Tyrosine-sulfated dermatopontin shares multiple binding sites and recognition determinants on triple-helical collagens with proteins implicated in cell adhesion and collagen folding, fibrillogenesis, cross-linking, and degradation.

Dermatopontin (DPT), a small extracellular matrix protein that stimulates collagen fibrillogenesis, contains sulfotyrosine residues but neither its level of sulfation nor its binding sites on fibrillar collagens are known. Here, we discovered that DPT is present in a relatively high mass concentration (~ 0.02%) in porcine corneal stroma, from which we purified five DPT charge variants (A-E) containing up to six sulfations. The major variant (C), containing four sulfotyrosine residues, was used to locate binding sites for DPT on triple-helical collagens II and III using the Collagen Toolkits. DPT-binding loci included the triple helix crosslinking sites and collagenase cleavage site. We find that strong DPT-binding sites on triple-helical collagen comprise an arginine-rich, positively-charged sequence that also contains hydrophobic residues. This collagen-binding signature of DPT is similar to that of the chaperone HSP47. Thus, we propose that DPT assumes the role of HSP47 as a collagen chaperone during and after the secretion. Peptide II-44, harbouring the conserved collagenase cleavage site, shows the strongest DPT-binding of the Collagen Toolkit II peptides. Substituting any of the three arginine residues (R) with alanine in the sequence GLAGQRGIVGLOGQRGER of II-44 resulted in almost complete loss of DPT binding. Since osteogenesis imperfecta, spondyloepiphyseal dysplasia, and spondyloepimetaphyseal dysplasia congenita are associated with missense mutations that substitute the corresponding arginine residues in collagens alpha-1(I) and alpha-1(II), we suggest that disrupted DPT binding to fibrillar collagens may contribute to these connective tissue disorders. In conclusion, the present work provides a cornerstone for further elucidation of the role of DPT.

Inhibition of urease activity by different compounds provides insight into the modulation and association of bacterial nickel import and ureolysis.

The nickel-dependent urease enzyme is responsible for the hydrolysis of urea to ammonia and carbon dioxide. A number of bacteria produce urease (ureolytic bacteria) and are associated with various infectious diseases and ammonia emissions from agriculture. We report the first comprehensive comparison of the inhibition of urease activity by compounds analysed under the same conditions. Thus, 71 commercially available compounds were screened for their anti-ureolytic properties against both the ureolytic bacterium Klebsiella pneumoniae and purified jack bean urease. Of the tested compounds, 30 showed more than 25% inhibition of the ureolytic activity of Klebsiella pneumoniae or jack bean urease, and among these, carbon disulfide, N-phenylmaleimide, diethylenetriaminepentaacetic acid, sodium pyrrolidinedithiocarbamate, 1,2,4-butanetricarboxylic acid, tannic acid, and gallic acid have not previously been reported to possess anti-ureolytic properties. The diverse effects of metal ion chelators on ureolysis were investigated using a cellular nickel uptake assay. Ethylenediaminetetraacetic acid (EDTA) and dimethylglyoxime (DMG) clearly reduced the nickel import and ureolytic activity of cells, oxalic acid stimulated nickel import but reduced the ureolytic activity of cells, 1,2,4-butanetricarboxylic acid strongly stimulated nickel import and slightly increased the ureolytic activity of cells, while L-cysteine had no effect on nickel import but efficiently reduced the ureolytic activity of cells.

A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies.

The SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to-express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of the catalytic domain, residues 403-621, is used for obtaining soluble and biologically active Ulp1. However, we have observed that the soluble and catalytically active Ulp1403-621 inhibits the growth of E. coli host cells. In the current study, we demonstrate an alternative route for producing active Ulp1 catalytic domain from a His-tagged N-terminally truncated variant, residues 416-621, which is expressed in E. coli inclusion bodies and subsequently refolded. Expressing the insoluble Ulp1416-621 variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1416-621 was recovered from inclusion bodies isolated from 1 L of high cell-density E. coli batch fermentation culture. After Ni2+-affinity purification of inactive and denatured Ulp1416-621 in 7.5 M urea, different refolding conditions with varying l-arginine concentration, pH, and temperature were tested. We have successfully refolded the enzyme in 0.25 M l-arginine and 0.5 M Tris-HCl (pH 7) at room temperature. Approximately 80 mg of active Ulp1416-621 catalytic domain can be produced from 1 L of high cell-density E. coli culture. We discuss the applicability of inclusion body-directed expression and considerations for obtaining high expression yields and efficient refolding conditions to reconstitute the active protein fold.

Serine protease HtrA1 accumulates in corneal transforming growth factor beta induced protein (TGFBIp) amyloid deposits.

PURPOSE: Specific mutations in the transforming growth factor beta induced (TGFBI) gene are associated with lattice corneal dystrophy (LCD) type 1 and its variants. In this study, we performed an in-depth proteomic analysis of human corneal amyloid deposits associated with the heterozygous A546D mutation in TGFBI. METHODS: Corneal amyloid deposits and the surrounding corneal stroma were procured by laser capture microdissection from a patient with an A546D mutation in TGFBI. Proteins in the captured corneal samples and healthy corneal stroma were identified with liquid chromatography-tandem mass spectrometry and quantified by calculating exponentially modified Protein Abundance Index values. Mass spectrometry data were further compared for identifying enriched regions of transforming growth factor beta induced protein (TGFBIp/keratoepithelin/ßig-h3) and detecting proteolytic cleavage sites in TGFBIp. RESULTS: A C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin 1 domain (FAS1-4), serum amyloid P-component, apolipoprotein A-IV, clusterin, and serine protease HtrA1 were significantly enriched in the amyloid deposits compared to the healthy cornea. The proteolytic cleavage sites in TGFBIp from the diseased cornea are in accordance with the activity of serine protease HtrA1. We also identified small amounts of the serine protease kallikrein-14 in the amyloid deposits. CONCLUSIONS: Corneal amyloid caused by the A546D mutation in TGFBI involves several proteins associated with other varieties of amyloidosis. The proteomic data suggest that the sequence 571-YHIGDEILVSGGIGALVR-588 contains the amyloid core of the FAS1-4 domain of TGFBIp and point at serine protease HtrA1 as the most likely candidate responsible for the proteolytic processing of amyloidogenic and aggregated TGFBIp, which explains the accumulation of HtrA1 in the amyloid deposits. With relevance to identifying serine proteases, we also found glia-derived nexin (protease-nexin 1) in the amyloid deposits, making this serine protease inhibitor a good candidate for the physiologically relevant inhibitor of one of the amyloid-associated serine proteases in the cornea and probably in other tissues. Noteworthy, the present results are in accordance with our findings from a previous study of corneal amyloid deposits caused by the V624M mutation in TGFBI, suggesting a common mechanism for lattice corneal dystrophies (LCDs) associated with mutations in the TGFBIp FAS1-4 domain.

The protein composition of the digestive fluid from the venus flytrap sheds light on prey digestion mechanisms.

The Venus flytrap (Dionaea muscipula) is one of the most well-known carnivorous plants because of its unique ability to capture small animals, usually insects or spiders, through a unique snap-trapping mechanism. The animals are subsequently killed and digested so that the plants can assimilate nutrients, as they grow in mineral-deficient soils. We deep sequenced the cDNA from Dionaea traps to obtain transcript libraries, which were used in the mass spectrometry-based identification of the proteins secreted during digestion. The identified proteins consisted of peroxidases, nucleases, phosphatases, phospholipases, a glucanase, chitinases, and proteolytic enzymes, including four cysteine proteases, two aspartic proteases, and a serine carboxypeptidase. The majority of the most abundant proteins were categorized as pathogenesis-related proteins, suggesting that the plant's digestive system evolved from defense-related processes. This in-depth characterization of a highly specialized secreted fluid from a carnivorous plant provides new information about the plant's prey digestion mechanism and the evolutionary processes driving its defense pathways and nutrient acquisition.

Composition and proteolytic processing of corneal deposits associated with mutations in the TGFBI gene.

Different types of granular corneal dystrophy (GCD) and lattice corneal dystrophy (LCD) are associated with mutations in the transforming growth factor beta induced gene (TGFBI). These dystrophies are characterized by the formation of non-amyloid granular deposits (GCDs) and amyloid (LCD type 1 and its variants) in the cornea. Typical corneal non-amyloid deposits from GCD type 2 (R124H), amyloid from a variant of LCD type 1 (V624M) and disease-free tissue controls were procured by laser capture microdissection and analyzed by tandem mass spectrometry. Label-free quantitative comparisons of deposits and controls suggested that the non-amyloid sample (R124H) specifically accumulated transforming growth factor beta induced protein (TGFBIp/keratoepithelin/ßig-h3), serum amyloid P-component, clusterin, type III collagen, keratin 3, and histone H3-like protein. The amyloid (V624M) similarly accumulated serum amyloid P-component and clusterin but also a C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin-1 domain (FAS1-4), apolipoprotein E and apolipoprotein A-IV. Significantly, analyses of the amyloid sample also revealed the presence of the serine protease Htr (High-temperature requirement) A1 and a number of proteolytic cleavage sites in the FAS1-4 domain of TGFBIp. These cleavage sites were consistent with the ligand binding and proteolytic activity of HtrA1 suggesting that it plays a role in the proteolytic processing of the amyloidogenic FAS1-4 domain. Taken together, the data suggest that the amyloidogenic-prone region of the fourth FAS1 domain of TGFBIp encompasses the Y571-R588 peptide and that HtrA1 is involved in the proteolytic processing of TGFBIp-derived amyloid in vivo.

The TSG-6 and I alpha I interaction promotes a transesterification cleaving the protein-glycosaminoglycan-protein (PGP) cross-link.

During co-incubation of human inter-alpha-inhibitor (IalphaI) and human tumor necrosis factor-stimulated gene 6 protein (TSG-6) SDS-stable interactions are formed between the two proteins. We have analyzed the products of this reaction and characterized the mechanism of complex formation. Following the incubation seven new bands not previously identified were apparent in SDS-PAGE. Three of these bands did not contain TSG-6, including heavy chain (HC)1.bikunin, HC2.bikunin, and free bikunin. In addition high molecular weight complexes composed of the same components as I alpha I, including HC1, HC2, and bikunin, were formed. The formation of these complexes was prevented by the addition of hyaluronan. The cross-links stabilizing these complexes displaying properties similar to the protein-glycosaminoglycan-protein (PGP) cross-link. The TSG-6-containing SDS-stable complexes were composed of HC1.TSG-6 or HC2.TSG-6 exclusively. Both glycosylated and non-glycosylated TSG-6 participated in the complex formation. The HC.TSG-6 cross-links were different from the PGP cross-link and were determined to be ester bonds between the alpha-carbonyl of the C-terminal Asp of the heavy chain and most likely a hydroxyl group containing the TSG-6 residue. The mechanism involved cleaving the PGP cross-link of I alpha I during a transesterification reaction. A TSG-6 hydroxyl group reacts with the ester bond between the alpha-carbonyl of the C-terminal Asp residues of HC1 or HC2 and carbon-6 of an internal N-acetylgalactosamine of the chondroitin-4-sulfate chain. An intermediate is formed resulting in a partitioning of the reaction between HC(1 or 2).TSG-6 complexes and transfer of HC(1 or 2) to the chondroitin via competing pathways.