Propeptide Cleavage Site Research Articles

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Engineered pro-peptide enhances the catalytic activity of keratinase to improve the conversion ability of feather waste.

Keratinase is an attractive industrial enzyme that can specifically catalyze keratin waste to obtain value-added products. A challenge to the application of keratinase is improving catalytic capacity to achieve efficient hydrolysis. In this study, we effectively expressed the keratinase gene from Bacillus licheniformis BBE11-1 in Bacillus subtilis WB600 based on pro-peptide engineering. Partial deletion of the pro-peptide sequence and the substitution of amino acid at the pro-peptide cleavage site (P1) suggested that the "chaperone effect"and "cleavage efficiency"of the pro-peptide determine the activity of the mature enzyme. Subsequently, seven target sites that can increase the activity of the mature enzyme by 16%-66% were obtained through the multiple sequence alignment of pro-peptides and site-directed mutation. We further performed combinatorial mutations at six sites based on the design principle of three-codon saturation mutations and obtained mutant 2-D12 (236.8 KU/mg) with a mature enzyme activity of 186% of the original (127.6 KU/mg). Finally, continuous fermentation was carried out in a 5-L bioreactor for 22 h, and the activity of the 2-D12 mature enzyme was increased to 391.6 KU/mg. Most importantly, 2-D12 could degrade more than 90% of feather waste into amino acids and peptides within 12 h with the aid of sulfite.

Desmoglein-2 mutations in propeptide cleavage-site causes arrhythmogenic right ventricular cardiomyopathy/dysplasia by impairing extracellular 1-dependent desmosomal interactions upon cellular stress

Desmoglein-2 (DSG2) mutations, which encode a heart-specific cadherin crucial for desmosomal adhesion, are frequent in arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D). DSG2 mutations have been associated with higher risk of biventricular involvement. Among DSG2 mutations, mutations of the inhibitory propeptide consensus cleavage-site (Arg-X-Arg/Lys-Arg), are particularly frequent. In the present work, we explored the functional consequences of DSG2 propeptide cleavage site mutations p.Arg49His, p.Arg46Trp, and p.Arg46Gln on localization, adhesive properties, and desmosome incorporation of DSG2. We studied the expression of mutant-DSG2 in human heart and in epithelial and cardiac cellular models expressing wild-type or mutant (p.Arg49His, p.Arg46Trp, and p.Arg46Gln) proDSG2-GFP fusion proteins. The consequences of the p.Arg46Trp mutation on DSG2 adhesiveness were studied by surface plasmon resonance. Incorporation of mutant p.Arg46Trp DSG2 into desmosomes was studied under low-calcium culture conditions and cyclic mechanical stress. We demonstrated in human heart and cellular models that all three mutations prevented N-terminal propeptide cleavage, but did not modify intercellular junction targeting. Surface plasmon resonance experiments showed a propeptide-dependent loss of interaction between the cadherin N-terminal extracellular 1 (EC1) domains. Additionally, proDSG2 mutant proteins were abnormally incorporated into desmosomes under low-calcium culture conditions or following mechanical stress. This was accompanied by an epidermal growth factor receptor-dependent internalization of proDSG2, suggesting increased turnover of unprocessed proDSG2. Our results strongly suggest weakened desmosomal adhesiveness due to abnormal incorporation of uncleaved mutant proDSG2 in cellular stress conditions. These results provide new insights into desmosomal cadherin regulation and ARVC/D pathophysiology, in particular, the potential role of mechanical stress on desmosomal dysfunction.

Combining Pro-peptide Engineering and Multisite Saturation Mutagenesis To Improve the Catalytic Potential of Keratinase.

Keratinases are becoming biotechnologically important since they have shown potential in hydrolysis of recalcitrant keratins with highly rigid and strongly cross-linked structures. However, the large-scale application of keratinases has been limited by the inefficient expression level and low enzyme activity. In this work, we employed pro-peptide engineering and saturation mutagenesis to construct excellent keratinase variants with improved activities. It turned out that amino acid substitutions at the pro-peptide cleavage site (P1) could accelerate the release of active mature enzymes, resulting in a 3-fold activity increase. Eighteen sites of the pro-peptide area were targeted for codon mutagenesis, and a multisite saturation mutagenesis library of the six potential sites was generated, achieving a significant improvement of keratinase activity from 179 to 1114 units/mL. Also, the mutants exhibited alterant catalytic properties. Finally, fermentation for keratinase production in a 15 L fermenter was carried out, and the enzyme activity reached up to over 3000 units/mL. Our results demonstrated that pro-peptide engineering played a crucial role in high expression and engineering of proteases. This study provides a universal route toward improvement of industrial enzymes that were first synthesized as precursors in the form of pre-pro-protein.

Mutations That Alter the Carboxy-Terminal-Propeptide Cleavage Site of the Chains of Type I Procollagen Are Associated With a Unique Osteogenesis Imperfecta Phenotype

Osteogenesis imperfecta (OI) is a genetic bone disorder characterized by fractures, low bone mass, and skeletal fragility. It most commonly arises from dominantly inherited mutations in the genes COL1A1 and COL1A2 that encode the chains of type I collagen. A number of recent reports have suggested that mutations affecting the carboxyl-terminal propeptide cleavage site in the products of either COL1A1 or COL1A2 give rise to a form of OI characterized by unusually dense bones. We have assembled clinical, biochemical, and molecular data from 29 individuals from 8 families with 7 different mutations affecting the C-propeptide cleavage site. The phenotype was generally mild: The median height was ∼33th centile. Eighty percent of subjects had their first fracture by the age of 10 years, and one-third had a femoral or tibial fracture by the age of 25 years. Fractures continued into adulthood, though rates varied considerably. Healing was normal and rarely resulted in long bone deformity. One-third of subjects older than 15 years had scoliosis. The teeth and hearing were normal in most, and blue sclerae were not observed. Other features noted included fibro-osseous dysplasia of the mandible and Achilles tendon calcification. The mean spinal bone mineral density Z-score was +2.9 (SD 2.1) compared with -2.2 (0.7) in subjects with COL1A1 haploinsufficiency mutations. Bone mineral density distribution, assessed by quantitative backscattered electron imaging in bone showed higher levels of mineralization than found in any other disorder. Bone histology showed high trabecular volume and increased cortical thickness, with hyperosteoidosis and delayed mineralization. In vitro studies with cultured skin fibroblasts suggested that these mutations interfere with processing of the chain in which the sequence alteration occurs, but the C-propeptide is eventually cleaved (and detectable in blood), suggesting there are alternative sites of cleavage. The precise mechanism of the bony pathology is not yet clear. © 2018 American Society for Bone and Mineral Research.

Molecular characterization, tissue distribution and feeding related changes of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti)

The protein nucleobindin-2 (NUCB2) was identified over a decade ago and recently raised great interest as its derived peptide nesfatin-1 was shown to reduce food intake and body weight in rodents. However, the involvement of NUCB2 in feeding behavior has not well been studied in fish. In the present study, we characterized the structure, distribution, and meal responsive of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti) for the first time. The full length cDNA of Ya-fish was 2140base pair (bp), which encoded a polypeptide of 487 amino acid residues including a 23 amino acid signal peptide. A high conservation in NUCB2 sequences was found in vertebrates, however the proposed propeptide cleavage site (Arg–Arg) conserved among other species is not present in Ya-fish NUCB2A sequence. Tissue distribution analysis revealed that Ya-fish NUCB2A mRNA was ubiquitously expressed in all test tissues, and abundant expression was detected in several regions including the hypothalamus, hepatopancreas, ovary and intestines. NUCB2A mRNA expression respond to feeding status change may vary and be tissue specific. NUCB2A mRNA levels significantly increased (P<0.05) in the hypothalamus and intestines after feeding and substantially decreased (P<0.01) during a week food deprivation in the hypothalamus. Meanwhile, NUCB2A mRNA in the hepatopancreas was significantly elevated (P<0.001) during food deprivation, and a similar increase was also found after short-time fasting. This points toward a potential hepatopancreas specific local role for NUCB2A in the regulation of metabolism during food deprivation. Collectively, these results provide the molecular and functional evidence to support potential anorectic and metabolic roles for NUCB2A in Ya-fish.

Genotype/phenotype analysis and pathophysiology of Desmoglein-2 propeptide cleavage-site mutations in Arrhythmogenic right ventricular cardiomyopathy

Purpose: Desmoglein-2 (DSG2) mutations are common causes of Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). A hot spot of missense mutations targets the consensus cleavage-site of DSG2 pro-peptide (Arg-X-Arg/Lys-Arg) by Kex2-like proprotein-convertases (PCs). Through mutational screening in 200 independent ARVC probands we identified heterozygous missense DSG2 mutations of the cleavage site (p.Arg46Trp, p.Arg46Gln, p.Arg49His and p.Lys48Asn) in ten probands and seven additional relatives. In the present work we analysed the genotype/phenotype correlations associated with these specific mutations and experimentally explored their molecular consequences. Methods and results: The pro-DSG2 cleavage site mutations were associated with a severe phenotype in 53% of cases with diffuse right ventricular (RV) dilatation, severe RV dysfunction and frequent left ventricular involvement leading to end-stage heart failure in 3 out of 17 mutation-carriers. To understand molecular dysfunctions associated with the observed severe phenotype, we performed ex vivo analysis on heart samples obtained from two mutation carriers and in vitro analysis by expressing a wild-type (WT) or mutant DSG2-GFP fusion protein in cellular models. First, we demonstrated, by using an antibody specifically directed against the propeptide, that all mutations prevented efficient propeptide cleavage. However pro-DSG2 mutants were correctly addressed to the intercellular junctions (cells) or at the intercalated disk (human tissue). Although co-immunoprecipitation analysis and extracellular crosslinking studies showed the persistence of homophilic interactions of mutant pro-DSG2, the presence of propeptide led to the abolition of interactions between the EC1 (N-terminal) domains of the cadherins. This was accompanied with a mis-incorporation of pro-DSG2 into desmosomes as revealed by immunofluorescent labelling at low calcium concentration compared to WT DSG2, due to an EGFR-dependant internalization of mutant pro-DSG2 and partners (Plakophillin-2 and Plakoglobin). Finally, we showed by western-blot an increase in the soluble pool of mutant pro-DSG2 compared to the DSG2 WT when cells were submitted to mechanical stress. Conclusion: DSG2 propeptide cleavage-site mutations are associated with a severe phenotype and an increased risk of heart failure. Our experimental results indicated a loss of DSG2 adhesiveness properties and a decrease of the incorporation of mutant DSG2 into desmosomes due to propeptide cleavage abolition, that could play an important role in ARVC pathophysiology.

SVM-Based Prediction of Propeptide Cleavage Sites in Spider Toxins Identifies Toxin Innovation in an Australian Tarantula

Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin transcripts, without resorting to protein sequencing, the propeptide cleavage site in the toxin precursor must be predicted bioinformatically. We evaluated different machine learning strategies (support vector machines, hidden Markov model and decision tree) and developed an algorithm (SpiderP) for prediction of propeptide cleavage sites in spider toxins. Our strategy uses a support vector machine (SVM) framework that combines both local and global sequence information. Our method is superior or comparable to current tools for prediction of propeptide sequences in spider toxins. Evaluation of the SVM method on an independent test set of known toxin sequences yielded 96% sensitivity and 100% specificity. Furthermore, we sequenced five novel peptides (not used to train the final predictor) from the venom of the Australian tarantula Selenotypus plumipes to test the accuracy of the predictor and found 80% sensitivity and 99.6% 8-mer specificity. Finally, we used the predictor together with homology information to predict and characterize seven groups of novel toxins from the deeply sequenced venom gland transcriptome of S. plumipes, which revealed structural complexity and innovations in the evolution of the toxins. The precursor prediction tool (SpiderP) is freely available on ArachnoServer (http://www.arachnoserver.org/spiderP.html), a web portal to a comprehensive relational database of spider toxins. All training data, test data, and scripts used are available from the SpiderP website.

Detection of VWF Copy Number Variations by Microarray Analysis in Von Willebrand Disease Type 3 Index Cases

Abstract 380▪FN2▪This icon denotes a clinically relevant abstractAs part of the TS Zimmerman Program for the Molecular and Clinical Biology of Von Willebrand disease (VWD), we performed complete laboratory phenotyping and full gene DNA sequence analysis on VWD index cases. VWD type 3 is an autosomal recessive disorder characterized by undetectable levels of von Willebrand factor (VWF). These patients are expected to have VWF mutations that abolish expression of the protein on both VWF alleles. We identified a group of VWD type 3 patients in which one or both alleles did not have a VWF mutation identified by DNA sequence analysis. Although DNA sequencing is considered to be the gold standard for mutation detection, this technique does not detect copy number variations (CNVs). Microarrays have been successfully used to detect CNVs missed by DNA sequencing in other disorders such as Duchenne muscular dystrophy. In order to investigate the role of VWF deletion and duplication in VWD, we designed a targeted gene high-resolution oligonucleotide comparative genomic hybridization (CGH) array on an Oxford Gene Technologies (OGT) 60K platform that permits high resolution analysis of the VWF gene using long oligonucleotides probes (∼45–60mer). The average spacing was 65 bp within coding regions and 178 bp within intronic regions. The advantages of CGH array over techniques such as multiplex ligation-dependent probe amplification (MLPA) is that many probes interrogate each region so detection is less likely to be affected by sequence variants and polymorphisms. In addition, the intronic oligonucleotide probes allowed us to detect dosage changes within the entire genomic region of the gene and determine the approximate breakpoint location. From the approximate breakpoints position, PCR primers can be designed to confirm the copy number variant and identify the exact breakpoint by sequencing. We analyzed a total of fourteen type 3 patients using the VWF CGH array. We identified deletions in the VWF gene in four patients. Three of the patients also had new mutations detected by DNA sequencing.PatientSequencing Mutation ResultCGH Array Result1c.1722_1723ins ACCTExon 4–5 Deletion2None DetectedExon 1–3 DeletionExon 4–5 Deletion3c. 2072delCExon 18 Deletion4c.2438_2439insGExon 35–38 DeletionIn patients 1 and 2 we identified an in-frame deletion of exons 4–5. This deletion was previously reported as recurrent mutation in type 1 and type 3 patients by Sutherland et al, Blood (2009) 114:1091. Patient 2 also had a novel deletion in exons 1–3. This deletion would remove the initiating ATG in exon 2. In patient 3, we detected an out of frame deletion of exon 18. Exon 18 contains the propeptide cleavage site. Finally, in patient 4 we detected an out of frame deletion of exons 35–38. These deletions are expected to be type 3 alleles. The breakpoints of each deletion are intronic. PCR will confirm the variant and identify the breakpoint. Our study demonstrates that deletions in the VWF gene may be a common mutation mechanism. We detected deletions in 29% of type 3 patients that did not have two VWF mutations identified by sequencing. CGH arrays are an effective way of identifying deletion and duplication in the VWF gene and mapping their approximate breakpoint locations. Disclosures:No relevant conflicts of interest to declare.

Detection and Structural Characterization of Natural Ara h 7, the Third Peanut Allergen of the 2S Albumin Family

In recent years, several novel relevant peanut allergens have been identified. Among those, a new member of the conglutin family was cloned by a phage display approach and initially annotated as Ara h 7.0101. Later, however, recloning of Ara h 7 revealed an alternate isoform, termed Ara h 7.0201. Because the natural Ara h 7 counterpart had not been found at the protein level in peanut extracts, the aim of the present study was to search for authentic natural Ara h 7 protein(s). To this end, enriched low molecular mass proteins (<20 kDa) from peanut extracts were separated by 2D electrophoresis and subjected to mass spectrometric analyses. Fifty of 65 analyzed spots were identified. Interestingly, Ara h 7.0101 was not identified, but Ara h 7.0201 and Ara h 7.0202, a different Ara h 7 isoallergen containing an additional pro-peptide cleavage site, were. In accordance with the conserved cysteine pattern of conglutins, Ara h 7.0201 possesses eight cysteine residues, in contrast to the six cysteines present in the previously cloned Ara h 7.0101. Moreover, a putative cleavage site in the Ara h 7.0202 isoform points to the characteristic biological function of conglutins as amylase/trypsin inhibitors.

Pattern analysis for the prediction of fungal pro-peptide cleavage sites

Support vector machines (SVMs) have many applications in investigating biological data from gene expression arrays to understanding EEG signals of sleep stages. In this paper, we have developed an application that will support the prediction of the pro-peptide cleavage site of fungal extracellular proteins which display mostly a monobasic or dibasic processing site. Many of the secretory proteins and peptides are synthesized as inactive precursors and they become active after post-translational processing. A collection of fungal pro-protein sequences are used as a training data set. A specifically designed kernel is expressed as an application of the well-known Gaussian kernel via feature spaces defined for our problem. Rather than fixing the kernel parameters with cross validation or other methods, we introduce a novel approach that simultaneously performs model selection together with the test of accuracy and testing confidence levels. This leads us to higher accuracy at significantly reduced training times. The results of the server ProP1.0 which predicts pro-peptide cleavage sites are compared with the results of this study. A similar mathematical approach may be adapted to pro-peptide cleavage prediction in other eukaryotes.

Glycosaminoglycans Facilitate Procathepsin B Activation through Disruption of Propeptide-Mature Enzyme Interactions

Lysosomal cysteine cathepsin B participates in numerous diverse cellular processes. In acquiring its activity, the proregion, which blocks the substrate-binding site in the proenzyme, needs to be cleaved off. Here we demonstrate that polyanionic polysaccharides, glycosaminoglycans (GAGs), can accelerate the autocatalytic removal of the propeptide and subsequent activation of cathepsin B. We show that naturally occurring GAGs such as chondroitin sulfates and heparin, as well as the synthetic analog dextran sulfate, accelerate the processing in a concentration-dependent manner. Heparin oligosaccharides down to the size of tetrasaccharides were efficient in accelerating the procathepsin B processing, whereas disaccharides were without effect. Further, the ability of the GAGs to accelerate procathepsin B processing was sensitive to increasing NaCl concentrations, indicating that electrostatic interaction between the GAGs and procathepsin B are operative in the accelerating effect. Also the processing of the catalytic procathepsin B mutant by wild type cathepsin B was enhanced in the presence of GAGs, suggesting that GAGs induce a conformational change in procathepsin B, converting it into a better substrate. Site-directed mutagenesis showed that His(28), Lys(39), and Arg(40), located within the procathepsin B propeptide, have significant roles in the acceleration of procathepsin B activation induced by short GAGs. Because procathepsin B and GAGs often co-localize in vivo, we propose that GAGs may play a physiological role in the activation of procathepsin B.

Evolutionary conservation of P-selectin glycoprotein ligand-1 primary structure and function

BackgroundP-selectin glycoprotein ligand-1 (PSGL-1) plays a critical role in recruiting leukocytes in inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L- and P-selectin binding. Little information is available on the intra- and inter-species evolution of PSGL-1 primary structure. In addition, the evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail. Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human, chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog) and examined mammalian PSGL-1 interactions with human selectins.ResultsA signal peptide was predicted in each sequence and a propeptide cleavage site was found in 9/14 species. PSGL-1 N-terminus is poorly conserved. However, each species exhibits at least one tyrosine sulfation site and, except in horse and dog, a T [D/E]PP [D/E] motif associated to the core-2 O-glycosylation of a N-terminal threonine. A mucin-like domain of 250–280 amino acids long was disclosed in all studied species. It lies between the conserved N-terminal O-glycosylated threonine (Thr-57 in human) and the transmembrane domain, and contains a central region exhibiting a variable number of decameric repeats (DR). Interspecies and intraspecies polymorphisms were observed. Transmembrane and cytoplasmic domain sequences are well conserved. The moesin binding residues that serve as adaptor between PSGL-1 and Syk, and are involved in regulating PSGL-1-dependent rolling on P-selectin are perfectly conserved in all analyzed mammalian sequences. Despite a poor conservation of PSGL-1 N-terminal sequence, CHO cells co-expressing human glycosyltransferases and human, bovine, pig or rat PSGL-1 efficiently rolled on human L- or P-selectin. By contrast, pig or rat neutrophils were much less efficiently recruited than human or bovine neutrophils on human selectins. Horse PSGL-1, glycosylated by human or equine glycosyltransferases, did not interact with P-selectin. In all five species, tyrosine sulfation of PSGL-1 was required for selectin binding.ConclusionThese observations show that PSGL-1 amino acid sequence of the transmembrane and cytoplasmic domains are well conserved and that, despite a poor conservation of PSGL-1 N-terminus, L- and P-selectin binding sites are evolutionary conserved. Functional assays reveal a critical role for post-translational modifications in regulating mammalian PSGL-1 interactions with selectins.

Molecular genetics of hereditary prothrombin deficiency in Indian patients: identification of a novel Ala362 --> Thr (Prothrombin Vellore 1) mutation.

Prothrombin deficiency is a rare (1:200 000) autosomal recessive disorder caused by diverse mutations in prothrombin gene. We have studied the molecular basis of this disorder in four unrelated Indian patients. The diagnosis was based on prolonged prothrombin (PT) and activated partial thromboplastin times and low factor II coagulant activity (FII: C) measured using a PT based assay. FII: C levels ranged between 4.7% and 17.5%. Mutations were identified in all the four patients. Five different causative mutations including four (80%) missense and an in-frame deletion (20%) were identified. One of them was a novel, Ala362 --> Thr amino acid change affecting 'B' chain of -thrombin. This mutation was present in a compound heterozygous state with a previously reported Arg-1 --> Gln missense change affecting pro-peptide cleavage site. Ala362 --> Thr occurred at a codon, evolutionarily conserved in all the 24 different prothrombins or its related serine proteases studied. Molecular modeling of this mutation was found to cause a conformational change around the region involving a catalytic triad residue His363 and a cysteine residue at codon 364. The FII: C level in this patient was 17.5%. Three other previously reported mutations were also detected in the homozygous state: Arg271 --> Cys in Kringle-2 region, a Glu309 --> Lys in "A" chain of -thrombin and an in-frame deletion of 3 bp (AAG) leading to Del Lys301/302 in "A" chain of -thrombin. This is the first report of the molecular basis of prothrombin deficiency in Indian patients and we suggest the eponym 'Prothrombin Vellore 1' for Ala362 --> Thr mutation.

Bacillolysin MA, a Novel Bacterial Metalloproteinase That Produces Angiostatin-like Fragments from Plasminogen and Activates Protease Zymogens in the Coagulation and Fibrinolysis Systems

We isolated a novel protease that converts plasminogen to angiostatin-like fragments (BL-angiostatins) from a culture of Bacillus megaterium A9542 through a single-step chromatography on CM-cellulose. The protease, designated bacillolysin MA (BL-MA), belongs to a family of neutral metalloproteinases based on the nucleotide sequence of its gene. At an enzyme:substrate ratio of 1:540, BL-MA cleaved human plasminogen mainly at Ser441-Val442 to form BL-angiostatin and miniplasminogen with a K(m) of 3.0 +/- 0.8 microM and a k(cat) of 0.70 +/- 0.09 s(-1). The resulting BL-angiostatins inhibited the proliferation, migration, and tube formation of vascular endothelial cells at concentrations of 1-10 microg/ml. Although BL-MA failed to activate plasminogen, it increased urokinase-catalyzed activation of plasminogen caused by production of miniplasminogen, which is highly susceptible to activation. In addition, BL-MA was active in converting prourokinase, prothrombin, coagulation factor X, and protein C to their active forms. BL-MA enhanced both the clotting of human plasma and clot dissolution in the presence of prourokinase. Thus, BL-MA affects blood coagulation and fibrinolysis systems and can be used to produce angiostatin-like plasminogen fragments and active serine proteases of human plasma.

Molecular Genetics of Hereditary Prothrombin Deficiency in Indian Patients: Identification of a Novel Ala362→Thr (Prothrombin Vellore 1) Mutation by Conformation Sensitive Gel Electrophoresis.

Prothrombin deficiency is a rare (1:200000) autosomal recessive disorder caused by diverse mutations in prothrombin gene. We have studied the molecular basis of this disorder in 4 unrelated Indian patients. The clinical features of these patients included easy bruisability, post-traumatic bleeds, hemarthroses (50% each) and epistaxis, gum bleedings, menorrhagia, hematemesis and post-surgical bleeding (25% each). The diagnosis was based on prolonged prothrombin and activated partial thromboplastin times and low factor II coagulant activity (FII: C) measured using a prothrombin time based assay. FII: C levels ranged between 4.7–17.5%. Genomic DNA was screened for prothrombin gene mutations by a novel PCR and conformation sensitive gel electrophoresis (CSGE) strategy. Fourteen exonic and their flanking intronic regions and 3′ untranslated region of prothrombin gene were amplified by 12 pairs of primers designed by Primer3 software. CSGE was performed in a mildly denaturing gel containing 10% acrylamide. Samples displaying abnormal CSGE profiles were sequenced by the Big Dye Terminator cycle sequencing kit to confirm the nature of nucleotide change. A novel missense mutation was studied based on the coordinates (identifier 1ppb) for the human ∞-thrombin (1.92Å resolution) three-dimensional structure. The evolutionary conservation of this aminoacid, mutated by missense change, was studied in the prothrombin sequence in 17 different species and 7 related proteases obtained from SwissProt and Trembl databases using PSI-BLAST. Mutations were identified in all the four patients. Five different causative mutations including 4 (80%) missense and an in-frame deletion (20%) were identified. One of them was a novel, Ala362→Thr aminoacid change affecting ‘B' chain of ∞-thrombin. This mutation was identified in a compound heterozygous state with a previously reported Arg-1→Gln missense change affecting pro-peptide cleavage site. Ala362→Thr occurred at a codon, evolutionarily conserved in all the 24 different prothrombins or its related serine proteases studied. This indicates its importance to the structure of α-thrombin. Molecular modeling of this Ala362→Thr mutation was found to cause a conformational change around the region involving a catalytic triad residue His363 and a cysteine residue at codon 364 due to the accommodation of a larger and polar side chain of threonine. The FII: C level in this patient was 17.5%. Three other previously reported mutations were also detected in the homozygous state: Arg271→Cys missense mutation in Kringle-2 region, a Glu309→Lys missense mutation in ‘A' chain of ∞-thrombin and an in-frame deletion of 3bp (AAG) leading to Del Lys301/302 in ‘A' chain of ∞-thrombin. This is the first report of the molecular basis of prothrombin deficiency in Indian patients and we suggest the eponym ‘Prothrombin Vellore 1′ for Ala362→Thr mutation.

Identification of a novel Fasciola hepatica cathepsin L protease containing protective epitopes within the propeptide

Cathepsin L (CL)-like proteases are important candidate vaccine antigens for protection against helminth infections. We previously identified an immunogenic 32 kDa protein specifically present in newly excysted juveniles (NEJs) of Fasciola hepatica. Here we show by N-terminal protein sequencing that this protein represents a CL-like protease still containing the propeptide. Two cDNAs encoding this CL were subsequently isolated from NEJs by RT-PCR. The predicted amino acid sequences of these cDNAs showed approximately 70% sequence homology to both CL1 and CL2 sequences isolated from adult stage F. hepatica and are, therefore, referred to as CL3. The CL3 clones encoded asparagine at position P1 of the propeptide cleavage site, suggesting a dependence on asparaginyl endopeptidases for maturation. Recombinant expression of a CL3 cDNA in Saccharomyces cerevisiae resulted in secretion of the proenzyme form. The propeptide of CL-like proteins was predicted to contain important B-cell epitopes. To determine the contribution of the propeptide to protective immunity, rats were vaccinated with Keyhole Limpet Haemocyanin-conjugated synthetic peptides encoding these putative B-cell epitopes derived from the CL1 or CL3 sequence. A subsequent challenge infection resulted in a significant ( P<0.05) reduction of fluke load compared to adjuvant controls. We conclude that the propeptide of CL3 plays an important role in inducing immunity against F. hepatica infection.

An Arg760Cys mutation in the consensus sequence of the von Willebrand factor propeptide cleavage site is responsible for a new von Willebrand disease variant

AbstractWe describe a von Willebrand disease (VWD) variant characterized by the persistence of von Willebrand factor (VWF) propeptide as a result of a C>T transition at nucleotide 2527 in exon 17 of the VWF gene. This mutation, which was present in the proband and his father, predicts the substitution of Cys for Arg at position 760 of pre–pro-VWF, 4 residues before the propeptide cleavage site belonging to a consensus sequence for substrate recognition by the processing enzyme paired dibasic amino acid–cleaving enzyme (PACE)/furin. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) documented the presence of both processed and unprocessed VWF in the patient's plasma, with unprocessed VWF relatively less represented. The patient's hemostatic phenotype was characterized by a mild decrease in plasma factor VIII (FVIII) and VWF, a decrease in plasma VWF multimers, and a mild reduction in the FVIII binding capacity of VWF. The FVIII binding defect was more pronounced in the proband than in the father because he also inherited the type 2N Arg91Gln mutation from his mother. The persistence of VWF propeptide did not impair VWF synthesis because platelet VWF content was normal, nor did it compromise VWF storage in endothelial cells, because of the normal post–1-deamino-8-D-arginine vasopressin (DDAVP) increase in plasma VWF. Coexpression of wild-type and Arg760Cys VWF into a Furin-producing BHK cell line resulted in decreased VWF secretion and a defect in the FVIII binding capacity of VWF, together with the persistence of VWF propeptide. These findings confirm that a normal consensus sequence for VWF propeptide cleavage and efficient cleavage are required in vivo for normal FVIII binding capacity of VWF.

Eubacterial HslV and HslU subunits homologs in primordial eukaryotes.

ATP-dependent protease complexes are present in all three kingdoms of life, where they rid the cell of misfolded or damaged proteins and control the level of certain regulatory proteins. They include the proteasome in Eukaryotes, Archea, and Actinomycetales and the HslVU (ClpQY) complex in other eubacteria. We showed that genes homologous to eubacterial HslV (ClpQ) and HslU (ClpY) are present in the genome of trypanosomatid protozoa and are expressed. The features of the cDNAs indicated that bona fide trypanosomatid messengers had been cloned and ruled out bacterial contamination as the source of the material. The N-terminal microsequence of HslV from Leishmania infantum (Protozoa: Kinetoplastida) permitted the identification of the propeptide cleavage site and indicated that an active protease is present. High similarities (> or =57.5%) with the prototypical HslV and HslU from Escherichia coli and conservation of residues essential for biochemical activity suggested that a functional HslVU complex is present in trypanosomatid protozoa. The structure of the N-termini of HslV and HslU further suggested mitochondrial localization. Phylogenetic analysis indicated that HslV and HslU from trypanosomatids clustered with eubacterial homologs but did not point to any particular bacterial lineage. Because typical eukaryotic 20S proteasomes are present in trypanosomatids, we concluded that the eubacterial HslVU and the eukaryotic multicatalytic protease are simultaneously present in these organisms. To our knowledge this is the first report of a eubacterial HslVU complex in eukaryotes and, consequently, of the simultaneous occurrence of both a proteasome and HslVU in living cells.

Characterization of a cDNA encoding a cathepsin L-like protein of Rhodnius prolixus.

The interaction of Rhodnius prolixus digestive enzymes with Trypanosoma cruzi could be important for parasite survival. We report herein the complete sequence of the messenger of a cathepsin L-like molecule (RpCat). The cDNA has 5'- and 3'- end UTRs and a methionine codon that corresponds likely to a translation initiation codon. In the deduced amino acid sequence, a region corresponding to an ERFININ domain, diagnostic of L-cathepsins, and a possible pro-peptide cleavage site were observed. At the C-terminus, a nine-amino acid sequence, almost identical to a secretion signal of human cathepsin L was found. RpCat messenger was expressed in intestines of R. prolixus adults, and from 1st to 4th but not in 5th instar nymph stages. In a similarity analysis, RpCat was grouped with L cathepsins forming a clear group separate of the B cathepsins.