The ubiquitin-like protein PLIC-2 is a negative regulator of G protein-coupled receptor endocytosis.

The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and beta-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics.</p

SdgB glycosylation protects SDR proteins from cleavage by human neutrophil-derived cathepsin G.

<p>(<b>A</b>) Live, in tact WT or Δ<i>sdgB</i> USA300 bacteria were incubated in the presence or absence of human neutrophil lysosomal extracts (NLE). Culture supernatants were immunoblotted with a mAb against the A-domain of ClfA (9E10) to detect cleaved ClfA fragments released from the bacteria. (<b>B</b>) Live, in tact WT or Δ<i>sdgB</i> cells were incubated in the presence or absence of lysosomal extracts from human THP1 cells or mouse RAW cells and culture supernatants were immunoblotted with anti-ClfA. (<b>C</b>) Live, intact WT or Δ<i>sdgB</i> cells were incubated with a panel of purified human neutrophil serine proteases, ie. neutrophil elastase (NE), cathepsin G (CatG), proteinase-3 (P3), and neutrophil serine protease-4 (NSP4). (<b>D</b>) <b>Δ</b><i>sdgB</i> cells were treated with human neutrophil lysosomal extract in the presence or absence of a biochemical inhibitor of cathepsin G. (<b>E</b>) WT or various Sdg-mutant strains were treated with purified human cathepsin G. (<b>B-E</b>) Culture supernatants were analyzed by immunoblotting as in (A) to detect released ClfA fragments. (<b>F</b>) Live bacteria of WT, <b>Δ</b><i>sdgB</i>, or Δ<i>sdgB</i> complemented with exogenous SdgB (p<i>sdgB</i>) were treated with purified human cathepsin G. Culture supernatants (Sup) or cell wall preparations (CWP) were immunoblotted with mAb against the A-domain of ClfA (S4675), SdrD (17H4), or IsdA (2D3). In addition to S4675, another mAb against the A-domain of ClfA (9E10) showed similar results (not shown). (<b>G</b>) Human cathepsin G inhibits adherence of glycosylation-deficient <i>S. aureus</i> to human fibrinogen. Live WT or Δ<i>sdgB</i> USA300 bacteria were pre-incubated with cathepsin G, and allowed to adhere to fibrinogen-precoated plates. Bacterial adhesion was quantified by measuring the amount of bacterial ATP associated with the plates.</p

SdgB and SdgA sequentially modify the SDR-domain with GlcNAc moieties.

<p>(<b>A</b>) SdgB generates rF1 epitopes on SDR protein. A combination of MBP-SDR-His and SdgA or SdgB was co-expressed in <i>E. coli</i>, and cell lysates were immunoblotted with mAb rF1, or with mAb against unmodified SDR (9G4) or anti-His. (<b>B</b>) Cell-free system to reconstitute SDR glycosylation using purified components. Recombinant MBP-SDR-His was incubated with purified SdgA or SdgB, and in the presence or absence of UDP-GlcNAc; rF1 reactivity was induced only in the presence of SdgB and UDP-GlcNAc. (<b>C</b>) Final model for step-wise glycosylation of SDR proteins by SdgA and SdgB. First, SdgB appends GlcNAc moieties onto the SD-region on SDR proteins, followed by additional GlcNAc modification by SdgA. The epitope for mAb rF1 includes the SdgB-dependent GlcNAc moieties. (<b>D</b>) Mass spectrometry analysis to identify the SDR-sugar moieties using purified MBP-SDR-His expressed in <i>E. coli</i>. (Upper panel) Deconvoluted mass spectrum of purified MBP-SDR-His protein, showing the expected intact mass of 58719 Da. (Middle panel) MBP-SDR-His protein was treated with purified SdgB enzyme in the presence of UDP-GlcNAc for 2 h at 37°C. After incubation, the mass of the MBP-SDR-His protein showed several peaks, each peak being separated from the others by the mass of additional GlcNAc residues. (Bottom panel) The above-mentioned reaction mixture of MBP-SDR-His and SdgB (middle panel) was additionally treated with purified SdgA enzyme. After further incubation for 2 hrs at 37°C, up to an additional 47 GlcNAc groups were found to be added. Thus, most of the serines in the DSD motifs in MBP-SD can be modified with these disaccharide sugar moieties.</p

Recognition of SdgB-dependent epitope by human antibodies.

<p>(<b>A</b>) Four different human IgG preparations were reacted with plate-bound CWP from WT or Δ<i>sdgB</i> USA300 by ELISA. To calculate the specific anti-staphylococcal IgG content, data were normalized using a calibration curve with known IgG concentrations of a mAb against peptidoglycan, which has the same reactivity with both USA300 strains by ELISA. Data are expressed as µg/mL of anti-staphylococcal IgG in the serum. The reduction in reactivity observed for CWP from Δ<i>sdgB</i> (red bars) as compared to wild-type CWP (black bars) reflects IgG specific for SdgB-dependent epitopes. Asterisks indicate significant differences (p < 0.05) from WT CWP. (<b>B</b>) CWP from WT, Δ<i>sdgA</i>, or Δ<i>sdgB</i>, Δ<i>sdgAΔsdgB</i> USA300 were immunoblotted with rF1 and three additional human mAbs (SD2, SD3, and SD4) from different patients. All four mAbs showed similar epitope specificity.</p

SdgB is the key rF1 epitope-modifying enzyme.

<p>(<b>A</b>) SdgB is necessary for rF1 reactivity. Cell wall lysates from WT and various putative glycosyltransferase mutants were immunoblotted with mAbs rF1, anti-ClfA (9E10), anti-SdrD (17H4) or anti-panSDR (9G4 α-SDR; recognizes the unmodified SDR-domain. (<b>B</b>) Complementation of Δ<i>sdgB</i> with exogenous SdgB confers rF1 reactivity. Cell wall lysates from WT, glycosyltransferase mutants, and the SdgB-complemented strain were immunoblotted with rF1, anti-ClfA, and anti-SDR mAbs as in (A). (<b>C</b>) Binding of rF1 to whole USA300 bacteria requires SdgB. Binding of mAbs to Δ<i>sdgB</i> USA300 was assessed by flow cytometry as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat-1003653-g001" target="_blank">Figure 1A</a>. (<b>D</b>) rF1-mediated killing of USA300 activity requires SdgB. Wild-type USA300 bacteria preopsonized with rF1 (closed square) or anti-gD (closed circle), and Δ<i>sdgB</i> preopsonized with rF1 (closed triangle) or anti-gD (open circle), were incubated with PMN, and bacterial killing was determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat-1003653-g001" target="_blank">Figure 1C</a>. (<b>E</b>) MBP-SDR-His construct was expressed in WT, Δ<i>sdgA</i>, Δ<i>sdgB</i>, or Δ<i>sgdAΔsdgB S. aureus</i>, and whole cell lysates were immunoblotted with rF1, anti-His and anti-SDR. (<b>F</b>) Preliminary model for step-wise glycosylation of SDR-proteins by SdgB and SdgA. SDR-domains are first glycosylated by SdgB, which appends sugar modifications creating the epitope of mAb rF1. SdgA further modifies these epitopes with additional sugar moieties (left panel). The Δ<i>sdgA S. aureus</i> mutant shows that SdgA-mediated modifications do not influence rF1-binding activity (middle panel). In Δ<i>sdgB or</i> Δ<i>sgdAΔsdgB S. aureus</i>, the unmodified SDR-region is now recognized by the anti-pan-SDR mAb (9G4).</p

mAb rF1 binds to a family of serine-aspartate-repeat (SDR)-proteins.

<p>(<b>A</b>) rF1-reactivity with USA300 CWP is sensitive to proteinase-K (PK) treatment. Lysostapahin-derived CWP from WT (USA300-Δ<i>spa</i>) bacteria was left untreated (lane 1) or treated with 10 µg/mL PK for 1 hour (lane 2), and immunoblotted with rF1. (<b>B</b>) rF1-reacitivty is dependent on the presence of SDR-proteins. CWPs from WT, indicated deletion strains of various combinations of SDR-family proteins <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat.1003653-Fitzgerald1" target="_blank">[12]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat.1003653-McAleese1" target="_blank">[40]</a>, and a Δ<i>spa</i> strain as control for non-specific binding, were immunoblotted with rF1. The lower molecular weight bands (∼50 kDa) were due to non-specific IgG binding to protein A. (<b>C</b>) rF1 also binds to additional SDR-proteins from <i>S. epidermidis</i>. Cell lysates from <i>S. epidemidis</i> were immunoprecipitated with rF1 (lane 1) or an isotype-control mAb (lane 2) and immunoblotted with rF1 mAb. Identities of rF1-reactive bands were revealed by mass-spectrometry of the same lysates (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat.1003653.s002" target="_blank">Figure S2</a>). (<b>D</b>) Alignment of SDR-proteins revealed by mass-spectrometry from <i>S. aureus</i> and <i>S. epidermidis</i>. SDR-regions are indicated by red hatches. Three truncation mutants of clumping factor A (ClfA) that were fused with maltose-binding protein (MBP) are also shown. (<b>E</b>) SDR-region is sufficient for rF1 reactivity. CWPs from <i>S. aureus</i> expressing truncated recombinant constructs were immunoblotted with anti-MBP mAb or rF1 mAb.</p

mAb rF1 exhibits robust binding to and killing of <i>S. aureus</i> bacteria.

<p>(<b>A-C</b>) Bacteria were preopsonized with huIgG1 mAbs rF1 (squares), 4675 anti-ClfA (triangles), or anti-herpes virus gD (circles). (<b>A</b>) Binding of mAbs to WT (USA300-Δ<i>spa</i>) bacteria was assessed by flow cytometry, and expressed as mean fluorescent intensity (MFI). (<b>B</b>) CFSE-labeled, preopsonized WT (USA300-Δ<i>spa</i>) bacteria were incubated with human PMN. Bacterial uptake was expressed as % of CFSE-positive PMN, after gating for CD11b-positive cells by flow cytometry. (<b>C</b>) Preopsonized WT (USA300-Δ<i>spa</i>) bacteria were incubated with PMN to assess bacterial killing. Numbers of viable CFU per mL are representative of at least three experiments. (<b>D</b>) Flow cytometry analysis of binding of rF1 to <i>S. aureus</i> from various infected tissues. Homogenized tissues were double stained with mAb rF1 (X-axis), and with anti-peptidoglycan mAb 702 to distinguish bacteria from tissue debris (Y-axis) (left panel; gate indicated by arrow), followed by gating of bacteria to generate histogram figures. (<b>E</b>) Binding of rF1 to various staphylococcal and non-staphylococcal Gram-positive bacterial species by flow cytometry. <i>Red lines</i>, rF1; <i>blue lines</i>, isotype control mAb anti-gD; <i>green lines</i>, control without mAb. (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat.1003653.s001" target="_blank">Figure S1</a>).</p

Strains, plasmids, and antibodies.

<p>Abbreviations: MRSA, methicillin-resistant <i>Staphylococcus aureus</i>; MSSA, methicillin-sensitive <i>Staphylococcus aureus</i>; VISA, vancomycin intermediate-resistant <i>Staphylococcus aureus</i>; mAb, monoclonal antibody. *NARSA, network on Antibiotic Resistance in <i>Staphylococcus aureus</i> (NARSA) Program supported under NIAID/NIH Contract No. HHSN272200700055C.</p