles--immobilized by high-pressure freezing--are associated with blebbing in walker carcinosarcoma cells

By applying high pressure freezing and freeze-substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2-5 mum in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine-rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high-pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron-transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS-PAGE of lysine-labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium-specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.

Activation of cyclic AMP response element-binding protein (CREB) in aggressive periodontal disease

OBJECTIVES: To report a novel observation of neutrophil signal transduction abnormalities in patients with localized aggressive periodontitis (LAP) that are associated with an enhanced phosphorylation of the nuclear signal transduction protein cyclic AMP response element-binding factor (CREB). METHOD AND MATERIALS: Peripheral venous blood neutrophils of 18 subjects, 9 patients with LAP and 9 race-, sex-, and age-matched healthy controls, were isolated and prepared using the Ficoll-Hypaque density-gradient technique. Neutrophils (5.4 x 10(6)/mL) were stimulated with the chemoattractant FMLP (10(-6) mol/L) for 5 minutes and lysed. Aliquots of these samples were separated by SDS-PAGE (60 microg/lane) on 9.0% (w/v) polyacrylamide slab gels and transferred electrophoretically to polyvinyl difluoride membranes. The cell lysates were immunoblotted with a 1:1,000 dilution of rabbit-phospho-CREB antibody that recognizes only the phosphorylated form of CREB at Ser133. The activated CREB was visualized with a luminol-enhanced chemoluminescence detection system and evaluated by laser densitometry. RESULTS: In patients with LAP, the average activation of CREB displayed an overexpression for the unstimulated peripheral blood neutrophils of 80.3% (17.5-fold) compared to healthy controls (4.6%). CONCLUSION: LAP neutrophils who express their phenotype appear to be constitutively primed, as evidenced by activated CREB in resting cells compared to normal individuals. The genetically primed neutrophil phenotype may contribute to neutrophil-mediated tissue damage in the pathogenesis of LAP.

Protein changes and proteolytic degradation in red and white clover plants subjected to waterlogging

Red (Trifolium pratense L., cv. “Start”) and white clover varieties (Trifolium repens L., cv. “Debut” and cv. “Haifa”) were waterlogged for 14 days and subsequently recovered for the period of 21 days. Physiological and biochemical responses of the clover varieties were distinctive, which suggested different sensitivity toward flooding. The comparative study of morphological and biochemical parameters such as stem length, leaflet area, dry weight, protein content, protein pattern and proteolytic degradation revealed prominent changes under waterlogging conditions. Protease activity in the stressed plants increased significantly, especially in red clover cv. “Start”, which exhibited eightfold higher azocaseinolytic activity compared to the control. Changes in the protein profiles were detected by SDS-PAGE electrophoresis. The specific response of some proteins (Rubisco, Rubisco-binding protein, Rubisco activase, ClpA and ClpP protease subunits) toward the applied stress was assessed by immunoblotting. The results characterized the red clover cultivar “Start” as the most sensitive toward waterlogging, expressing reduced levels of Rubisco large and small subunits, high content of ClpP protease subunits and increased activity of protease isoforms.

Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp. mycoides small colony type.

With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II-membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the 'mycoides cluster' or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the "mycoides cluster'.

Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects

The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.

Demonstration of species-specific and cross-reactive components of Taenia solium metacestode antigens.

Analysis of human serum reactivities to antigenic components of soluble Taenia solium metacestode proteins showed the predominant presence of determinants shared by T. solium, Echinococcus multilocularis and E. granulosus. Two polypeptides were demonstrated by SDS-PAGE and Western blot or enzyme-linked immunoelectrotransfer blot (EITB) assay to bind serum and CSF antibodies only from T. solium cysticercosis patients. These species-specific antigenic polypeptides focused between pH 4.6 and 3.9 after resolution by isoelectric focusing followed by EITB. The high species-specificity demonstrated by the present techniques offers the opportunity to confirm serologically an infection by T. solium metacestode.

A collaborative study on larval excretory/secretory antigens of Toxocara canis for the immunodiagnosis of human toxocariasis with ELISA.

Two batches of excretory/secretory (E/S) antigens from second stage larvae of Toxocara canis maintained in vitro were prepared independently in two different laboratories (Zürich and Basel) and analysed in order to obtain information for future efforts to standardize the enzyme-linked immunosorbent assay (ELISA) used for the serodiagnosis of human toxocariasis. SDS-PAGE and "Western-blotting" revealed at least 10 different antigenic components common to the two antigen preparations. However, distinct qualitative and quantitative differences among the two E/S-antigens were observed, since one antigen had a more complex composition than the other. Despite these differences, an accordance of serodiagnosis was obtained in 80% of 25 sera from patients with suspected Toxocara infection tested independently in two different ELISA systems (Basel and Zürich) with the corresponding E/S-antigens. The specificity was 93% as determined (BS-antigen, BS-ELISA) by testing 46 out of 3396 sera from patients with parasitologically proven extra-intestinal helminthic infections. Cross-reactions occurred mainly with sera from patients infected with filariae (5 from 13 cases) exhibiting very high extinction values in their homologous ELISA-system. The reproducibility (intra- and inter-test variations) of two ELISA systems using the corresponding E/S-antigens varied from 5-15%. The results demonstrate that T. canis E/S-antigens may well be applicable for standardization of the ELISA used for the serodiagnosis of human toxocariasis.

Purification and characterization of a specific antigen from Echinococcus multilocularis.

A polypeptide (Em2a) purified by affinity chromatography from the Echinococcus multilocularis metacestode showed a high degree of purity as assayed by SDS-PAGE and analytical isoelectrical focusing. A minor contamination with host albumin was revealed. Estimation of relative mol. mass gave a value of 54,000. The isoelectric point was found to be 4.8. Antigenic activity of the polypeptide was demonstrated by immunoprecipitation and western blotting. In these assays the protein was recognized only by homologous sera from patients infected with larval E. multilocularis. This antigen (Em2a) did not react in the ELISA with sera from patients infected with heterologous helminths; these sera were highly cross-reacting with antigen from E. granulosus hydatid fluid. Seventy-three (94%) from 78 investigated patients (alveolar echinococcosis) showed a seropositive reaction with the polypeptide Em2a.

MASP-1 of the complement system promotes clotting via prothrombin activation.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity, and it has been shown to activate coagulation factors. Here we studied the effects of MASP-1 on clot formation in whole blood (WB) and platelet-poor plasma (PPP) by thrombelastography and further elucidated the underlying mechanism. Cleavage of prothrombin by MASP-1 was investigated by SDS-PAGE and N-terminal sequencing of cleavage products. Addition of MASP-1 or thrombin to WB and PPP shortened the clotting time and clot formation time significantly compared to recalcified-only samples. The combination of MASP-1 and thrombin had additive effects. In a purified system, MASP-1 was able to induce clotting only in presence of prothrombin. Analysis of MASP-1-digested prothrombin confirmed that MASP-1 cleaves prothrombin at three cleavage sites. In conclusion, we have shown that MASP-1 is able to induce and promote clot formation measured in a global setting using the technique of thrombelastography. We further confirmed that MASP-1-induced clotting is dependent on prothrombin. Finally, we have demonstrated that MASP-1 cleaves prothrombin and identified its cleavage sites, suggesting that MASP-1 gives rise to an alternative active form of thrombin by cleaving at the cleavage site R393.

Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.

Effect of mannitol and cold treatments on phosphate uptake and protein phosphorylation in Lemna minor (L.) plants

This report is aimed at elucidating the effect of mannitol and cold treatments on P uptake and protein phosphorylation in Lemna minor plants. Duckweed p lants were incu bated in the presence of [32P]or [33P]Pi in half-strength phosphate deprived E-medium under constant light regime for 1.5 h. Total plant protein extracts (pellet and supernatant) were then prepared and subjected to IEF x SDS-PAGE. To analyse the effect of the stresses on P uptake and protein labelling, Lemna minor plants were preincubated with 0.1, 0.5 mol · L-1 mannitol and at 4°C respectively, for 4 hours, before adding labelled orthophosphate. The results show that the general protein phosphorylation (including LHCII) is related to the level of P uptake. Radioactive phosphate incorporation is stimulated by a low concentration of mannitol (0.1 mol · L-1) but reduced by 0.5 mol · L-1 mannitol and cold stress in planta. The labelling into proteins is affected neither when stresses were applied to the plants after incubation with labelled orthophosphate, nor after in vitro protein phosphorylation. This indicates that general protein kinase activities in vivo are strictly limited by P uptake. A marked accumulation of soluble hexoses (mainly sucrose, glucose, and fructose) is observed under imposed stress, suggesting that the inhibition of P uptake in response to hyperosmotic and cold stresses is mediated by sugar accumulation in situ. However, metabolisable sugars like glucose did not alter the entry of phosphate at concentrations of 0.5 mol · L-1, showing that the chemical nature of the osmoticum influences P uptake.

CO2, light and temperature influence senescence and protein degradation in wheat leaf segments

Effects of environmental conditions influencing photosynthesis and photorespiration on senescence and net protein degradation were investigated in segments from the first leaf of young wheat (Triticum aestivum L. cv. Arina) plants. The segments were floated on H2O at 25, 30 or 35°C in continuous light (PAR: 50 or 150 µmol m−2 s−1) in ambient air and in CO2-depleted air. Stromal enzymes, including phosphoglycolate phosphatase, glutamine synthetase, ferredoxin-dependent glutamate synthase, phosphoribulokinase, and the peroxisomal enzyme, glycolate oxidase, were detected by SDS-PAGE followed by immunoblotting with specific antibodies. In general, the net degradation of proteins and chlorophylls was delayed in CO2-depleted air. However, little effect of CO2 on protein degradation was observed at 25°C under the lower level of irradiance. The senescence retardation by the removal of CO2 was most pronounced at 30°C and at the higher irradiance. The stromal enzymes declined in a coordinated manner. Immunoreactive fragments from the degraded polypeptides were in most cases not detectable. However, an insolubilized fragment of glycolate oxidase accumulated in vivo, especially at 25°C in the presence of CO2. Detection of this fragment was minimal after incubation at 30°C and completely absent on blots from segments kept at 35°C. In CO2-depleted air, the fragment was only weakly detectable after incubation at 25°C. The results from these investigations indicate that environmental conditions that influence photosynthesis may interfere with senescence and protein catabolism in wheat leaves.

MASP-1 Induced Clotting--The First Model of Prothrombin Activation by MASP-1.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R) 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous pathways, either cleaving at R271 or R393 first. Both pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.