A unique anther-mucilage in the pollination biology of Tylosema esculentum

Summary Tylosema esculentum, a perennial geophyte bearing yellow distylous flowers in racemes, maintains a high degree of outbreeding through reciprocal herkogamy. In addition, a viscous liquid, the anther-mucilage, is produced by the anther connective tissue and released concurrently with the pollen. The polysaccharide- and lipid-rich mucilage, which is functional in the shedding and transfer of pollen, is available for more than 1 day due to the gradual solidification of the mucilage. The assimilation of the pollen with the liquid substance significantly affects the pollination biology of T. esculentum. This is the first report on the unique phenomenon of wet pollen in the Caesalpiniaceae.     

Distinct Roles of N-Glycosylation at Different Sites of Corin in Cell Membrane Targeting and Ectodomain Shedding

Corin is a membrane-bound protease essential for activating natriuretic peptides and regulating blood pressure. Human corin has 19 predicted N-glycosylation sites in its extracellular domains. It has been shown that N-glycans are required for corin cell surface expression and zymogen activation. It remains unknown, however, how N-glycans at different sites may regulate corin biosynthesis and processing. In this study, we examined corin mutants, in which each of the 19 predicted N-glycosylation sites was mutated individually. By Western analysis of corin proteins in cell lysate and conditioned medium from transfected HEK293 cells and HL-1 cardiomyocytes, we found that N-glycosylation at Asn-80 inhibited corin shedding in the juxtamembrane domain. Similarly, N-glycosylation at Asn-231 protected corin from autocleavage in the frizzled-1 domain. Moreover, N-glycosylation at Asn-697 in the scavenger receptor domain and at Asn-1022 in the protease domain is important for corin cell surface targeting and zymogen activation. We also found that the location of the N-glycosylation site in the protease domain was not critical. N-Glycosylation at Asn-1022 may be switched to different sites to promote corin zymogen activation. Together, our results show that N-glycans at different sites may play distinct roles in regulating the cell membrane targeting, zymogen activation, and ectodomain shedding of corin.     

The Cytoplasmic Domain of A Disintegrin and Metalloproteinase 10 (ADAM10) Regulates Its Constitutive Activity but Is Dispensable for Stimulated ADAM10-dependent Shedding

The membrane-anchored metalloproteinase a disintegrin and metalloprotease 10 (ADAM10) is required for shedding of membrane proteins such as EGF, betacellulin, the amyloid precursor protein, and CD23 from cells. ADAM10 is constitutively active and can be rapidly and post-translationally enhanced by several stimuli, yet little is known about the underlying mechanism. Here, we use ADAM10-deficient cells transfected with wild type or mutant ADAM10 to address the role of its cytoplasmic and transmembrane domain in regulating ADAM10-dependent protein ectodomain shedding. We report that the cytoplasmic domain of ADAM10 negatively regulates its constitutive activity through an ER retention motif but is dispensable for its stimulated activity. However, chimeras with the extracellular domain of ADAM10 and the transmembrane domain of ADAM17 with or without the cytoplasmic domain of ADAM17 show reduced stimulated shedding of the ADAM10 substrate betacellulin, whereas the ionomycin-stimulated shedding of the ADAM17 substrates CD62-L and TGFα is not affected. Moreover, we show that influx of extracellular calcium activates ADAM10 but is not essential for its activation by APMA and BzATP. Finally, the rapid stimulation of ADAM10 is not significantly affected by incubation with proprotein convertase inhibitors for up to 8 h, arguing against a major role of increased prodomain removal in the rapid stimulation of ADAM10. Thus, the cytoplasmic domain of ADAM10 negatively influences constitutive shedding through an ER retention motif, whereas the cytoplasmic domain and prodomain processing are not required for the rapid activation of ADAM10-dependent shedding events.     

ADAMs, a disintegrin and metalloproteinases, mediate shedding of oxytocinase

Placental leucine aminopeptidase (P-LAP), a type-II transmembrane protease responsible for oxytocin degradation during pregnancy, is converted to a soluble form through proteolytic cleavage. The goal of this study was to determine the nature of the P-LAP secretase activity. The hydroxamic acid-based metalloprotease inhibitors GM6001 and ONO-4817 as well as the TNF-alpha protease inhibitor-2 (TAPI-2) reduced P-LAP release, while tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, which are matrix metalloproteinase inhibitors, had no effect on P-LAP release in Chinese hamster ovary (CHO) cells stably overexpressing P-LAP, thus indicating possible involvement of ADAM (a disintegrin and metalloproteinase) members in P-LAP shedding. Furthermore, overexpression of ADAM9 and ADAM12 increased P-LAP release in P-LAP-CHO transfectants. Immunohistochemical analysis in human placenta demonstrated strong expression of ADAM12 in syncytiotrophoblasts, while little expression of ADAM9 was detected throughout the placenta. Our results suggest ADAM members, at least including ADAM12, are involved in P-LAP shedding in human placenta.     

Mapping of the Interaction Site between Sortilin and the p75 Neurotrophin Receptor Reveals a Regulatory Role for the Sortilin Intracellular Domain in p75 Neurotrophin Receptor Shedding and Apoptosis

Neurotrophins comprise a group of neuronal growth factors that are essential for the development and maintenance of the nervous system. However, the immature pro-neurotrophins promote apoptosis by engaging in a complex with sortilin and the p75 neurotrophin receptor (p75^NTR). To identify the interaction site between sortilin and p75^NTR, we analyzed binding between chimeric receptor constructs and truncated p75^NTR variants by co-immunoprecipitation experiments, surface plasmon resonance analysis, and FRET. We found that complex formation between sortilin and p75^NTR relies on contact points in the extracellular domains of the receptors. We also determined that the interaction critically depends on an extracellular juxtamembrane 23-amino acid sequence of p75^NTR. Functional studies further revealed an important regulatory function of the sortilin intracellular domain in p75^NTR-regulated intramembrane proteolysis and apoptosis. Thus, although the intracellular domain of sortilin does not contribute to p75^NTR binding, it does regulate the rates of p75^NTR cleavage, which is required to mediate pro-neurotrophin-stimulated cell death.     

An Emerging Role of Sonic Hedgehog Shedding as a Modulator of Heparan Sulfate Interactions

Major developmental morphogens of the Hedgehog (Hh) family act at short range and long range to direct cell fate decisions in vertebrate and invertebrate tissues. To this end, Hhs are released from local sources and act at a distance on target cells that express the Hh receptor Patched. However, morphogen secretion and spreading are not passive processes because all Hhs are synthesized as dually (N- and C-terminal) lipidated proteins that firmly tether to the surface of producing cells. On the cell surface, Hhs associate with each other and with heparan sulfate (HS) proteoglycans. This raises the question of how Hh solubilization and spreading is achieved. We recently discovered that Sonic hedgehog (Shh) is solubilized by proteolytic processing (shedding) of lipidated peptide termini in vitro. Because unprocessed N termini block Patched receptor binding sites in the cluster, we further suggested that their proteolytic removal is required for simultaneous Shh activation. In this work we confirm inactivity of unprocessed protein clusters and demonstrate restored biological Shh function upon distortion or removal of N-terminal amino acids and peptides. We further show that N-terminal Shh processing targets and inactivates the HS binding Cardin-Weintraub (CW) motif, resulting in soluble Shh clusters with their HS binding capacities strongly reduced. This may explain the ability of Shh to diffuse through the HS-containing extracellular matrix, whereas other HS-binding proteins are quickly immobilized. Our in vitro findings are supported by the presence of CW-processed Shh in murine brain samples, providing the first in vivo evidence for Shh shedding and subsequent solubilization of N-terminal-truncated proteins.     

The cytosolic domain of protein-tyrosine kinase 7 (PTK7), generated from sequential cleavage by a disintegrin and metalloprotease 17 (ADAM17) and γ-secretase, enhances cell proliferation and migration in colon cancer cells

Protein-tyrosine kinase 7 (PTK7) is a member of the defective receptor protein-tyrosine kinases and is known to function as a regulator of planar cell polarity during development. Its expression is up-regulated in some cancers including colon carcinomas. A 100-kDa fragment of PTK7 was detected in the culture media from colon cancer cells and HEK293 cells. The shed fragment was named sPTK7-Ig1-7 because its molecular mass was very similar to that of the entire extracellular domain of PTK7 that contains immunoglobulin-like loops 1 to 7 (Ig1-7). The shedding of sPTK7-Ig1-7 was enhanced by treatment with phorbol 12-myristate 13-acetate. In addition to the sPTK7-Ig1-7 found in the culture medium, two C-terminal fragments of PTK7 were detected in the cell lysates: PTK7-CTF1, which includes a transmembrane segment and a cytoplasmic domain, and PTK7-CTF2, which lacks most of the transmembrane segment from PTK7-CTF1. Analysis of PTK7 processing in the presence of various protease inhibitors or after knockdown of potential proteases suggests that shedding of PTK7 into sPTK7-Ig1-7 and PTK7-CTF1 is catalyzed by ADAM17, and further cleavage of PTK7-CTF1 into PTK7-CTF2 is mediated by the γ-secretase complex. PTK7-CTF2 localizes to the nucleus and enhances proliferation, migration, and anchorage-independent colony formation. Our findings demonstrate a novel role for PTK7 in the tumorigenesis via generation of PTK7-CTF2 by sequential cleavage of ADAM17 and γ-secretase.     

Ectodomain shedding of E-cadherin and c-Met is induced by Helicobacter pylori infection

Helicobacter pylori, a microaerophilic gram-negative bacterium, colonizes the human stomach. About 50% of the world's population is infected, and this infection is considered as the major risk factor for the development of gastric adenocarcinomas in 1% of infected subjects. Carcinogenesis is characterized by the process of epithelial-to-mesenchymal transition (EMT), in the course of which fully differentiated epithelial cells turn into depolarized and migratory cells. Concomitant disruption of adherence junctions (AJ) is facilitated by growth factors like hepatocyte growth factor 1 (HGF-1), but has been also shown to depend on ectodomain shedding of E-cadherin. The aim of this study was to investigate the impact of infection with H. pylori of NCI-N87 gastric epithelial cells on the shedding of E-cadherin and HGF-receptor c-Met. Our results show that infection with H. pylori provokes shedding of the surface proteins c-Met and E-cadherin. Evidence is provided that ADAM10 contributes to the shedding of c-Met and E-cadherin.     

Syndecan-1 promotes Staphylococcus aureus corneal infection by counteracting neutrophil-mediated host defense

Many microbial pathogens subvert cell surface heparan sulfate proteoglycans (HSPGs) to infect host cells in vitro. The significance of HSPG-pathogen interactions in vivo, however, remains to be determined. In this study, we examined the role of syndecan-1, a major cell surface HSPG of epithelial cells, in Staphylococcus aureus corneal infection. We found that syndecan-1 null (Sdc1(-/-)) mice significantly resist S. aureus corneal infection compared with wild type (WT) mice that express abundant syndecan-1 in their corneal epithelium. However, syndecan-1 did not bind to S. aureus, and syndecan-1 was not required for the colonization of cultured corneal epithelial cells by S. aureus, suggesting that syndecan-1 does not mediate S. aureus attachment to corneal tissues in vivo. Instead, S. aureus induced the shedding of syndecan-1 ectodomains from the surface of corneal epithelial cells. Topical administration of purified syndecan-1 ectodomains or heparan sulfate (HS) significantly increased, whereas inhibition of syndecan-1 shedding significantly decreased the bacterial burden in corneal tissues. Furthermore, depletion of neutrophils in the resistant Sdc1(-/-) mice increased the corneal bacterial burden to that of the susceptible WT mice, suggesting that syndecan-1 moderates neutrophils to promote infection. We found that syndecan-1 does not affect the infiltration of neutrophils into the infected cornea but that purified syndecan-1 ectodomain and HS significantly inhibit neutrophil-mediated killing of S. aureus. These data suggest a previously unknown bacterial subversion mechanism where S. aureus exploits the capacity of syndecan-1 ectodomains to inhibit neutrophil-mediated bacterial killing mechanisms in an HS-dependent manner to promote its pathogenesis in the cornea.     

Adamantyl glycosphingolipids provide a new approach to the selective regulation of cellular glycosphingolipid metabolism

Mammalian glycosphingolipid (GSL) precursor monohexosylceramides are either glucosyl- or galactosylceramide (GlcCer or GalCer). Most GSLs derive from GlcCer. Substitution of the GSL fatty acid with adamantane generates amphipathic mimics of increased water solubility, retaining receptor function. We have synthesized adamantyl GlcCer (adaGlcCer) and adamantyl GalCer (adaGalCer). AdaGlcCer and adaGalCer partition into cells to alter GSL metabolism. At low dose, adaGlcCer increased cellular GSLs by inhibition of glucocerebrosidase (GCC). Recombinant GCC was inhibited at pH 7 but not pH 5. In contrast, adaGalCer stimulated GCC at pH 5 but not pH 7 and, like adaGlcCer, corrected N370S mutant GCC traffic from the endoplasmic reticulum to lysosomes. AdaGalCer reduced GlcCer levels in normal and lysosomal storage disease (LSD) cells. At 40 μM adaGlcCer, lactosylceramide (LacCer) synthase inhibition depleted LacCer (and more complex GSLs), such that only GlcCer remained. In Vero cell microsomes, 40 μM adaGlcCer was converted to adaLacCer, and LacCer synthesis was inhibited. AdaGlcCer is the first cell LacCer synthase inhibitor. At 40 μM adaGalCer, cell synthesis of only Gb(3) and Gb(4) was significantly reduced, and a novel product, adamantyl digalactosylceramide (adaGb(2)), was generated, indicating substrate competition for Gb(3) synthase. AdaGalCer also inhibited cell sulfatide synthesis. Microsomal Gb(3) synthesis was inhibited by adaGalCer. Metabolic labeling of Gb(3) in Fabry LSD cells was selectively reduced by adaGalCer, and adaGb(2) was produced. AdaGb(2) in cells was 10-fold more effectively shed into the medium than the more polar Gb(3), providing an easily eliminated "safety valve" alternative to Gb(3) accumulation. Adamantyl monohexosyl ceramides thus provide new tools to selectively manipulate normal cellular GSL metabolism and reduce GSL accumulation in cells from LSD patients.