Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice

Globální myšlení žáků: srovnání vybraných evropských zemí

In the past decades, when the life on the Earth became interrelated and mutually influenced, the education of the citizens of the world has been pushed to the foreground of the priorities pursued by educational objectives. Should the teaching of geography pursue this educational demand? Do our students think in global dimensions? Do they realise that the life on the Earth is interrelated? Is this knowledge translated into their attitudes and values? The asked questions outline the topic that has become a focus of international research (Béneker et al. 2013). It is the main goal of this paper to highlight the level of Czech students’ world-mindedness as compared to students from selected European countries (the Netherlands, Finland, Germany, Hungary and Serbia)

Transabdominal two-cavity approach for radical nephrectomy combined with inferior vena cava thrombectomy for malignant thrombus caused by renal cell carcinoma: a case series

Abstract Background Advanced renal cell carcinoma in some cases causes malignant intravascular thrombus with the potential for growth into the inferior vena cava or even the right atrium. Renal cell carcinoma is accompanied by malignant intravascular thrombus in up to 10% of cases. We present an overview of three patients diagnosed as having renal cell carcinoma with malignant intravascular thrombus requiring radical nephrectomy combined with inferior vena cava thrombectomy. Case presentation Three patients diagnosed as having renal cell carcinoma were indicated for renal cell carcinoma combined with inferior vena cava thrombectomy between 2014 and 2017 at our department: a 69-year-old white Caucasian woman, a 74-year-old white Caucasian woman, and a 58-year-old white Caucasian woman. According to the Novick classification of inferior vena cava tumor thrombus, there was one infrahepatic (level II) and two supradiaphragmatic (level IV) malignant intravascular thrombi. The average age of these patients was 67 years (range 58–74 years). All patients underwent radical nephrectomy combined with inferior vena cava thrombectomy through transabdominal approach. In patients with level IV malignant intravascular thrombus, transesophageal echocardiogram was used to guide the placement of the inferior vena cava cross-clamp above the diaphragm. In one patient the pericardium was opened to place a cross-clamp above a tumor just below the right atrium. There were no postoperative mortalities to date with an average follow-up of 23 months (range 2–48 months). To date, no patient has demonstrated recurrent inferior vena cava malignant intravascular thrombus requiring secondary inferior vena cava thrombectomy or any other treatment. A comparison of estimated blood loss and transfusion rate was not significantly different in all three cases. Conclusion Despite the technical complexity of the procedure, caval thrombectomy combined with radical nephrectomy currently represents the only radical treatment for renal cell carcinoma accompanied by malignant intravascular thrombus with good mid-term oncological outcomes

Crossing borders to facilitate live donor kidney transplantation: the Czech-Austrian kidney paired donation program – a retrospective study

Kidney paired donation (KPD) is a valuable tool to overcome immunological barriers in living donor transplantation. While small national registries encounter difficulties in finding compatible matches, multi-national KPD may be a useful strategy to facilitate transplantation. The Czech (Prague) and Austrian (Vienna) KPD programs, both initiated in 2011, were merged in 2015. A bi-national algorithm allowed for ABO- and low-level HLA antibody-incompatible exchanges, including the option of altruistic donor-initiated domino chains. Between 2011 and 2019, 222 recipients and their incompatible donors were registered. Of those, 95.7% (Prague) and 67.9% (Vienna) entered into KPD registries, and 81 patients received a transplant (95% 3-year graft survival). Inclusion of ABO-incompatible pairs in the Czech program contributed to higher KPD transplant rates (42.6% vs. 23.6% in Austria). After 2015 (11 bi-national match runs), the median pool size increased to 18 pairs, yielding 33 transplants (8 via cross-border exchanges). While matching rates doubled in Austria (from 9.1% to 18.8%), rates decreased in the Czech program, partly due to implementation of more stringent HLA antibody thresholds. Our results demonstrate the feasibility of merging small national KPD programs to increase pool sizes and may encourage the implementation of multi-national registries to expand the full potential of KPD

N213 glycosylation partially compensates for N336 N-glycan loss.

<p><b>A.</b> The indicated mutants were expressed in Cl8 cells. Each of the mutant proteins migrated more quickly in SDS-PAGE than wild type Smo, but not as quickly as SmoNQ4 or NQ5. <b>B.</b> Glycosylation at N213 partially compensates for N336 glycan loss. The rescue reporter assay was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005473#pgen.1005473.g002" target="_blank">Fig 2A</a>. Each of the indicated double mutants, with the exception of N213Q,N336Q, was able to rescue reporter gene expression in the <i>smo</i> knockdown background to a level similar to that of N336Q. dSmoN213Q,N336Q demonstrated a level of activity similar to dSmoNQ5. Significance was determined using Student’s t-test. <b>C.</b> N336Q-containing double mutants are retained in the ER. Cl8 cells expressing the indicated Smo proteins (anti-Myc, magenta), the Cal-EGFP-KDEL marker (green) and Hh were examined by immunofluorescence microscopy. Whereas wild type Smo reached the plasma membrane, the double mutants overlapped with the ER marker. ActinRed (red) marks F-actin. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right). <b>D.</b> Treatment of lysates from WT or N213Q,N336Q expressing cells with deglycosylating enzymes reveals that the N213,336Q mutant is present in the EndoH sensitive ER fraction, arrowhead. <b>E-E’</b>. N336Q and N213Q,N336Q mutants have disulfide bond defects. Biotin-maleimide was used to tag free thiol groups in cellular lysates prepared from Cl8 cells expressing WT, N336Q, N213Q,N336Q and C320A dSmo proteins. WT dSmo is not well captured on NeutrAvadin beads (lane 2, bound). N-glycan mutants are captured similarly to the disulfide bond mutant C320A (lanes 3–5, bound), indicating that at least one disulfide bridge is disrupted by N-glycan loss. E’ shows the ratio of bound to unbound dSmo proteins normalized to kinesin. Relative binding was determined by densitometry analysis of two independent binding assays. C320A, which has an established disulfide bond defect served as positive control. It’s binding ratio was arbitrarily set to 1.0 and other values are shown relative to it. Error bars are provided to show the standard deviation between the two experiments. <b>F.</b> dSmoN213Q,N336Q fails to rescue <i>smo</i> knockdown <i>in vivo</i>. <i>UAS-dsmoN213Q</i>, <i>N336Q</i> was co-expressed with <i>UAS-dicer</i> and <i>UAS-smo</i>^{<i>3’UTR</i>} using the <i>nubbin-Gal4</i> driver. Its expression did not modify the <i>smo</i> knockdown phenotype (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005473#pgen.1005473.g002" target="_blank">Fig 2F</a>). Multiple progeny were analyzed over two crosses and a representative wing is shown.</p

Identification of Smo N-linked glycosylation sites.

<p><b>A</b>. A multiple sequence alignment of Smo proteins from different phyla are shown. Consensus sequences for N-linked glycosylation are highlighted in gray and the Asn acceptor residues are bold. Sites conserved across vertebrate proteins are indicated as N1-N4. The predicted <i>D</i>. <i>melanogaster</i> sites are not tightly conserved with vertebrates. <b>B</b>. Drosophila Smo is N-glycosylated. Cell lysates prepared from Cl8 cells expressing Hh with wild type or NQ5 dSmo proteins were treated with the indicated deglycosylating enzymes. Wild type Smo demonstrated ER (arrow) and post-ER (arrowhead) glycosylation species. NQ5 migrated similarly to the fully deglycosylated species under all conditions (arrow). <b>C</b>. Mouse Smo is N-glycosylated. Cellular lysates from <i>Smo-/-</i> cells stably expressing mSmoWT or mSmoNQ4 were treated with deglycosylating enzymes and subjected to SDS-PAGE and western blot. mSmoWT resolves as two distinct forms (lane 2). The arrow marks the ER form and the arrowhead indicates the post-ER form. mSmoNQ4 migrates in SDS-PAGE similarly to the PNGase-treated wild type protein (lanes 4–5). <b>C’</b>. mSmoNQ4 is O-glycosylated. Lysates were prepared from NIH3T3 cells expressing mSmoNQ4 and subjected to lambda phosphatase, PNGase and O-glycosidase/neuraminidase treatments. The upper band collapsed upon O-glycosidase/neuraminidase treatment. <b>D</b>. Expression of individual N to Q dSmo mutants. The indicated N to Q dSmo mutants were expressed in Cl8 cells and cell lysates were analyzed by SDS-PAGE and western blot against the Myc tag. Kin serves as loading control. <b>E</b>. Extracellular mSmo consensus sites are N-glycosylated. Mutation of individual extracellular mSmo glycosylation sites induced faster mobility on SDS-PAGE. mSmoN450Q migrated similarly to SmoWT. For western blots, mSmo was detected using anti-Smo and dSmo with anti-Myc. Kinesin (Kin) and Tubulin (Tub) were blotted for loading controls.</p

N-glycosylation status correlates with signal bias.

<p>A model for modulation of mSmo signal bias. SAG (yellow) binding to N-glycosylated mSmo stabilizes a conformation that effectively signals through both canonical and non-canonical routes. SAG binding to the mSmo mutant stripped of N-glycans (red) silences non-canonical signaling, but is highly permissive for canonical signaling.</p

N336Q impacts dSmo trafficking and activity.

<p><b>A.</b> N336 possesses an essential glycan modification. The rescue reporter assay was performed in <i>smo</i> knockdown Cl8 cells as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005473#pgen.1005473.g002" target="_blank">Fig 2</a>. Each of the single N to Q mutants was able to rescue reporter gene activity in the <i>smo</i> knockdown background with the exception of N336Q. This mutant rescued to ~50% of the wild type activity. <b>B.</b> dSmoN336Q is mislocalized. Cl8 cells expressing Hh and the indicated dSmo proteins were imaged by immunofluorescence microscopy. Whereas each of the active single glycosylation site N to Q mutants (anti-Myc, magenta) reached the plasma membrane (indicated by F-actin stain, green) in Hh-expressing cells, dSmoN336Q failed to do so. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right). <b>C.</b> SmoN336Q is retained in the ER. Myc-SmoN336Q expressed in Cl8 cells overlaps with the ER marker protein Calreticulin-KDEL-GFP. Scale bar is 5 μm (upper right). <b>D.</b> Treatment with deglycosylating enzymes demonstrates that the majority of dSmoN336Q is present in the EndoH sensitive, ER resident fraction (black arrowhead). WT and N213Q proteins are equally distributed between ER and post-ER fractions (white arrowhead).</p