The origins and developments of sulfation-prone tyrosine-rich and acidic N- and C-terminal extensions of class ll and lll small leucine-rich repeat proteins shed light on connective tissue evolution in vertebrates.

BACKGROUND: Small leucine-rich repeat protein (SLRP) family members contain conserved leucine-rich repeat motifs flanked by highly variable N- and C-terminal regions. Most class II and III SLRPs have tyrosine-rich N-terminal regions and some of these are sulfated. However, the evolutionary origin and conservation of the tyrosine-rich and acidic terminal regions remain undetermined. In this study, we present the most comprehensive multiple sequence alignment (MSA) analyses of all eight class II and III SLRPs to date. Based on the level of conservation of tyrosine residues and adjacent sequences, we predict which tyrosine residues are most likely to be sulfated in the terminal regions of human class II and III SLRPs. RESULTS: Using this novel approach, we predict a total of 22 tyrosine sulfation sites in human SLRPs, of which only 8 sites had been experimentally identified in mammals. Our analyses suggest that sulfation-prone, tyrosine-rich and acidic terminal regions of the class II and III SLRPs emerged via convergent evolution at different stages of vertebrate evolution, coinciding with significant evolutionary events including the development of endochondral bones and articular cartilage, the aquatic to terrestrial transition, and the formation of an amnion. CONCLUSIONS: Our study suggests that selective pressures due to changes in life conditions led to the formation of sulfotyrosine-rich and acidic terminal regions. We believe the independent emergence and evolution of sulfotyrosine-rich and acidic N- and C-terminal regions have provided each class II and III SLRP member with novel vital functions required to develop new specialized extracellular matrices and tissues in vertebrate species.

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.

Synergistic Tannic Acid-Fluoride Inhibition of Ammonia Emissions and Simultaneous Reduction of Methane and Odor Emissions from Livestock Waste.

Gaseous emissions from livestock production are complex mixtures including ammonia, methane, volatile organic compounds (VOC), and H2S. These contribute to eutrophication, reduced air quality, global warming, and odor nuisance. It is imperative that these gases are mitigated in an environmentally sustainable manner. We present the discovery of a microbial inhibitor combo consisting of tannic acid and sodium fluoride (TA-NaF), which exhibits clear synergistic inhibition of ammonia production in pure bacteria culture and in pig manure while simultaneously inhibiting methane and odorant (H2S and VOC) emissions. In laboratory headspace experiments on pig manure, we used proton-transfer-reaction mass spectrometry and cavity ring-down spectroscopy to measure the effect of TA-NaF on gaseous emissions. Ammonia emission was reduced by more than 95%, methane by up to ∼99%, and odor activity value by more than 50%. Microbial community analysis and gas emission data suggest that TA-NaF acts as an efficient generic microbial inhibitor, and we hypothesize that the synergistic inhibitory effect on ammonia production is related to tannic acid causing cell membrane leakage allowing fluoride ions easy access to urease.

Fibril Core of Transforming Growth Factor Beta-Induced Protein (TGFBIp) Facilitates Aggregation of Corneal TGFBIp.

Mutations in the transforming growth factor beta-induced (TGFBI) gene result in a group of hereditary diseases of the cornea that are collectively known as TGFBI corneal dystrophies. These mutations translate into amino acid substitutions mainly within the fourth fasciclin 1 domain (FAS1-4) of the transforming growth factor beta-induced protein (TGFBIp) and cause either amyloid or nonamyloid protein aggregates in the anterior and central parts of the cornea, depending on the mutation. The A546T substitution in TGFBIp causes lattice corneal dystrophy (LCD), which manifests as amyloid-type aggregates in the corneal stroma. We previously showed that the A546T substitution renders TGFBIp and the FAS1-4 domain thermodynamically less stable compared with the wild-type (WT) protein, and the mutant FAS1-4 is prone to amyloid formation in vitro. In the present study, we identified the core of A546T FAS1-4 amyloid fibrils. Significantly, we identified the Y571-R588 region of TGFBIp, which we previously found to be enriched in amyloid deposits in LCD patients. We further found that the Y571-R588 peptide seeded fibrillation of A546T FAS1-4, and, more importantly, we demonstrated that native TGFBIp aggregates in the presence of fibrils formed by the core peptide. Collectively, these data suggest an involvement of the Y571-R588 peptide in LCD pathophysiology.

Secreted major Venus flytrap chitinase enables digestion of Arthropod prey.

Predation plays a major role in energy and nutrient flow in the biological food chain. Plant carnivory has attracted much interest since Darwin's time, but many fundamental properties of the carnivorous lifestyle are largely unexplored. In particular, the chain of events leading from prey perception to its digestive utilization remains to be elucidated. One of the first steps after the capture of animal prey, i.e. the enzymatic breakup of the insects' chitin-based shell, is reflected by considerable chitinase activity in the secreted digestive fluid in the carnivorous plant Venus flytrap. This study addresses the molecular nature, function, and regulation of the underlying enzyme, VF chitinase-I. Using mass spectrometry based de novo sequencing, VF chitinase-I was identified in the secreted fluid. As anticipated for one of the most prominent proteins in the flytrap's "green stomach" during prey digestion, transcription of VF chitinase-I is restricted to glands and enhanced by secretion-inducing stimuli. In their natural habitat, Venus flytrap is exposed to high temperatures. We expressed and purified recombinant VF chitinase-I and show that the enzyme exhibits the hallmark properties expected from an enzyme active in the hot and acidic digestive fluid of Dionaea muscipula. Structural modeling revealed a relative compact globular form of VF chitinase-I, which might contribute to its overall stability and resistance to proteolysis. These peculiar characteristics could well serve industrial purposes, especially because of the ability to hydrolyze both soluble and crystalline chitin substrates including the commercially important cleavage of α-chitin.

Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization.

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/ßig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3' containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.

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.

Synergistic inhibition of ureolytic activity and growth of Klebsiella pneumoniae in vitro suggests cobinding of fluoride and acetohydroxamic acid at the urease active site and provides a novel strategy to combat ureolytic bacteria.

The ability of ureolytic bacteria to break down stable urea to alkaline ammonia leads to several environmental and health challenges. Ureolytic bacteria such as Helicobacter pylori, Klebsiella pneumoniae, and Proteus mirabilis can become pathogenic and cause persistent infections that can be difficult to treat. Inhibiting urease activity can reduce the growth and pathogenicity of ureolytic bacteria. In the present in vitro study, we investigated the synergistic effects of tannic acid (TA) and the urease inhibitors fluoride (F-) and acetohydroxamic acid (AHA). The concentration of AHA needed for efficient inhibition of the ureolytic activity of K. pneumoniae can be significantly reduced if AHA is coapplied with tannic acid and sodium fluoride (NaF). Thus, only 1.20 µmol l-1 AHA in combination with 0.30 mmol l-1 tannic acid and 0.60 mmol l-1 NaF delayed the onset of ureolytic pH increase by 95.8 % and increased the growth lag phase by 124.3 % relative to untreated K. pneumoniae. At these concentrations, without AHA, TA and NaF increased the onset of the ureolytic pH change by only 37.0 % and the growth lag phase by 52.5 %. The strong inhibition obtained with low concentrations of AHA in triple-compound treatments suggests cobinding of F- and AHA at the urease active site and could reduce the side effects of AHA when it is employed as a drug against e.g. urinary tract infections (UTIs) and blocked catheters. This study reports the basis for a promising novel therapeutic strategy to combat infections caused by ureolytic bacteria and the formation of urinary tract stones and crystalline biofilms on catheters.

The insoluble TGFBIp fraction of the cornea is covalently linked via a disulfide bond to type XII collagen.

TGFBIp, also known as keratoepithelin and ßig-h3, is among the most abundant proteins in the human cornea, and approximately 60% is associated with the insoluble fraction following extraction in sodium dodecyl sulfate (SDS) sample buffer. TGFBIp is of particular interest because a wide range of mutations causes amyloid or fuchsinophilic crystalloid deposits in the cornea leading to visual impairment. We show that the SDS-insoluble fraction of TGFBIp from porcine and human corneas is covalently linked via a reducible bond to the NC3 domain of type XII collagen in a TGFBIp:type XII collagen stoichiometric ratio of 2:1. Because type XII collagen is anchored to striated collagen fibers of the extracellular matrix, its interaction with TGFBIp is likely to provide anchoring for cells to the extracellular matrix through the integrin binding capability of TGFBIp. Furthermore, the TGFBIp-type XII collagen molecule will affect our understanding of the molecular pathogenesis of the TGFBI-linked corneal dystrophies.

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.

Whole-Genome Sequences of Streptococcus alactolyticus Strain An1F4 and Escherichia coli Strains Ae3A3 and Ae3B3, Isolated from Feces of Domestic Pigs.

Here, we present the genome sequences of a strain of Streptococcus alactolyticus and two strains of Escherichia coli that were isolated from feces samples from domestic pigs in Denmark. The genome sequences contribute to a better understanding of the microbiological processes in the feces and manure of domestic pigs.

Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp.

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.

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.

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.

Differential expression and processing of transforming growth factor beta induced protein (TGFBIp) in the normal human cornea during postnatal development and aging.

Transforming growth factor beta induced protein (TGFBIp, also named keratoepithelin) is an extracellular matrix protein abundant in the cornea. The purpose of this study was to determine the expression and processing of TGFBIp in the normal human cornea during postnatal development and aging. TGFBIp in corneas from individuals ranging from six months to 86 years of age was detected and quantified by immunoblotting. The level of TGFBIp in the cornea increases about 30% between 6 and 14 years of age, and adult corneas contain 0.7-0.8 microg TGFBIp per mg wet tissue. Two-dimensional (2-D) immunoblots of the corneal extracts showed a characteristic "zig-zag" pattern formed by different lower-molecular mass TGFBIp isoforms (30-60 kDa). However, the relative abundance of the different isoforms was different between infant corneas (<1 year) and the child/adult corneas (>6 years). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) data of TGFBIp isoforms separated on large 2-D gels show that TGFBIp is proteolytically processed from the N-terminus. This observation was supported by in silico 2-D gel electrophoresis showing that sequential proteolytical trimming events from the N-terminus of mature TGFBIp generate TGFBIp isoforms which form a similar "zig-zag" pattern when separated by 2-D polyacrylamide gel electrophoresis (PAGE). This study shows that in humans TGFBIp is more abundant in mature corneas than in the developing cornea and that the processing of TGFBIp changes during postnatal development of the cornea. In addition, TGFBIp appears to be degraded in a highly orchestrated manner in the normal human cornea with the resulting C-terminal fragments being retained in the cornea. The age-related changes in the expression and processing of corneal TGFBIp suggests that TGFBIp may play a role in the postnatal development and maturation of the cornea. Furthermore, these observations may be relevant to the age at which mutant TGFBIp deposits in the cornea in those dystrophies caused by mutations in the transforming growth factor beta induced gene (TGFBI) as well as the mechanisms of corneal protein deposition.

Development of a M9-based urea medium (M9U) for sensitive and real-time monitoring of ureolytic activity of bacteria and cell-free urease.

The enzyme urease is widespread in nature and catalyzes the hydrolysis of urea to form ammonia and carbonic acid. The high proficiency of the enzyme is associated with a wide range of societal challenges. In agriculture, bacterial urease activity leads to loss of fertilizer through NH3 emission, which has a negative impact on the environment and human health. Urease is also an essential virulence factor for several pathogenic bacteria. To screen for potential urease inhibitors, efficient, sensitive, and accurate urease activity assays are needed. However, most urease activity assays are labor-intensive and become time-consuming when used to screen multiple samples. Based on systematic optimization, we have developed a urea-containing growth medium and method for continuous real-time monitoring and screening of urease activity from both bacterial cells and pure urease in a plate reader setup. The defined M9-based urea (M9U) medium was found to be more sensitive and suitable for a plate reader setup than both Christensen's urea broth (CUB) and Stuart's urea broth (SUB), which are established and well-known complex urea media that formed the principle foundation of M9U. Furthermore, we show that urease activity measurements using the M9U medium in our plate reader-based method allow reliable high-throughput screening of urease inhibitors.

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.

Evidence against a blood derived origin for transforming growth factor beta induced protein in corneal disorders caused by mutations in the TGFBI gene.

PURPOSE: Several inherited corneal disorders in humans result from mutations in the transforming growth factor beta induced gene (TGFBI), which encodes for the extracellular transforming growth factor beta induced protein (TGFBIp) that is one of the most abundant proteins in the cornea. We previously reported a significant amount of TGFBIp in plasma by immunoblotting using the only TGFBIp antiserum (anti-p68(beta ig-h3)) available at that time (anti-p68(beta ig-h3) was generated against residues Val210-His683 of TGFBIp). This observation raised the possibility that a fraction of corneal TGFBIp may originate from the plasma. However, recent experiments in our laboratory indicated that the anti-p68(beta ig-h3) antiserum cross-reacts with an environmental protein contaminant. Therefore, we investigated the specificity of the originally utilized anti-p68(beta ig-h3) antiserum and re-evaluated the amount of TGFBIp in human plasma by immunoblotting using a new specific antiserum. METHODS: The observed cross-reactivity of the previously utilized anti-p68(beta ig-h3) antiserum was tested by immunoblotting and the antigen identity was determined by mass spectrometry. A part of human TGFBI encoding an NH2-terminal 11.4 kDa fragment of TGFBIp (residues Gly134-Ile236) was amplified by polymerase chain reaction (PCR) and cloned in E. coli. The TGFBIp fragment was expressed in E. coli, purified by Ni2+-affinity chromatography, and used to immunize rabbits to produce a specific antiserum (anti-TGFBIp(134-236)). To enhance the detection of possible TGFBIp in plasma by allowing a higher sample load, albumin and immunoglobulin G (IgG) were specifically depleted from normal human plasma by affinity chromatography. The presence of TGFBIp in plasma was investigated by immunoblotting using the anti-TGFBIp(134-236) antiserum. Purified TGFBIp from porcine corneas was used for estimation of the TGFBIp detection limit. RESULTS: The previously utilized TGFBIp antiserum, anti-p68(beta ig-h3), cross-reacted with human keratin-1, a common environmental protein contaminant. Thus, the anti-p68(beta ig-h3) antiserum recognizes both TGFBIp and keratin-1. In contrast, the anti-TGFBIp(134-236) antiserum reacted with TGFBIp but showed no indication of reactivity with other proteins in plasma. Using this antiserum, TGFBIp was not detected in crude or albumin/IgG-depleted human plasma and the detection limit of TGFBIp using immunoblotting was estimated to be 10 ng. CONCLUSIONS: Our failure to detect TGFBIp in human plasma using a highly specific antiserum suggests that TGFBIp is not present in a physiologically relevant concentration in human plasma. The previous impression that normal human plasma contains a significant amount of TGFBIp by immunoblotting was due to the utilization of a less specific antiserum that recognizes both TGFBIp and human keratin-1. Together with other results, our observation makes it unlikely that TGFBIp is imported into the cornea from the circulation as reported for other abundant extracellular corneal proteins and suggests corneal origin of TGFBIp deposits in individuals with inherited corneal diseases caused by mutations in the TGFBI gene.

The human cornea proteome: bioinformatic analyses indicate import of plasma proteins into the cornea.

Increased biochemical knowledge of normal and diseased corneas is essential for the understanding of corneal homeostasis and pathophysiology. In a recent study, we characterized the proteome of the normal human cornea and identified 141 distinct proteins. This dataset represents the most comprehensive protein study of the cornea to date and provides a useful reference for further studies of normal and diseased human corneas. The list of identified proteins is available at the Cornea Protein Database. In the present paper, we review the utilized procedures for extraction and fractionation of corneal proteins and discuss the potential roles of the identified proteins in relation to homeostasis, diseases, and wound-healing of the cornea. In addition, we compare the list of identified proteins with high quality gene expression libraries (cDNA libraries) and Serial Analysis of Gene Expression (SAGE) data. Of the 141 proteins, 86 (61%) were recognized in cDNA libraries from the corneas of dogs and rabbits, or humans with keratoconus, and 98 (69.5%) were recognized in SAGE data of mouse and human corneas. However, the percentages of identified genes in each of the protein functional groups differed markedly. Thus, exceptionally few of the traditional blood/plasma proteins and immune defense proteins that were identified in the human cornea were recognized in the gene expression libraries of the cornea. This observation strongly indicates that these abundant corneal proteins are not expressed in the cornea but originate from the surrounding pericorneal tissue.

Isolation, crystallisation, and preliminary X-ray analysis of the bovine mitochondrial EF-Tu:GDP and EF-Tu:EF-Ts complexes.

Previous studies have shown that when bovine mitochondrial elongation factor Ts (EF-Ts) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli elongation factor Tu (EF-Tu). In contrast to earlier experiments, purification of free mitochondrial EF-Ts was accomplished under nondenaturing conditions since only about 60% of the expressed EF-Ts copurified with E. coli EF-Tu. The bovine mitochondrial EF-Tu:GDP complex, the homologous mitochondrial EF-Tu:EF-Ts complex, and the heterologous E. coli/mitochondrial EF-Tu:EF-Ts complex were isolated and crystallised. The crystals of the EF-Tu:GDP complex diffract to 1.94 A and belong to space group P2(1) with cell parameters a=59.09 A, b=119.78 A, c=128.89 A and beta=96.978 degrees. The crystals of the homologous mitochondrial EF-Tu:EF-Ts complex diffract to 4 A and belong to space group C2 with cell parameters a=157.7 A, b=151.9 A, c=156.9 A, and beta=108.96 degrees.