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PubMed1994

Characterization of tolloid-related-1: a BMP-1-like product that is required during larval and pupal stages of Drosophila development.

The Drosophila tolloid (tld) gene product belongs to a family of developmentally important proteins that includes bone morphogenetic protein-1 (BMP-1). In Drosophila, tld is required at the blastoderm stage to establish pattern within the dorsal half of the embryo. Genetic analysis suggests that the major function of tld is to augment the activity of the decapentaplegic gene product, a close relative of the TGF-beta superfamily members, BMP-2 and BMP-4. In this report, we describe a new gene called tolloid-related-1 (tlr-1) that maps immediately proximal to tld. Sequence analysis indicates that tlr-1 has a large N-terminal extension relative to tld, but otherwise shows the same general organization of sequence motifs found in tld and other BMP-1 family members. These include a region of similarity to astacin, a crayfish metalloprotease, five copies of a repeat first found in complement proteins C1r and C1s, and two copies of an epidermal growth factor-like sequence. In situ hybridization experiments show that tlr-1 expression partially overlaps tld expression in early embryos, but shows unique transcriptional patterns in late stage embryos that are not seen with tld. In larval stages, both genes are expressed in identical patterns in imaginal discs and in the optic lobes of the brain, but tlr-1 is more abundant than tld. Deletions that eliminate tlr-1 expression cause lethality during larval and pupal stages of development. A small proportion of homozygous mutant flies eclose and show wing veination defects. Transgenic animals in which a tlr-1 cDNA is driven by the tld promoter fail to rescue tld mutations, and extra copies of tld fail to rescue tlr-1 mutations, implying that these genes have evolved functionally distinct features. We propose that tld and tlr-1 arose by gene duplication and that each has evolved independently to acquire distinct tissue specific roles in Drosophila development.

Amino Acid Sequence↗
PubMed1990

Ecdysteroids increase the yield of recombinant protein produced in baculovirus insect cell expression system.

Spodoptera frugiperda cells are the hosts of wild type and recombinant virus in the baculovirus insect cell expression system. The expression of the foreign gene could be enhanced by the addition of ecdysteroids and increased amount of recombinant protein was secreted into the medium. The time and concentration dependence of this effect was followed in the case of 20-hydroxyecdysone-, makisterone and ecdysone. 20-hydroxyecdysone proved to be the most efficient, producing a three fold increase in the level of recombinant protein secreted into the medium, as it was measured by ELISA. This effect was also confirmed by tracing the L-(35S)methionine incorporation into the gene product. Makisterone was also effective in stimulation, while ecdysone proved to be ineffective.

Animals↗
PubMed1984

Detection of intermediary Clr with complete active site, using a synthetic proteinase inhibitor.

The synthetic proteinase inhibitor, FUT-175 (6-amidino-2-naphthyl-4-guanidinobenzoate), strongly suppressed activation of Clr at 37 degrees C, causing 50% inhibition at 0.03 mM. To clarify whether the inhibitor was incorporated into the active site of intermediary Clr formed during the incubation, determination of the active site was tried using this inhibitor. Consequently, release of amidinonaphthol equimolar with the amount of Clr used was observed in the early period of incubation, in which the activation to Clr- was about 5%. These results indicate that intermediary Clr already has a complete active site.

Benzamidines↗
PubMed1991

The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1.

Mutations in the Drosophila tolloid (tld) gene lead to a partial transformation of dorsal ectoderm into ventral ectoderm. The null phenotype of tld is similar to, but less severe than decapentaplegic (dpp), a TGF-beta family member required for the formation of all dorsal structures. We have cloned the tld locus by P element tagging. At the blastoderm stage, tld RNA is expressed dorsally, similar to that described for dpp. Analysis of a tld cDNA reveals three sequence motifs: an N terminal region of similarity to a metalloprotease, two EGF-like repeats, and five copies of a repeat found in human complement proteins C1r and C1s. tld sequence is 41% identical to human bone morphogenetic protein 1 (BMP-1); the closest members to dpp within the TGF-beta superfamily are BMP-2 and BMP-4, two other bone morphogenetic proteins. These findings suggest that these genes are members of a signal generating pathway that has been conserved between insects and mammals.

Amino Acid Sequence↗
PubMedDate not supplied

Activities of the MBL-associated serine proteases (MASPs) and their regulation by natural inhibitors.

There has been rapid progress in determining the mechanism by which complement is activated by the complex formed between Mannose-Binding Lectin and its associated proteases (MASPs). MBL and the MASPs are of low abundance, but are similar to the more abundant C1q-C1r2s2 complex (C1), which has been extensively investigated. In this review we summarise recent findings on MBL-MASPs' structure. enzymic activity and regulation, and compare MBL-MASPs with C1.

Animals↗
PubMed1983

Complete amino acid sequence of the catalytic chain of human complement subcomponent C1-r.

The amino acid sequence of human C1-r b chain hs been determined, from sequence analysis performed on fragments obtained by CNBr cleavage, dilute acid hydrolysis, tryptic cleavage of the succinylated protein, and subcleavages by staphylococcal protease. The polypeptide chain contains 242 amino acids (Mr 27 096), and the sequence shows strong homology with other mammalian serine proteases. The histidine, aspartic acid, and serine residues of the active site (His-57, Asp-102, and Ser-195 in bovine chymotrypsinogen) are located at positions 39, 94, and 191, respectively. The chain which lacks the "histidine-loop" disulfide bridge, contains five half-cystine residues, of which four (positions 157-176 and 187-217) are homologous to residues involved in disulfide bonds generally conserved in serine proteases, whereas the half-cystine residue at position 114 is likely to be involved in the single disulfide bridge connecting the catalytic b chain to the n-terminal a chain. Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 51 and 118.

Amino Acid Sequence↗
PubMed2006

The interaction of recombinant subdomains of the procollagen C-proteinase with procollagen I provides a quantitative explanation for functional differences between the two splice variants, mammalian tolloid and bone morphogenetic protein 1.

The procollagen C-proteinase (PCP) is a zinc peptidase of the astacin family and the metzincin superfamily. The enzyme removes the C-terminal propeptides of fibrillar procollagens and activates other matrix proteins. Besides its catalytic protease domain, the procollagen C-proteinase contains several C-terminal CUB modules (named after complement factors C1r and C1s, the sea urchin UEGF protein, and BMP-1) and EGF-like domains. The two major splice forms of the C-proteinase differ in their overall domain composition. The longer variant, termed mammalian tolloid (mTld, i.e., PCP-2), has the protease-CUB1-CUB2-EGF1-CUB3-EGF2-CUB4-CUB5 composition, whereas the shorter form termed bone morphogenetic protein 1 (BMP-1, i.e., PCP-1) ends after the CUB3 domain. Two related genes encode proteases similar to mTld in humans and have been termed mammalian tolloid like-1 and -2 (mTll-1 and mTll-2, respectively). For mTll-1, it has been shown that it has C-proteinase activity. We demonstrate that recombinant EGF1-CUB3, CUB3, CUB3-EGF2, EGF2-CUB4, and CUB4-CUB5 modules of the procollagen C-proteinase can be expressed in bacteria and adopt a functional antiparallel beta-sheet conformation. As shown by surface plasmon resonance analysis, the modules bind to procollagen I in a 1:1 stoichiometry with dissociation constants (K(D)) ranging from 622.0 to 1.0 nM. Their binding to mature collagen I is weaker by at least 1 order of magnitude. Constructs containing EGF domains bind more strongly than those consisting of CUB domains only. This suggests that a combination of CUB and EGF domains serves as the minimal functional unit. The binding affinities of the EGF-containing modules for procollagen increase in the order EGF1-CUB3 < CUB3-EGF2 < EGF2-CUB4. In the context of the full length PCP, this implies that a given module has an affinity that continues to increase the more C-terminally the module is located within the PCP. The tightest binding module, EGF2-CUB4 (K(D) = 1.0 nM), is only present in mTld, which might provide a quantitative explanation for the different efficiencies of BMP-1 and mTld in procollagen C-proteinase activity.

Animals↗
PubMed2006

IFN-gamma upregulates expression of the mouse complement C1rA gene in keratinocytes via IFN-regulatory factor-1.

We examined the expression of the mouse complement component C1rA (mC1rA) in IFN-gamma-stimulated mouse keratinocytes (Pam 212) and found that it was upregulated. To analyze the mechanism involved, we cloned the 2,150 bp 5'-flanking region of mC1rA by the vectorette-PCR technique, and identified the transcription start site of mC1rA by rapid amplification of complementary DNA ends. Analysis of the 5' sequence revealed putative binding sites for activator protein 1, CCAAT/enhancer binding protein (C/EBP), signal transducer and activator of transcription 1 (STAT-1), IFN-regulatory factor-1 (IRF-1), and others. We detected transcriptional activation dependent on this upstream region in reporter gene assays and Northern blots. To identify the cis-acting regulatory elements involved, we analyzed serial deletion constructs of the promoter using luciferase reporters. The -80 to -19 bp region, which contains a putative IRF-1 binding site, was required for both basal promoter activity and responses to IFN-gamma. The use of site-directed point mutations, electrophoresis mobility shift assays, and supershift assays indicated that the putative IRF-1 binding site was essential for both IFN-gamma-dependent and -independent transcriptional activity of the mC1rA promoter. We conclude that IFN-gamma stimulates mC1rA gene expression via IRF-1 in mouse keratinocytes.

Animals↗
PubMed1994

Enterokinase, the initiator of intestinal digestion, is a mosaic protease composed of a distinctive assortment of domains.

Enterokinase is a protease of the intestinal brush border that specifically cleaves the acidic propeptide from trypsinogen to yield active trypsin. This cleavage initiates a cascade of proteolytic reactions leading to the activation of many pancreatic zymogens. The full-length cDNA sequence for bovine enterokinase and partial cDNA sequence for human enterokinase were determined. The deduced amino acid sequences indicate that active two-chain enterokinase is derived from a single-chain precursor. Membrane association may be mediated by a potential signal-anchor sequence near the amino terminus. The amino terminus of bovine enterokinase also meets the known sequence requirements for protein N-myristoylation. The amino-terminal heavy chain contains domains that are homologous to segments of the low density lipoprotein receptor, complement components C1r and C1s, the macrophage scavenger receptor, and a recently described motif shared by the metalloprotease meprin and the Xenopus A5 neuronal recognition protein. The carboxyl-terminal light chain is homologous to the trypsin-like serine proteases. Thus, enterokinase is a mosaic protein with a complex evolutionary history. The amino acid sequence surrounding the amino terminus of the enterokinase light chain is ITPK-IVGG (human) or VSPK-IVGG (bovine), suggesting that single-chain enterokinase is activated by an unidentified trypsin-like protease that cleaves the indicated Lys-Ile bond. Therefore, enterokinase may not be the "first" enzyme of the intestinal digestive hydrolase cascade. The specificity of enterokinase for the DDDDK-I sequence of trypsinogen may be explained by complementary basic-amino acid residues clustered in potential S2-S5 subsites.

Amino Acid Sequence↗
PubMed1999

Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity.

A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease.

Amino Acid Sequence↗
PubMed2003

A recombinant chimeric epidermal growth factor-like module with high binding affinity for integrins.

Integrins are cell surface receptors involved in numerous pathological processes such as metastasis invasion and abnormal angiogenesis. To target these receptors, the epidermal growth factor (EGF)-like domain of human complement protease C1r was used as a natural scaffold to design chimeric modules containing the RGD motif. Here we report a high yield bacterial expression system and its application to the production of two such modules, EGF-RGD and V2, the latter variant mimicking the RGD-containing domain of disintegrins. These modules were characterized chemically, and their biological activity was investigated by cellular assays using various Chinese hamster ovary cell lines expressing beta1 and beta3 integrins and by surface plasmon resonance spectroscopy. Remarkably, the modifications leading to the V2 variant had differential effects on the interaction with beta3 and beta1 integrins. The disintegrin-like V2 module exhibited enhanced binding affinities compared with EGF-RGD, with KD values of 7.2 nm for alpha5beta1 (a 4-fold decrease) and 3.5 nm for alphavbeta3 (a 1.5-fold decrease), comparable with the values determined for natural integrin ligands. Analysis by NMR spectroscopy also revealed a differential dynamic behavior of the RGD motif in the EGF-RGD and V2 variants, providing insights into the structural basis of their relative binding efficiency. These novel RGD-containing EGF modules open the way to the design of improved variants with selective affinity for particular integrins and their use as carriers for other biologically active modules.

Amino Acid Sequence↗
PubMed2005

High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays.

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.

Amino Acid Sequence↗
PubMed2003

Optimized protocol for linear RNA amplification and application to gene expression profiling of human renal biopsies.

Gene expression analysis using high-density cDNA or oligonucleotide arrays is a rapidly emerging tool for transcriptomics, the analysis of the transcriptional state of a cell or organ. One of the limitations of current methodologies is the requirement of a relatively large amount of total or polyadenylated RNA as starting material. Standard array hybridization protocols require 5-15 micrograms labeled RNA. To obtain these quantities from small amounts of starting RNA material, RNA can be amplified in a linear fashion. Here we introduce an optimized protocol for rapid and easy-to-use amplification of as little as 1 ng total RNA. Our analysis shows that this method is linear and highly reproducible and that it preserves similarities as well as dissimilarities between normal and disease-related samples. We applied this technique to the RNA expression profiling of human renal allograft biopsies with normal histology and compared them to the profiles of renal biopsies with histological evidence of chronic transplant nephropathy or chronic rejection. Among others, complement component C1r was found to be significantly up-regulated in chronic rejection and chronic transplant nephropathy biopsies compared to normal samples, while fructose-1,6-biphosphatase showed lower-than-normal expression.

Gene Expression Profiling↗
PubMed1988

Selective synthesis of mRNA and proteins by human peripheral blood neutrophils.

Human peripheral blood polymorphonuclear neutrophils (PMN) have been considered to be capable of little if any protein biosynthesis. We evaluated the ability of PMN to synthesize both mRNA and proteins. Using in vitro [35S]methionine pulse-chase labeling of purified PMN, followed by immunoprecipitation of cell lysates with immobilized mAb and analysis by gel electrophoresis, PMN were shown to synthesize CR1, FcR, CR3 alpha-chain, MHC class I, and actin. In contrast, incorporation of [35S]methionine into either CR3 beta-chain or the secondary granule protein lactoferrin was not detected. Purification of mRNA from PMN and analysis by Northern blots demonstrated the presence in PMN of CR1, actin, and MHC class I transcripts. However, despite the apparent lack of CR3 beta-chain biosynthesis, specific beta-chain message was detectable in PMN RNA. Inhibition of mRNA synthesis in PMN with actinomycin D resulted in decreased synthesis of nascent CR1, FcR, MHC class I, and actin compared with control cells. Thus, PMN continue to transcribe and translate the genes for certain membrane and cytoskeletal proteins. In contrast, the lack of detectable synthesis of either lactoferrin or CR3 beta-chain suggested that biosynthesis in circulating PMN is selective.

Blood Proteins↗
PubMed1981

[Isolation and crystallization of the Clr subunit of the first component of human complement (author's transl)].

Isolation of Clr from Cohn-Fraction I of human plasma is accomplished by a series of steps which include affinity chromatography on IgG-p-azobenzyloxyethylsulfonylethoxy-Sepharose CL-4B, ion exchange chromatography and preparative zone electrophoresis. The protein is easily soluble in 0.1 M Tris/HCl buffer, pH 10.0 and is crystallized readily by means of dialysis against 0.15 M saline, pH 7.0 in the form of hexagonal dipyramides. The structural elucidation using X-ray analysis is now possible.

Chromatography, Affinity↗
PubMed1996

Impaired production of both normal and mutant C1 inhibitor proteins in type I hereditary angioedema with a duplication in exon 8.

In the autosomal dominant disorder type I hereditary angioedema, reduced levels of C1 inhibitor may be due in part to increased turnover and decreased synthesis of normal C1 inhibitor protein. A type I hereditary angioedema patient was recently described in whom the C1 inhibitor mutation consisted of a 20-bp duplication of nucleotides 1414 to 1433 in exon 8 that introduced a frame shift predicting the loss of a normal stop codon and the translation of a protein 52 amino acids longer than normal. In this study, we analyzed the expression of C1 inhibitor in fibroblasts obtained from a skin biopsy of this patient. Two proteins of approximately 78 and 94 kDa were found intracellularly, corresponding to the products of normal and mutated alleles, respectively. Pulse-chase analysis showed a complete lack of secretion of the mutated form. In addition, there was decreased extracellular production of the normal C1 inhibitor, suggesting either decreased secretion or increased intracellular catabolism of the normal protein because of the presence of the mutant allele. The production of other complement proteins was normal. This study provides a model for further analysis of autosomal dominant genetic disorders in which production of the functional protein may be affected by the product of the mutated allele.

Alleles↗
PubMed1984

Structure and activity of C1r and C1s.

During activation of the first component of the classical complement pathway the two zymogen subcomponents, C1r and C1s are converted to active proteolytic enzymes. Activated C1r cleaves C1s which then becomes the activator of C4 and C2. Amino acid sequence studies of the proteolytic chains of C1r and C1s, carried out in Oxford and Aberdeen respectively, have shown that they belong to the serine proteinase family. Modelling of these sequences to the three-dimensional coordinates of chymotrypsin (Birktoft & Blow 1972) reveals that both molecules have a conserved structural core, and that most of the differences lie in the external loops. Catalytically functional residues (Ile-16, His-57, Asp-102, Ser-195) are conserved, and residue 189 is aspartic acid, consistent with the known trypsin-like specificity of cleavage. Examination of the amino acid sequences of C4a, and comparison with those of the homologous molecules C3a and C5a, shows that there is a marked difference in the distribution of basic residues near the C-terminal arginine residue which is the site of action of C1s. When these amino acid sequences are modelled to the coordinates of C3a (Huber et al. 1980) and docked to the active site of C1s, the basic residues of C4a appear to interact with two glutamate residues peculiar to C1s, suggesting that this interaction may contribute to the ability of C1s to discriminate C4 from C3 and C5.

Amino Acid Sequence↗