Characterization of proADAMTS5 processing by proprotein convertases

ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in arthritis, is synthesized as a zymogen, proADAMTS5. We report a detailed characterization of the propeptide excision mechanism and demonstrate that it is a major regulatory step with unusual characteristics. Using furin-deficient cells and a furin inhibitor, we found that proADAMTS5 was processed by proprotein convertases, specifically furin and PC7, but not PC6B. Mutagenesis of three sites containing basic residues within the ADAMTS5 propeptide (RRR(46), RRR(69) and RRRRR(261)) suggested that proADAMTS5 processing occurs after Arg(261). That furin processing was essential for ADAMTS5 activity was illustrated using the known ADAMTS5 substrate aggrecan, as well as a new substrate, versican, an important regulatory proteoglycan during mammalian development. When compared to other ADAMTS proteases, proADAMTS5 processing has several distinct features. In contrast to ADAMTS1, whose furin processing products were clearly present intracellularly, cleaved ADAMTS5 propeptide and mature ADAMTS5 were found exclusively in the conditioned medium. Despite attempts to enhance detection of intracellular proADAMTS5 processing, such as by immunoprecipitation of total ADAMTS5, overexpression of furin, and secretion blockade by monensin, neither processed ADAMTS5 propeptide nor the mature enzyme were found intracellularly, which was strongly suggestive of extracellular processing. Extracellular ADAMTS5 processing was further supported by activation of proADAMTS5 added exogenously to HEK293 cells stably expressing furin. Unlike proADAMTS9, which is processed by furin at the cell-surface, to which it is bound, ADAMTS5 does not bind the cell-surface. Thus, the propeptide processing mechanism of ADAMTS5 has several points of distinction from those of other ADAMTS proteases, which may have considerable significance in the context of osteoarthritis.     

Tissue distribution and processing of proSAAS by proprotein convertases

The conversion of inactive precursor proteins into bioactive neuropeptides and peptide hormones involves regulated secretory proteins such as prohormone convertases PC1 and PC2. The neuroendocrine protein 7B2 represents a specific binding protein for PC2, and the protein proSAAS, which interacts with PC1, exhibits certain structural and functional homologies with 7B2. With the intention of better understanding the physiological role of proSAAS and its derived peptides, we investigated its tissue localization using a new radioimmunoassay (RIA) to a C-terminal proSAAS-derived peptide. Immunoreactivity corresponding to this SAAS-derived peptide is mostly localized to the brain and gut. Analysis of the brain distribution of the proSAAS-derived peptides indicates that the hypothalamus and pituitary are the two richest areas, consistent with the previously described high expression of PC1 in these two areas. In order to investigate the cleavage of proSAAS by prohormone convertases, we incubated recombinant His-tagged proSAAS with recombinant mouse proPC2 or furin, separated the cleavage products using high-pressure gel permeation chromatography and analyzed the products by RIA. Our results indicate that either PC2 or furin can accomplish in vitro rapid removal and efficient internal processing of the C-terminal peptide, exposing the inhibitory hexapeptide to possible further digestion by carboxypeptidases. Finally, we also studied proSAAS processing in the brains of wild-type and PC2 null mice and found that proSAAS is efficiently processed in vivo. Whereas the C-terminal peptide is mostly internally cleaved in wild-type mouse brain, it is not processed as efficiently in the brain of PC2 null mice, suggesting that PC2 is partially responsible for this cleavage in vivo.     

Proteolytic processing of angiopoietin-like protein 4 by proprotein convertases modulates its inhibitory effects on lipoprotein lipase activity

Angiopoietin-like protein 4 (ANGPTL4) has been associated with a variety of diseases. It is known as an endogenous inhibitor of lipoprotein lipase (LPL), and it modulates lipid deposition and energy homeostasis. ANGPTL4 is cleaved by unidentified protease(s), and the biological importance of this cleavage event is not fully understood with respect to its inhibitory effect on LPL activity. Here, we show that ANGPTL4 appears on the cell surface as the full-length form, where it can be released by heparin treatment in culture and in vivo. ANGPTL4 protein is then proteolytically cleaved into several forms by proprotein convertases (PCs). Several PCs, including furin, PC5/6, paired basic amino acid-cleaving enzyme 4, and PC7, are able to cleave human ANGPTL4 at a consensus site. PC-specific inhibitors block the processing of ANGPTL4. Blockage of ANGPTL4 cleavage reduces its inhibitory effects on LPL activity and decreases its ability to raise plasma triglyceride levels. In summary, the cleavage of ANGPTL4 by these PCs modulates its inhibitory effect on LPL activity.     

Morphological boundary forms by a novel inductive event mediated by Lunatic fringe and Notch during somitic segmentation

Boundary formation plays a central role in differentiating the flanking regions that give rise to discrete tissues and organs during early development. We have studied mechanisms by which a morphological boundary and tissue separation are regulated by examining chicken somite segmentation as a model system. By transplanting a small group of cells taken from a presumptive border into a non-segmentation site, we have found a novel inductive event where posteriorly juxtaposed cells to the next-forming border instruct the anterior cells to become separated and epithelialized. We have further studied the molecular mechanisms underlying these interactions by focusing on Lunatic fringe, a modulator of Notch signaling, which is expressed in the region of the presumptive boundary. By combining DNA in ovo electroporation and embryonic transplantation techniques we have ectopically made a sharp boundary of Lunatic fringe activity in the unsegmented paraxial mesoderm and observed a fissure formed at the interface. In addition, a constitutive active form of Notch mimics this instructive phenomenon. These suggest that the boundary-forming signals emanating from the posterior border cells are mediated by Notch, the action of which is confined to the border region by Lunatic fringe within the area where mRNAs of Notch and its ligand are broadly expressed in the presomitic mesoderm.     

Implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS. Analysis of SARS-CoV spike glycoprotein (S) using recombinant plasmid and virus infections demonstrated that the S-precursor (proS) exists as a approximately 190 kDa endoplasmic reticulum form and a approximately 210 kDa Golgi-modified form. ProS is subsequently processed into two C-terminal proteins of approximately 110 and approximately 80 kDa. The membrane-bound proprotein convertases (PCs) furin, PC7 or PC5B enhanced the production of the approximately 80 kDa protein. In agreement, proS processing, cytopathic effects, and viral titers were enhanced in recombinant Vero E6 cells overexpressing furin, PC7 or PC5B. The convertase inhibitor dec-RVKR-cmk significantly reduced proS cleavage and viral titers of SARS-CoV infected cells. In addition, inhibition of processing by dec-RVKR-cmk completely abrogated the virus-induced cellular cytopathicity. A fluorogenically quenched synthetic peptide encompassing Arg(761) of the spike glycoprotein was efficiently cleaved by furin and the cleavage was inhibited by EDTA and dec-RVKR-cmk. Taken together, our data indicate that furin or PC-mediated processing plays a critical role in SARS-CoV spread and cytopathicity, and inhibitors of the PCs represent potential therapeutic anti-SARS-CoV agents.     

Lunatic fringe null female mice are infertile due to defects in meiotic maturation

We have demonstrated that Notch genes are expressed in developing mammalian ovarian follicles. Lunatic fringe is an important regulator of Notch signaling. In this study, data are presented that demonstrate that radical fringe and lunatic fringe are expressed in the granulosa cells of developing follicles. Lunatic fringe null female mice were found to be infertile. Histological analysis of the lunatic fringe-deficient ovary demonstrated aberrant folliculogenesis. Furthermore, oocytes from these mutants did not complete meiotic maturation. This is a novel observation because this is the first report describing a meiotic defect that results from mutations in genes that are expressed in the somatic granulosa cells and not the oocytes. This represents a new role for the Notch signaling pathway and lunatic fringe in mammalian folliculogenesis.     

Proteolytic cleavage of the rat heparan sulfate 6-O-endosulfatase SulfFP2 by furin-type proprotein convertases

Heparan sulfate 6-O-endosufatases Sulf1 and Sulf2 hydrolyze the 6-O-sulfate of the glucosamine residues in heparin and heparan sulfate, thereby regulating multiple signaling pathways. A previous study reported that human Sulf1 and Sulf2 were proteolytically processed in a manner sensitive to a furin inhibitor. However, the exact sites of cleavage, the sequence motifs for proteolysis, and the effect of the cleavage on enzyme activity remain unknown. Here we show that the cleavage of rat Sulf2 (also called SulfFP2) occurs at two arginine residues, 543 and 570, in the hydrophilic domain. Both sites reside in the consensus sequence for the cleavage by furin-type proprotein convertases, and the consensus motifs are essential for cleavages. The cleavage at arginine 570 is sensitive to a furin inhibitor. Furthermore, the uncleavable form of SulfFP2 shows sulfatase activity comparable to the cleavable SulfFP2, indicating that the cleavage is not indispensable for activation of SulfFP2.     

Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse.

BACKGROUND: The process of somitogenesis can be divided into three major events: the prepatterning of the mesoderm; the formation of boundaries between the prospective somites; and the cellular differentiation of the somites. Expression and functional studies have demonstrated the involvement of the murine Notch pathway in somitogenesis, although its precise role in this process is not yet well understood. We examined the effect of mutations in the Notch pathway elements Delta like 1 (Dll1), Notch1 and RBPJkappa on genes expressed in the presomitic mesoderm (PSM) and have defined the spatial relationships of Notch pathway gene expression in this region. RESULTS: We have shown that expression of Notch pathway genes in the PSM overlaps in the region where the boundary between the posterior and anterior halves of two consecutive somites will form. The Dll1, Notch1 and RBPJkappa mutations disrupt the expression of Lunatic fringe (L-fng), Jagged1, Mesp1, Mesp2 and Hes5 in the PSM. Furthermore, expression of EphA4, mCer 1 and uncx4.1, markers for the anterior-posterior subdivisions of the somites, is down-regulated to different extents in Notch pathway mutants, indicating a global alteration of pattern in the PSM. CONCLUSIONS: We propose a model for the mechanism of somite border formation in which the activity of Notch in the PSM is restricted by L-fng to a boundary-forming territory in the posterior half of the prospective somite. In this region, Notch function activates a set of genes that are involved in boundary formation and anterior-posterior somite identity.     

Biosynthetic processing of the pro-alpha 1(V)2pro-alpha 2(V) collagen heterotrimer by bone morphogenetic protein-1 and furin-like proprotein convertases.

The low abundance fibrillar collagen type V is incorporated into and regulates the diameters of type I collagen fibrils. Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays key roles in regulating formation of vertebrate extracellular matrix; it cleaves the C-propeptides of the major fibrillar procollagens I-III and processes precursors to produce the mature forms of the cross-linking enzyme prolysyl oxidase, the proteoglycan biglycan, and the basement membrane protein laminin 5. Here we have successfully produced recombinant pro-alpha1(V)(2)pro-alpha2(V) heterotrimers, and we have used these to characterize biosynthetic processing of the most prevalent in vivo form of type V procollagen. In addition, we have compared the processing of endogenous pro-alpha1(V) chains by wild type mouse embryo fibroblasts and by fibroblasts derived from embryos doubly homozygous null for the Bmp-1 gene and for a gene encoding the closely related metalloprotease mammalian Tolloid-like 1. Together, results presented herein indicate that within pro-alpha1(V)(2)pro-alpha2(V) heterotrimers, pro-alpha1(V) N-propeptides and pro-alpha2(V) C-propeptides are processed by BMP-1-like enzymes, and pro-alpha1(V) C-propeptides are processed by furin-like proprotein convertases in vivo.     

Sequence divergences between cyst nematode effector protein orthologs may contribute to host specificity

The soybean cyst nematode (Heterodera glycines) and the closely related sugar beet cyst nematode (Heterodera schachtii) are devastating pathogens of plant roots that use secreted effector proteins to engage in sophisticated host-parasite interactions. While H. schachtii infects and reproduces readily on the roots of Arabidopsis thaliana, H. glycines rarely is able to infect this model plant. The molecular basis for differing host ranges remains obscure but likely involves differences between nematode effector proteins and the recognition of host factors. Recently we reported that constitutive expression of the H. schachtii 10A06 effector protein gene (Hs-10A06) in Arabidopsis affected plant morphology and increased susceptibility to H. schachtii and that the 10A06 protein functions through its interaction with Arabidopsis spermidine synthase 2 (SPDS2). Therefore, we investigated whether differences between cyst nematode effector protein orthologs in two nematode species have a role in mediating host specificity. Here, we show that, similar to Hs-10A06, ectopic expression of H. glycines 10A06 (Hg-10A06) in Arabidopsis affected leaf number and root length, however, to a much lesser extent. More importantly, no effect of Hg-10A06 overexpression on Arabidopsis susceptibility to H. schachtii was observed. While we found that Hg-10A06 can weakly interact with Arabidopsis SPDS2 in yeast-two hybrid assays, this ability to interact with SPDS2 was decreased approximately five-fold compared with Hs-10A06. Collectively, these data suggest that sequence divergence between cyst nematode effector protein orthologs could contribute in determining cyst nematode host range.Key words: Heterodera schachtii, arabidopsis, 10A06 effector protein, spermidine synthase 2Cyst nematodes are sedentary pathogens of roots of many economically important crop plants and induce the formation of specialized feeding cells, so-called syncytia, that provide the nematodes with nourishment. The infection process is mediated through secretion of an array of nematode effector proteins inside plant tissues and cells. One of these effector proteins is 10A06, which was initially identified from a gland cell cDNA library from H. glycines, the soybean cyst nematode.¹ The 927 bp full-length H. glycines Hg-10A06 cDNA (GenBank Accession {"type":"entrez-nucleotide","attrs":{"text":"AF502391","term_id":"30315231","term_text":"AF502391"}}AF502391) encoded a predicted protein of 308 amino acids with an N-terminal signal peptide of 17 amino acids for secretion. Recently, we identified the orthologous 10A06 sequence from the sugar beet cyst nematode H. schachtii (Hs-10A06), which is able to infect the model plant Arabidopsis thaliana. The Hs-10A06 cDNA (GenBank Accession {"type":"entrez-nucleotide","attrs":{"text":"GQ373256","term_id":"255528984","term_text":"GQ373256"}}GQ373256) contained an open reading frame of 858 bp encoding a 285-amino acid protein with an N-terminal signal peptide for secretion.² Sequence alignment of H. glycines and H. schachtii 10A06 proteins revealed a strong homology between both orthologues with 86% identity and 87% similarity. The largest difference between the two proteins is the lack of a stretch of 23 amino acids in Hs-10A06. Additionally, a region of 15 amino acid residues located between amino acid 167 and 181 exhibited a high degree of divergence between both proteins. Constitutive expression of Hs-10A06 in Arabidopsis affected plant morphology and increased susceptibility to H. schachtii.² We uncovered in yeast two-hybrid assays that the Hs-10A06 protein interacts with Arabidopsis SPDS2, a key enzyme involved in polyamine biosynthesis, to mediate susceptibility. Here, we assessed the effects of ectopic Hg-10A06 expression in the non-host Arabidopsis on plant morphology and nematode susceptibility. Moreover, we assayed whether Hg-10A06 also is able to interact with SPDS2 from Arabidopsis.     

Schistosomiasis may contribute to goblet cell carcinoid of the appendix

Abstract : To investigate whether schistosomiasis can contribute to appendiceal goblet cell carcinoid, appendix samples were obtained from 3 patients with combined appendiceal schistosomiasis and goblet cell carcinoid (CSG), 6 patients with goblet cell carcinoid only (GCC), 12 patients with appendiceal schistosomiasis only (ASO), and 12 cases with normal appendix (NA), all of similar gender ratio and age distributions. Hematoxylin and eosin-(H&E) stained sections were studied in 3 CSGs and 12 ASOs to diagnose schistosomiasis by detecting schistosome eggs. H&E and alcian blue/PAS-stained sections and immunohistochemistry of CgA and CEA were employed to establish the diagnosis of GCC in the 3 CSGs and 6 GCCs. Then, to determine whether schistosomiasis can contribute to GCC, immunostaining patterns of CgA and Ki67 in mucosal crypt epithelia were investigated and compared among all 33 cases. Our results revealed typical histological and immunohistochemical phenotypes of GCC in the 3 CSGs and 6 GCCs and schistosome egg deposits in 3 CSGs and 12 ASOs. We found that the expression levels of both CgA and Ki67 in mucosal crypt epithelia were significantly higher in CSG than in GCC (P < 0.05 = 0.013 and P = 0.004, respectively). Moreover, high expression levels of both CgA and Ki67 in mucosal crypt epithelia favor ASO as compared to NA (P < 0.001 = 3.4 × 10(-6) and 3.1 × 10(-5), respectively). Our findings suggest that appendiceal schistosomiasis was associated with increased proliferation and neuroendocrine differentiation of mucosal pluripotent crypt cells and that it may contribute to GCC, which is documented to originate from mucosal pluripotent crypt cells in mucosal crypt epithelia.     

The period of the somite segmentation clock is sensitive to Notch activity

The number of vertebrae is defined strictly for a given species and depends on the number of somites, which are the earliest metameric structures that form in development. Somites are formed by sequential segmentation. The periodicity of somite segmentation is orchestrated by the synchronous oscillation of gene expression in the presomitic mesoderm (PSM), termed the "somite segmentation clock," in which Notch signaling plays a crucial role. Here we show that the clock period is sensitive to Notch activity, which is fine-tuned by its feedback regulator, Notch-regulated ankyrin repeat protein (Nrarp), and that Nrarp is essential for forming the proper number and morphology of axial skeleton components. Null-mutant mice for Nrarp have fewer vertebrae and have defective morphologies. Notch activity is enhanced in the PSM of the Nrarp(-/-) embryo, where the ~2-h segmentation period is extended by 5 min, thereby forming fewer somites and their resultant vertebrae. Reduced Notch activity partially rescues the Nrarp(-/-) phenotype in the number of somites, but not in morphology. Therefore we propose that the period of the somite segmentation clock is sensitive to Notch activity and that Nrarp plays essential roles in the morphology of vertebrae and ribs.     

Synchronized oscillation of the segmentation clock gene in vertebrate development

In vertebrate somitogenesis, “segmentation clock” genes (her in zebrafish, hes in mouse, and hairy in chick) show oscillation, synchronized over nearby cells through intercellular interaction. In zebrafish, neighboring cells interact by Delta-Notch signaling to realize synchronization. Under Delta-Notch, however, a cell with a high expression of the segmentation clock gene tends to suppress its expression in adjacent cells, which might produce spatial heterogeneity instead of synchronized oscillation. Here we studied the conditions under which pre-somitic mesoderm cells show synchronized oscillation of gene expression mathematically. We adopted a model that explicitly considers the kinetics of the mRNA and proteins of the segmentation clock gene and cell–cell interaction via Delta-Notch signaling. From statistical study of a model with randomly generated parameters, we revealed how the likelihood that the system generates stable synchronized oscillation depends on the rate of each reaction in the gene–protein kinetics.     

Multiple mechanisms may contribute to the adherence ofCandida yeasts to living cells

The adherence ofCandida yeasts to monolayers of human intestinal epithelium was studied in order to determine the specific and nonspecific mechanisms that might contribute to yeast adherence. Multiple factors were shown to significantly affect the adherence of yeasts to intestinal cells. It was demonstrated that hydrophobic yeasts adhered two times greater than normal yeasts, and positively charged yeasts adhered ten times greater than normal yeasts to monolayers of intestinal epithelium. The binding of yeasts to the intestinal cells was saturable and was most effectively blocked by mucin, which caused an 83% reduction in adherence, whereas the addition ofd-glucose caused a 41% reduction in adherence. Aggregation or coadherence of yeasts occurred as the yeast inocula were increased.Candida appears to possess the ability to adhere to living tissue by several mechanisms, such as adhesin-receptor interactions, nonspecific hydrophobic and ionic bonding, and aggregation or coadherence. This is the first demonstration of multiple forces that may act simultaneously in the process of adherence of yeasts to living cells.