Versican processing by a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats proteinases-5 and -15 facilitates myoblast fusion

Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy.

Quick and low cost immobilization of proteinases on polyesters: comparison of lactobacilli cell-envelope proteinase and trypsin for protein degradation

Cell-envelope proteinases (CEPs) are a class of proteolytic enzymes produced by lactic acid bacteria and have several industrially relevant applications. However, soluble CEPs are economically unfavorable for such applications due to their poor stability and lack of reusability. In a quest to prepare stable biocatalysts with improved performance, CEP from Lactobacillus delbrueckii subsp. lactis 313 and trypsin (as a model enzyme) were immobilized onto nonwoven polyester fabrics in a three-step protocol including ethylenediamine activation and glutaraldehyde crosslinking. Immobilization gave protein loading yields of 21.9% (CEP) and 67.7% (trypsin) while residual activity yields were 85.6% (CEP) and 4.1% (trypsin). The activity of the immobilized enzymes was dependent on pH, but was retained at elevated temperatures (40-70°C). An increase in Km values was observed for both enzymes after immobilization. After 70 days of storage, the immobilized CEP retained ca. 62% and 96% of initial activity when the samples were stored in a lyophilized form at -20°C or in a buffer at 4°C, respectively. Both immobilized CEP and trypsin were able to hydrolyze proteins such as casein, skimmed milk proteins and bovine serum albumin. This immobilization protocol can be used to prepare immobilized biocatalyst for various protein degradation processes.

Clozapine reverses schizophrenia-related behaviours in the metabotropic glutamate receptor 5 knockout mouse: association with N-methyl-d-aspartic acid receptor up-regulation

Abnormalities in glutamatergic signalling are proposed in schizophrenia in light of the schizophreniform psychosis elicited by NMDA antagonists. The metabotropic glutamate receptor 5 (mGluR5) interacts closely with the NMDA receptor and is implicated in several behavioural endophenotypes of schizophrenia. We have demonstrated that mice lacking mGluR5 have increased sensitivity to the hyperlocomotive effects of the NMDA antagonist MK-801. Mice lacking mGluR5 also show abnormal locomotor patterns, reduced prepulse inhibition (PPI), and deficits on performance of a short-term spatial memory task on the Y-maze. Chronic administration of the antipsychotic drug clozapine ameliorated the locomotor disruption and reversed the PPI deficit, but did not improve Y-maze performance. Chronic clozapine increased NMDA receptor binding ([3H]MK-801) but did not alter dopamine D2 ([3H]YM-09151), 5-HT2A ([3H]ketanserin), or muscarinic M1/M4 receptor ([3H]pirenzepine), binding in these mice. These results demonstrate behavioural abnormalities that are relevant to schizophrenia in the mGluR5 knockout mouse and a reversal of behaviours with clozapine treatment. These results highlight both the interactions between mGluR5 and NMDA receptors in the determination of schizophreniform behaviours and the potential for the effects of clozapine to be mediated by NMDA receptor regulation.
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Evaluation of cross-linked enzyme aggregates of Lactobacillus cell-envelope proteinases, for protein degradation

Enzymatic hydrolysis is a widely used approach to improve the functional, nutritionaland physiological properties of food proteins. In this study, cross-linked enzyme aggre-gates (CLEAs) have been prepared from cell-envelope proteinases (CEPs) of Lactobacillusdelbrueckii subsp. lactis 313 and their proteolytic properties have been evaluated using severalfood proteins. We have optimized cross-linking conditions including ammonium sulphateconcentration, incubation temperatures, agitation speed, glutaraldehyde cross-linker con-centration, reaction time and the addition of proteic feeders. Particularly, the presence ofBSA improves retained activity of cross-linked CEP aggregates (CLCEPAs) from 21.5% to 40.9%.Blocking unreacted cross-linking groups on aggregates is important to enhance recyclabil-ity of CLCEPAs. CLCEPAs had attractive thermal stability at 50◦C and it showed enhancedcatalytic activity over long-term storage after lyophilization. We have demonstrated thatCLCEPAs has proteolytic properties on different food proteins including complex (chickenegg albumin, skimmed-milk protein), fractionated (bovine casein, whey protein isolate), andpurified (bovine serum albumin) proteins. Being the first report of CLEAs from lactobacilliCEPs, this study demonstrates the feasibility of using LDL 313 CLCEPAs for degradation ofvarious food proteins.

Food utilisation and digestive ability of aquatic and semi-terrestrial crayfishes, Cherax destructor and Engaeus sericatus (Astacidae, Parastacidae)

Both Engaeus sericatus and Cherax destructor are omnivorous crayfishes consuming a variety of food items. Materials identified in the faeces of both E. sericatus and C. destructor consisted of mainly plant material with minor amounts of arthropod animals, algae and fungi. The morphology of the gastric mill of C. destructor suggests that it is mainly involved in crushing of food material while the gastric mill of E. sericatus appears to be better suited to cutting of food material. Given this, the gastric mill of E. sericatus may be better able to cut the cellulose and hemicellulose fibres associated with fibrous plant material. In contrast, the gastric mill of C. destructor appears to be more efficient in grinding soft materials such as animal protein and algae. Both species accumulated high amounts of lipids in their midgut glands (about 60% of the dry mass) which were dominated by triacylglycerols (81–82% of total lipids). The dominating fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:2(n-6), and 18:3(n-3). The two latter fatty acids can only be synthesised by plants, and are thus indicative of the consumption of terrestrial plants by the crayfishes. The similarity analysis of the fatty acid patterns showed three distinct clusters of plants and each of the crayfish species. The complement of digestive enzymes, proteinases, total cellulase, endo-β-1,4-glucanase, β-glucosidase, laminarinase and xylanase within midgut gland suggests that both C. destructor and E. sericatus are capable of hydrolysing a variety of substrates associated with an omnivorous diet. Higher activities of total cellulase, endo-β-1,4-glucanase and β-glucosidase indicate that E. sericatus is better able to hydrolyse cellulose within plant material than C. destructor. In contrast to E. sericatus, higher total protease and N-acetyl-β-d-glucosaminidase activity in the midgut gland of C. destructor suggests that this species is better able to digest animal materials in the form of arthropods. Differences in total cellulase and gastric mill morphology suggest that E. sericatus is more efficient at digesting plant material than C. destructor. However, the contents of faecal pellets and the fatty acid compositions seem to indicate that both species opportunistically feed on the most abundant and easily accessible food items.

New insights into protein export in malaria parasites

In order to survive and promote its virulence the malaria parasite must export hundreds of its proteins beyond an encasing vacuole and membrane into the host red blood cell. In the last few years, several major advances have been made that have significantly contributed to our understanding of this export process. These include: (i) the identification of sequences that direct protein export (a signal sequence and a motif termed PEXEL), which have allowed predictions of the exportomes of Plasmodium species that are the cause of malaria, (ii) the recognition that the fate of proteins destined for export is already decided within the parasite's endoplasmic reticulum and involves the PEXEL motif being recognized and cleaved by the aspartic protease plasmepsin V and (iii) the discovery of the Plasmodium translocon of exported proteins (PTEX) that is responsible for the passage of proteins across the vacuolar membrane. We review protein export in Plasmodium and these latest developments in the field that have now provided a new platform from which trafficking of malaria proteins can be dissected.

An aspartyl protease directs malaria effector proteins to the host cell

Plasmodium falciparum causes the virulent form of malaria and disease manifestations are linked to growth inside infected erythrocytes. To survive and evade host responses the parasite remodels the erythrocyte by exporting several hundred effector proteins beyond the surrounding parasitophorous vacuole membrane. A feature of exported proteins is a pentameric motif (RxLxE/Q/D) that is a substrate for an unknown protease. Here we show that the protein responsible for cleavage of this motif is plasmepsin V (PMV), an aspartic acid protease located in the endoplasmic reticulum. PMV cleavage reveals the export signal (xE/Q/D) at the amino terminus of cargo proteins. Expression of an identical mature protein with xQ at the N terminus generated by signal peptidase was not exported, demonstrating that PMV activity is essential and linked with other key export events. Identification of the protease responsible for export into erythrocytes provides a novel target for therapeutic intervention against this devastating disease.

Redox voltammetry of sub-parts per billion levels of Cu2+ at polyaspartate-modified gold electrodes

An electrochemical sensor for the detection of Cu2+ is reported which incorporates poly-l-aspartic acid (PLAsp) with 32–96 aspartate units as a selective ligand for the metal ion. PLAsp is covalently attached to a gold electrode modified with a monolayer of 3-mercaptopropionic acid using carbodiimide coupling via an N-hydroxysuccinimide (NHS) ester intermediate. The acid side groups and deprotonated peptide nitrogens on two aspartate moieties are thought to be primarily responsible for chelation of Cu2+, which remains bound when reduced to Cu+. A consequence of the multiple binding points that are available with a polypeptide is the low detection limit. The lowest concentration detected was 3 nM (0.2 ppb) achieved with Osteryoung square wave voltammetry. This detection limit compares favourably with that of ICP-OES and previously reported cysteine-modified electrodes. Analysis of tap and lake water samples using the PLAsp-modified electrode agreed well with ICP-OES analysis of the same samples.

Electrochemical metal ion sensors. Exploiting amino acids and peptides as recognition elements

Amino acids and peptides are known to bind metal ions, in some cases very strongly. There are only a few examples of exploiting this binding in sensors. The review covers the current literature on the interaction of peptides and metals and the electrochemistry of bound metal ions. Peptides may be covalently attached to surfaces. Of particular interest is the attachment to gold via sulfur linkages. Sulfur-containing peptides (eg cysteine) may be adsorbed directly, while any amino group can be covalently attached to a carboxylic acid-terminated thiol. Once at a surface, the possibility for using the attached peptide as a sensor for metal ions becomes realised. Results from the authors’ laboratory and elsewhere have shown the potential for selective monitoring of metal ions at ppt levels. Examples of the use of poly-aspartic acid and the copper binding peptide Gly-Gly-His for detecting copper ions are given.

Binding affinities of amino acid analogues at the charged aqueous titania interface : implications for titania-binding peptides

Despite the extensive utilization of biomolecule-titania interfaces, biomolecular recognition and interactions at the aqueous titania interface remain far from being fully understood. Here, atomistic molecular dynamics simulations, in partnership with metadynamics, are used to calculate the free energy of adsorption of different amino acid side chain analogues at the negatively-charged aqueous rutile TiO2 (110) interface, under conditions corresponding with neutral pH. Our calculations predict that charged amino acid analogues have a relatively high affinity to the titania surface, with the arginine analogue predicted to be the strongest binder. Interactions between uncharged amino acid analogues and titania are found to be repulsive or weak at best. All of the residues that bound to the negatively-charged interface show a relatively stronger adsorption compared with the charge-neutral interface, including the negatively-charged analogue. Of the analogues that are found to bind to the titania surface, the rank ordering of the binding affinities is predicted to be "arginine" > "lysine" ≈ aspartic acid > "serine". This is the same ordering as was found previously for the charge-neutral aqueous titania interface. Our results show very good agreement with available experimental data and can provide a baseline for the interpretation of peptide-TiO2 adsorption data.

Changes in intervertebral disc morphology persist 5 mo after 21-day bed rest

As part of the nutrition-countermeasures (NUC) study in Cologne, Germany in 2010, seven healthy male subjects underwent 21 days of head-down tilt bed rest and returned 153 days later to undergo a second bout of 21-day bed rest. As part of this model, we aimed to examine the recovery of the lumbar intervertebral discs and muscle cross-sectional area (CSA) after bed rest using magnetic resonance imaging and conduct a pilot study on the effects of bed rest in lumbar muscle activation, as measured by signal intensity changes in T(2)-weighted images after a standardized isometric spinal extension loading task. The changes in intervertebral disc volume, anterior and posterior disc height, and intervertebral length seen after bed rest did not return to prebed-rest values 153 days later. While recovery of muscle CSA occurred after bed rest, increases (P ≤ 0.016) in multifidus, psoas, and quadratus lumborum muscle CSA were seen 153 days after bed rest. A trend was seen for greater activation of the erector spinae and multifidus muscles in the standardized loading task after bed rest. Greater reductions of multifidus and psoas CSA muscle and greater increases in multifidus signal intensity with loading were associated with incidence of low back pain in the first 28 days after bed rest (P ≤ 0.044). The current study contributes to our understanding of the recovery of the lumbar spine after 21-day bed rest, and the main finding was that a decrease in spinal extensor muscle CSA recovers within 5 mo after bed rest but that changes in the intervertebral discs persist.

Incomplete recovery of lumbar intervertebral discs 2 years after 60-day bed rest

STUDY DESIGN: Prospective longitudinal study. OBJECTIVE: To evaluate the recovery of the lumbar intervertebral discs after bed rest. SUMMARY OF BACKGROUND DATA: Prolonged bed rest is a useful model to understand the modeling and remodeling of tissues due to disuse and reloading, yet this process in the lumbar intervertebral discs has not been examined in detail. METHODS: A total of 24 male subjects completed 60 days of head-down tilt bed rest as part of the 2nd Berlin BedRest Study and returned for magnetic resonance scanning 180 days (n = 22) and 2 years (n = 21) after bed rest. Lumbar disc volume, anterior and posterior disc height, disc signal intensity, intervertebral length, and lordosis were measured on sagittal plane magnetic resonance images. RESULTS.: Compared with prior to bed rest, increases in disc volume, disc height, and intervertebral length persisted 180 days (P ≤ 0.0004) and 720 days (P ≤ 0.024) after bed rest. Disc signal intensity remained increased 180 days (P = 0.034) after bed rest but was then decreased (P = 0.018) compared with baseline at the next measurement date. CONCLUSION: The recovery of the lumbar intervertebral discs after 60-day bed rest is a prolonged process and incomplete within 2 years.

Dissecting ADAMTS proteoglycanase biology in development and disease

 This research was focused upon members of the ADAMTS family of 'proteinases', proteins that have enzymatic activity. Their dysregulation can cause developmental defects and disease. This thesis describes the expression and regulation of ADAMTS15, as well as new developmental functions and inhibition of ADAMTS5 in arthritis.

Bioanalytical evaluation of Lactobacillus delbrueckii subsp. lactis 313 cell-envelope proteinase extraction

Lactobacilli cell-envelope proteinases (CEPs) have demonstrated numerous biopharmaceutical applications in the development of new streams of blockbuster nutraceuticals; thus, the development of efficient and commercially viable methods for CEP extraction will promote their full-scale application. In this study, the sub-cellular location of CEPs in Lactobacillus delbrueckii subsp. lactis 313 (LDL 313) was identified and the effects of different extraction methods were investigated for their ability to efficiently release CEPs from LDL 313. Significantly high relative proteinase activity of~95% was detected in cell-wall fractions and ~5% activity was observed for osmotic fluids, implying that proteinases in LDL 313 are cell-wall bound. CEPs were released from cell-wall via incubation in calcium-free buffer, indicating the enzyme is liable to self-digestion and ionic misfolding. Of the different extraction methods investigated, the use of 5 M LiCl was the most suitable, under the conditions of experimentation, for releasing high levels of CEPs from LDL 313.