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Inhibitor of ppGalNAc-T3-mediated O-glycosylation blocks cancer cell invasiveness and lowers FGF23 levels

  1. Lina Song
  2. Adam D Linstedt  Is a corresponding author
  1. Carnegie Mellon University, United States
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Cite this article as: eLife 2017;6:e24051 doi: 10.7554/eLife.24051

Abstract

Small molecule inhibitors of site-specific O-glycosylation by the polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) family are currently unavailable but hold promise as therapeutics, especially if selective against individual ppGalNAc-T isozymes. To identify a compound targeting the ppGalNAc-T3 isozyme, we screened libraries to find compounds that act on a cell-based fluorescence sensor of ppGalNAc-T3 but not on a sensor of ppGalNAc-T2. This identified a hit that subsequent in vitro analysis showed directly binds and inhibits purified ppGalNAc-T3 with no detectable activity against either ppGalNAc-T2 or ppGalNAc-T6. Remarkably, the inhibitor was active in two medically relevant contexts. In cell culture, it opposed increased cancer cell invasiveness driven by upregulated ppGalNAc-T3 suggesting the inhibitor might be anti-metastatic. In cells and mice, it blocked ppGalNAc-T3-mediated glycan-masking of FGF23 thereby increasing its cleavage, a possible treatment of chronic kidney disease. These findings establish a pharmacological approach for the ppGalNAc-transferase family and suggest that targeting specific ppGalNAc-transferases will yield new therapeutics.

https://doi.org/10.7554/eLife.24051.001

eLife digest

Complex cascades of interactions between different molecules regulate every process in the body. Enzymes are critical for this, because they act as ‘catalysts’ to speed up chemical reactions in a cell. Each type of enzyme has a specific role. The enzyme ppGalNAc-T3, for example, attaches sugar molecules onto certain proteins via a process called glycosylation. This modification fine-tunes the activity of the proteins.

The enzyme ppGalNAc-T3 is implicated in at least two medically important pathways. It increases the amount of the hormone FGF23, which regulates phosphate levels in the bloodstream. Hormones are messenger molecules that regulate most processes that are crucial for life, and too much or too little of a hormone can lead to diseases. High levels of FGF23, for example, can cause serious and often fatal problems in patients with chronic kidney disease.

The enzyme ppGalNAc-T3 is also known to encourage and stimulate some cancer cells to spread from their original location to other parts of the body – a process known as metastasis – which ultimately leads to the vast majority of cancer deaths every year. Thus, a drug or molecule that blocks ppGalNAc-T3 could be used to lower FGF23 levels in patients with kidney disease and potentially prevent cancer cells from spreading. However, until now, it was unknown if such a molecule existed.

To identify a compound that specifically regulates ppGalNAc-T3, Song and Linstedt engineered human cells grown in the laboratory to become fluorescent when ppGalNAc-T3 was blocked. Out of the over 20,000 compounds screened, one compound named T3Inh-1 selectively blocked ppGalNAc-T3. Further experiments showed that the T3Inh-1 compound reduced FGF23 hormone levels in both tissue cells grown in the laboratory and mice, without causing any toxic side effects. It also prevented breast cancer cells grown in the laboratory from spreading. The results demonstrated that T3Inh-1 is the first drug-like inhibitor that can target this kind of enzyme.

An important next step will be to test the compound in animal models for chronic kidney disease and cancer metastasis.

https://doi.org/10.7554/eLife.24051.002

Introduction

Glycosylation, which fine-tunes the function of proteins, is the most abundant and diverse posttranslational modification (Schjoldager and Clausen, 2012; van der Post et al., 2013; Moremen et al., 2012). Despite many documented roles in health and disease, the enzymes that mediate mucin-type O-glycosylation in the Golgi apparatus have yet to be discovered as druggable targets. The initiating enzymes, a family of 20 ppGalNAc-transferase isozymes, determine which substrates are modified and at which sites. Significant questions remain regarding their specificity, regulation, targets and functions and the lack of a pharmacological approach has been a critical limitation. The only confirmed inhibitor of mucin-type O-glycosylation, benzyl-N-acetyl-α-galactosaminide, blocks elongation rather than initiation and requires millimolar concentrations that can be toxic (Kuan et al., 1989; Patsos et al., 2009). Not only are pan-specific modulators of the ppGalNAc-transferases lacking, there is nothing isoform-specific. The latter will be essential to restrict effects to particular pathways and could possibly lead to a new basis for therapeutics conceptually related to the widespread use of drugs targeting individual protein kinases.

ppGalNAc-T3 serves as an important test case as it is implicated in at least two medically important pathways: cancer metastasis and stabilization of FGF23 (Chefetz and Sprecher, 2009; Schjoldager et al., 2011; Kato et al., 2006; Peng et al., 2012; Kohsaki et al., 2000; Kitada et al., 2013; Gao et al., 2013; Brockhausen, 2006; Brooks et al., 2007). ppGalNAc-T3 is overexpressed in cancerous tissue often correlating with shorter survival (Kitada et al., 2013; Brockhausen, 2006; Harada et al., 2016; Mochizuki et al., 2013). Knockdown of ppGalNAc-T3 expression in cultured ovarian cancer cells inhibits their invasive capacities arguing that ppGalNAc-T3 has potential as a therapeutic target (Wang et al., 2014). ppGalNAc-T3 mediates glycan-masking of FGF23 in bone as part of a control mechanism determining the form of FGF23 that is secreted (Kato et al., 2006; Tagliabracci et al., 2014). When present, the added O-glycan blocks FGF23 cleavage by the furin protease resulting in secretion of intact FGF23 that activates FGF23 receptor complexes at the kidney and intestine (Rowe, 2015). In contrast, non-glycosylated FGF23 is cleaved and the cleaved C-terminal product competitively blocks these same receptors (Goetz et al., 2010). Significantly, elevated intact FGF23 occurs in chronic kidney disease and upon kidney transplant where it is directly linked to poor prognosis due to its effects on renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis (Eckardt and Kasiske, 2009). Many studies have concluded that therapeutic control over FGF23 would be transformative in the clinic (Degirolamo et al., 2016; Fukumoto, 2016; Isakova et al., 2015; Smith, 2014).

As a step toward testing whether biologic discoveries and new therapeutics will result from developing small molecule modulators that control the activity of specific ppGalNAc-transferases we initiated high throughput cell-based screening of compound libraries. An inhibitor of ppGalNAc-T3 was identified and found to block breast cancer cell invasiveness as well as secretion of intact FGF23 promising new therapeutic approaches for cancer and chronic kidney disease, respectively.

Results and discussion

HEK cell lines were engineered to express fluorescent sensors that are specific to ppGalNAc-T2 or ppGalNAc-T3 activity (Song et al., 2014). For each sensor, glycosylation of its isozyme-specific target site prevents furin protease from removing a blocking domain (Figure 1A). Thus, fluorescence increases upon ppGalNAc-transferase inhibition because removal of the blocking domain allows dimerization of a fluorogen activating protein domain so that it binds and activates the fluorescence of malachite green. They are ratiometric because the sensor backbone contains a green fluorescent protein as an internal control for expression. Each sensor showed clear activation after mutation of its glycan acceptor sites and these mutated constructs served as positive controls in the screen (Figure 1B, ∆glycan). The T3 sensor exhibited a background level of activation due to incomplete glycosylation (Song et al., 2014) but this was considered advantageous for the possible identification of enzyme activators along with the desired inhibitors. Consistent with our previous report (Song et al., 2014), sensor expression in HEK cell lines depleted of either ppGalNAc-T2 or T3 via zinc finger nuclease editing resulted in specific activation of the corresponding sensor confirming their isozyme selectivity (Figure 1B, HEK∆T2, HEK∆T3). Our screen included compounds based on structural diversity (21,710 compounds in total) with 6 hr treatments at 10 µM prior to flow cytometry to assay MG and GFP fluorescence on a cell-by-cell basis (Figure 1C). Each compound was tested in duplicate and against both sensors (Figure 1—source data 1). Because each sensor requires essentially identical cellular reactions- the only difference being which ppGalNAc-transferase isoform modifies the sensor- most off-target hits (such as sugar nucleotide transporters, extending enzymes, or furin) will alter both sensors, whereas directly acting, isoform-specific candidates will be sensor-specific. Using cut-off parameters for the MG/GFP ratios that excluded >99% of the compounds (Q ≥ 3 or Q ≤ −2.5), the screen yielded 72 sensor-specific hits with 18 increasing and 35 decreasing the T2 sensor fluorescence and 11 increasing and 8 decreasing the T3 sensor fluorescence (Figure 1D).

Figure 1 with 3 supplements see all
Screen for modulators of ppGalNAc-T2/T3.

(A) Diagram showing sensor design and the linker sequences used. O-glycosylation of the linker masks the furin site but if an inhibitor blocks the ppGalNAc-transferase then furin cleaves the linker releasing the blocking domain (BD) allowing fluorescent activating protein (FAP) dimerization and dye activation. Linker furin sites are underlined and sites of glycosylation or mutation are in bold. (B) HEK cell lines with or without ppGalNAc-T2 or T3 stably expressing the WT or ∆glycan T2 or T3 sensor constructs (see linkers in A) were imaged in the presence of 110 nM of the dye MG11p (MG) to detect GFP or MG. Bar = 20 µm. (C) Schematic showing cell plating, drug treatment, cell release, fluorescence measurement and parallel analysis using both T2 and T3 sensors. Hits that activate both may be pan-specific or act on off-target pathways common to both sensors whereas sensor specific hits are likely acting directly on the corresponding ppGalNAc-transferase. (D) The plot shows Q values (Q=(R-RNeg)/SDNeg) for each compound (treatment at 10 µM for 6 hr) using the average of duplicate MG/GFP ratios for the compound (R), the vehicle-only control (RNeg), and the standard deviation of the vehicle-only controls (SDNeg). The cut-off values of +3 and −2.5 are indicated (*). Also indicated are the values for the positive controls (T2∆glycan and T3∆glycan) and the structure of the indicated T3-specific hit (inset). (E) Values (% enzyme activity relative to vehicle-only controls) in the in vitro assay using purified ppGalNAc-T2 or T3 as a secondary screen are shown for 20 hits from the primary screen. Compounds were present at 50 µM. Compound 1614 is T3Inh-1.

https://doi.org/10.7554/eLife.24051.003
Figure 1—source data 1

Primary screen data for HEK cells expressing T2 or T3 sensors.

The accompanying spreadsheet shows calculated Q values (see Methods) for each compound tested. Note that autofluorescent compounds are left blank.

https://doi.org/10.7554/eLife.24051.004
Figure 1—source data 2

Secondary screen data (in vitro enzyme assays).

https://doi.org/10.7554/eLife.24051.005

To determine which of these directly acted on the targeted enzymes, we carried out in vitro glycosylation assays in which the purified lumenal domains of ppGalNAc-T2 or ppGalNAc-T3 (containing catalytic and lectin domains) were incubated with peptide and UDP-GalNAc substrates in the presence of 50 µM of each compound. A second stage reaction (UDP-Glo) then converted the accumulated UDP product to ATP and then, via luciferase, to light. This resulted in 20 candidates that either reduced or increased the luminescence by a factor ≥50% relative to vehicle-only controls (Figure 1E). Of these, one compound (#1614) stood out as a strong and selective inhibitor of ppGalNAc-T3 and became the focus of this phase of the study. The compound is a quinoline of no known activity that we now refer to as ppGalNAc-T3 Inhibitor 1 or T3Inh-1 (Figure 1D, inset).

Importantly, T3Inh-1 exhibited no toxicity and did not affect HEK cell proliferation (Figure 1—figure supplement 1). Also, a 24 hr treatment did not affect staining intensity by the lectins Concanavalin A (ConA), Wheat germ agglutinin (WGA), Sambucus Nigra (SNA) or Vicia Villosa (VVA), which bind branched alpha-mannose, N-acetylglucosamine, sialic acid, or terminal GalNAc, respectively (Figure 1—figure supplement 2). This implies that the enzymes contributing to the abundant glycans of N- and O-glycosylation detected by these lectins were unaffected and is consistent with ppGalNAc-T3 modifying a relatively limited number of substrates (Schjoldager et al., 2015). Finally, there was no change in localization or expression level of ppGalNAc-T3, ppGalNAc-T2 or any other Golgi marker tested (Figure 1—figure supplement 3).

To determine the effective concentration of T3Inh-1 in cells and in vitro, it was retested at various doses against the sensors and against the purified enzymes. T3Inh-1 activated the T3 sensor with an apparent IC50 of 12 µM and showed little or no activity towards the T2 sensor (Figure 2A). Similarly, T3Inh-1 was a potent and selective direct inhibitor of ppGalNAc-T3 (Figure 2B). Inhibition of ppGalNAc-T3 occurred with an IC50 of 7 µM and was undetectable against ppGalNAc-T2. T3Inh-1 also lacked activity against ppGalNAc-T6 (Figure 2B), which is the isozyme considered most closely related to ppGalNAc-T3 (Bennett et al., 2012Revoredo et al., 2016).

T3Inh-1 is a direct mixed-mode inhibitor of ppGalNAc-T3.

(A) Comparison of T2 and T3 sensor activation at the indicated concentrations of T3Inh-1 (n = 3 ± SEM). MG/GFP ratio was determined for 20,000 cells by FACS and average value is plotted as percent of the positive control (i.e. the ∆glycan version of each sensor). (B) Comparison of effect of the indicated concentrations of T3Inh-1 on in vitro glycosylation mediated by purified ppGalNAc-T2, ppGalNAc-T3, or ppGalNAc-T6. Values are averages expressed as percentage of the control ‘vehicle-only’ reactions (n = 6 ± SEM for ppGalNAc-T3, n = 3 ± SEM for others). (C–D) The in vitro assay was carried out in the presence of 0, 7.5, or 15 µM T3Inh-1 at the indicated concentrations of peptide or UDP-GalNAc substrate. Values are averages expressed as percent of the control reactions with no inhibitor and saturating substrates (n = 3 ± SEM). (E) Representative fluorescence spectra are shown for T3Inh-1 alone or for purified ppGalNAc-T3 in the presence of the indicated concentrations of T3Inh-1. Note dose-dependent quenching of tryptophan fluorescence indicating direct binding. (F) Fluorescence quenching was quantified at each concentration using the peak value at 324 nm (n = 3 ± SEM). Note that all graphs have error bars but some are too small to be apparent.

https://doi.org/10.7554/eLife.24051.011
Figure 2—source data 1

Panel A: sensor signals versus T3Inh-1 concentration.

https://doi.org/10.7554/eLife.24051.012
Figure 2—source data 2

Panel B: enzyme activity versus T3Inh-1 concentration.

https://doi.org/10.7554/eLife.24051.013
Figure 2—source data 3

Panel C: inhibitor effect versus peptide concentration.

https://doi.org/10.7554/eLife.24051.014
Figure 2—source data 4

Panel D: inhibitor effect versus UDP-GalNAc concentration.

https://doi.org/10.7554/eLife.24051.015
Figure 2—source data 5

Panel E and F: Fluorescence change versus T3Inh-1 concentration.

https://doi.org/10.7554/eLife.24051.016

Towards characterizing the mechanism of inhibition we used the in vitro assay with purified ppGalNAc-T3 and individually varied both peptide and UDP-GalNAc substrate concentrations in the presence of 0, 7.5 or 15 µM T3Inh-1. The results were similar for both substrates (Figure 2C–D), where T3Inh-1 decreased the Vmax and increased the Km (Table 1) indicating a mixed-mode of inhibition in which the inhibitor most likely binds both free enzyme to reduce substrate binding and enzyme-substrate complexes to reduce turnover. Implied in this model of action is direct binding to the enzyme typically at an allosteric site. To test for direct binding, intrinsic tryptophan fluorescence of ppGalNAc-T3 was determined in the presence of increasing T3Inh-1 concentrations. At all concentrations, the compound itself yielded miniscule signals, whereas the compound had a profound and dose-dependent effect on the ppGalNAc-T3 emission spectrum (Figure 2E). These results confirmed direct binding with an apparent Kd of 17 µM (Figure 1F). The similarity in concentration dependence of sensor activation in cells, in vitro inhibition and direct binding argues that T3Inh-1 acts directly on cellular ppGalNAc-T3 and inhibits its activity.

Table 1

Inhibition by T3Inh-1 at varying substrate concentrations. Values shown were determined from the data in Figure 2 using Prism (see Materials and methods).

https://doi.org/10.7554/eLife.24051.017
SubstrateParameter0 µM7.5 µM15 µM
Peptide (EA2)Vmax100%82%36%
 Km (µM)173.7208.4210.3
 Ki (µM)9.9
UDP-GalNAcVmax100%71%56%
 Km (µM)74.9153.4448.3
 Ki (µM)2.9

Given its validation as a direct inhibitor of ppGalNAc-T3 without obvious off-target effects we turned to biologically relevant tests of T3Inh-1. As mentioned, overexpression of ppGalNAc-T3 is linked to cancer cell invasiveness as well as poor outcomes in patients (Kitada et al., 2013; Brockhausen, 2006; Harada et al., 2016; Mochizuki et al., 2013). Although no linkage to breast cancer has been reported, our analysis of publically available data for 1117 breast cancer patients (Szász et al., 2016) using Kaplan-Meier survival plots shows that high expression of ppGalNAc-T3 correlates with poor patient overall and metastasis-free survival (Figure 3—figure supplement 1A–B). Therefore, we carried out migration and invasion assays with the breast cancer cell line MDA-MB231, which expresses a relatively high level of ppGalNAc-T3 (Figure 3—figure supplement 1C), in the absence or presence of 5 µM T3Inh-1. Cells were cultured on uncoated (to assay migration) or Matrigel-coated (to assay invasiveness) Bioboat filters for 24 or 48 hr and those cells that moved to the underside of the filters were imaged and quantified. T3Inh-1 was strikingly effective, inhibiting migration by >80% (Figure 3A) and invasion by 98% (Figure 3B) while causing no discernable effect on cell proliferation (Figure 3C). To confirm that the effect was due to ppGalNAc-T3, the same experiment was carried out using MCF7 cells, which is a breast cancer cell line that expresses relatively low levels of ppGalNAc-T3 (Figure 3—figure supplement 1C). Critically, invasion by MCF7 cells was significantly increased by transfection with ppGalNAc-T3 and this increase was strongly blocked by T3Inh-1 (Figure 3D). Although O-glycosylation has been connected to cancer migration and invasion there is little evidence that it could be targetable for clinical purposes. Our results provide a ‘proof of concept’ and a strong starting point for developing the necessary tools.

Figure 3 with 1 supplement see all
T3Inh-1 inhibits cell invasion.

(A) Cell migration through uncoated filters was determined for the MDA-MB231 breast cancer cell line grown in the absence or presence of 5 µM T3Inh-1. The raw image of the filter shows both cells and the filter holes whereas a size-cut off was used in the thresholded image to specifically visualize the cells. Results were quantified by counting cells that migrated to the underside of the filter and each experiment was normalized using the average determined for controls at 24 hr (n = 3 ± SEM). (B) Identical analysis except that the filters were pre-coated with Matrigel so that the assay measures invasion not just migration and the 48 hr control was used for normalization (n = 3 ± SEM). (C) MDA-MB231 proliferation was determined for cells grown in the presence or absence of 5 µM T3Inh-1 by cell counting at 24 or 48 hr. Representative images before and after thresholding (no size cutoff) are shown as well as quantification normalized by the value determined for untreated cells at 48 hr (n = 3 ± SEM). (D) Mock and ppGalNAc-T3 transfected MCF7 cells were plated on Matrigel-coated filters in the absence or presence of 5 µM T3Inh-1 for 24 hr. Thresholded images show cells on underside of filters. Cell counts are shown relative to untreated controls after normalization using the total number of cells (determined using parallel wells 24 hr post-plating). For all panels, asterisks denote p<0.05 (two-tailed Student’s t test) for untreated to T3Inh-1 comparison.

https://doi.org/10.7554/eLife.24051.018
Figure 3—source data 1

Panel A: Cell counts in migration assay.

https://doi.org/10.7554/eLife.24051.019
Figure 3—source data 2

Panel B: Cell counts in invasion assay.

https://doi.org/10.7554/eLife.24051.020
Figure 3—source data 3

Panel C: Cell counts in proliferation assay.

https://doi.org/10.7554/eLife.24051.021
Figure 3—source data 4

Panel D: Cell counts in MCF7 invasion assay.

https://doi.org/10.7554/eLife.24051.022

As the relevant target(s) of ppGalNAc-T3 that drive metastatic-like cell behavior remain to be identified, we next tested whether T3Inh-1 could inhibit glycan masking of FGF23, a known ppGalNAc-T3 target. If so, we expected reduced secretion of intact FGF23. HEK cells co-expressing transfected FGF23 and ppGalNAc-T3 were treated with increasing concentrations of T3Inh-1 and secreted FGF23 was assayed by immunoblot. There was a clear dose-dependent loss of intact FGF23 (Figure 4A) and an increase in the ratio of cleaved/intact yielding a half-max of 14 µM for this effect (Figure 4B). For an unknown reason, perhaps related to its instability in media (Kato et al., 2006), the cleaved fragment did not show a corresponding increase. Rather, its recovery varied with the average over three experiments yielding a relatively small increase (Figure 4—figure supplement 1A–B). As expected, intact and cleaved FGF23 showed no change in cell lysates even for cells with lysosomal degradation inhibited by chloroquine (Figure 4—figure supplement 1C–E) arguing that T3Inh-1 affected cleavage just prior to secretion and did not affect FGF23 expression or cause intracellular routing to lysosomes. Importantly, we also tested the effect of T3Inh-1 on cleavage of ANGPTL3, which is controlled by ppGalNAc-T2-mediated glycan masking (Schjoldager et al., 2012, 2010). Secreted intact ANGPTL3 remained high at all concentrations, confirming the selectivity of T3Inh-1 towards ppGalNAc-T3 (Figure 4A–B). Reasoning that we might be able to see a similar effect on secreted FGF23 in an animal model, mice were injected intraperitoneally with T3Inh-1 and serum levels of cleaved FGF23 were determined. Three groups (0, 25 and 50 mg/kg T3Inh-1) of mice received either one or two injections separated by 24 hr, followed by blood collection after another 24 hr. There were no apparent ill effects on animal health. An ELISA assay with antibodies against the N- and C-terminal portions of FGF23 was used to determine the ratio of cleaved/intact FGF23 in the blood (Sun et al., 2015; Bai et al., 2016). Remarkably, T3Inh-1 caused a robust and statistically significant increase in this ratio at the tested 25 and 50 mg/kg concentrations (Figure 4C). These findings support the further development of T3Inh-1 toward mitigating the effects of elevated FGF23 signaling in chronic kidney disease patients.

Figure 4 with 1 supplement see all
T3Inh-1 increases cleavage of FGF23.

(A) Immunoblot of media collected from cells after a 6 hr period in the presence of the indicated concentrations of T3Inh-1. HEK cells were transfected with FLAG-FGF23 and ppGalNAc-T3 or Myc-ANGPTL3 and anti-FLAG and anti-Myc antibodies were used to assay intact (*) and cleaved (<) FGF23 and ANGPTL3, respectively. The identity and origin of the unmarked band (at approximately 25kD) is unknown and its presence was variable. (B) Quantified results showing the percent ratio change of cleaved/intact FGF23 or ANGPTL3 normalized to the amount present in untreated controls (n ≥ 3 ± SEM). (C) Serum ELISA assay results showing ratio of cleaved/intact FGF23 in mouse sera collected 24 hr after either 1 or 2 (consecutive day) intraperitoneal injections of the indicated amount of T3Inh-1 (averages of 4 animals ±SEM). P-values are from two-tailed Student’s t test.

https://doi.org/10.7554/eLife.24051.024
Figure 4—source data 1

Panel B: Cleaved/intact FGF23 and ANGPTL3 secreted at differing T3Inh-1 concentrations.

https://doi.org/10.7554/eLife.24051.025
Figure 4—source data 2

Panel C: Cleaved/intact FGF23 in mouse serum after one two T3Inh-1 injections.

https://doi.org/10.7554/eLife.24051.026

This study identifies an isozyme-selective inhibitor targeting ppGalNAc-T3. The compound binds directly conferring a mixed-mode of inhibition and is equally active in vitro and in cells. Its discovery paves the way for structural studies that will contribute to our understanding of the enzyme reaction mechanism and guide rational design of modified versions of T3Inh-1 to improve its binding affinity and efficacy. Forthcoming tests of disease models, possibly employing higher affinity versions, may strengthen the case for therapeutic uses of T3Inh-1. It is difficult to predict possible side effects because a full list of ppGalNAc-T3 substrates is not yet available. However, the known effects of its knockout in the mouse model are all attributed to FGF23 processing (Ichikawa et al., 2014). Thus, use of T3Inh-1 to reduce intact FGF23 (and increase the inhibitory cleaved fragment) to treat chronic kidney disease may have limited side effects. Clearly, the issue of multiple substrates has not been a major concern in successful therapies targeting protein kinases. To conclude, we anticipate rational design aided by T3Inh-1, as well as further screening using isozyme-specific sensors, to result in a panel of both isozyme- and pan-specific modulators targeting ppGalNAc-transferases. As individual ppGalNAc-transferase isozymes are associated with unique diseases (Schjoldager and Clausen, 2012; van der Post et al., 2013; Moremen et al., 2012), the result would be a new class of therapeutics capable of treating an array of differing diseases.

Materials and methods

Cell lines and antibodies

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HEK cell lines were previously described (Song et al., 2014). HeLa (Cat#ATCC-CCL-2, CVCL_0030), MDA-MB231 (Cat#ATCC-HTB-26, CVCL_0062), and MCF7 (Cat#ATCC-HTB-22, CVCL_0031) were purchased from ATCC (Manassas, VA). All cell lines were verified mycoplasma free every two months using Hoechst staining. Antibodies used were monoclonal antibodies 4C4 against ppGalNAc-T2 and UH5 against ppGalNAc-T3 (8, 31, 36), monoclonal 9e10 against the myc epitope (Evan et al., 1985; Jesch et al., 2001), a polyclonal against the FLAG epitope (Bethyl Labs, Cat#A190-102B, AB_1944186), a polyclonal against GPP130 (Puri et al., 2002), a purchased anti-ppGalNAc-T3 antibody (ThermoFisher, Cat#PA5-25217, AB_2542717), an anti-α-tubulin antibody (Biolegend, Clone TU-01, Cat#625902, AB_2210041), Alexa 488 anti-mouse (Cat#A28175, AB_2536161) and Alexa 555 anti-rabbit (Cat#A27039, AB_2536100) from Thermo Fisher (Pittsburgh, PA), and horse radish peroxidase-conjugated goat anti-mouse (Cat#170–6516, AB_11125547) and goat anti-rabbit (Cat#170–6515, AB_11125142) antibodies from Sigma-Aldrich (St. Louis, MO).

Primary screen

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HEK cells stably expressing the T2 sensor (containing ANGPTL3 linker sequence with T225G modification (Song et al., 2014)) or the T3 sensor (containing FGF23-based linker [Song et al., 2014]) were cultured in MEM (Corning, NY, Cat#10–010-CV) with 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA, Cat#S111150) and 100 IU/ml penicillin-streptomycin (Sigma-Aldrich, Cat#P4333) at 37°C, 5% CO2. Positive controls were cell lines expressing matched sensors with the glycosylation site mutated (∆glycan), specifically T225G/T226G and T178G for the T2 and T3 sensors, respectively (Song et al., 2014). Cells (50,000/well) were seeded in flat bottom 96-well-plates (Corning, NY, Cat#3997) and grown for 24 hr. Compounds (the diversity set from ChemBridge Corporation [Chicago, IL] and the approved oncology drugs set V and the diversity set II from the National Cancer Institute Developmental Therapeutics Program) were then added to achieve a 10 µM final concentration. After 6 hr, the medium was aspirated and the cells were released by adding 100 µl 5 mM EDTA/PBS containing 110 nM MG dye (Sharp Edge Labs, Pittsburgh, PA) for 5 min at 37°C. The plates were then transferred to an Accuri C6 flow cytometer (BD Biosciences) where GFP and MG fluorescence was measured using 488 nm and 640 nm for 10,000 cells per well. Data analysis used FlowJo software (www.flowjo.com, SCR_008520). For each well the geometric means of the MG and GFP signals were used to compute the MG/GFP ratio. Each compound was analyzed in two wells and the average of the two resulting ratios (R) was recorded. Each daily run included at least 16 wells of vehicle-only controls (sensor-expressing cells treated with a matching DMSO concentration [Fisher Scientific, Cat#BP231-100]) and a similar analysis was used to calculate their average MG/GFP ratio (RNeg) and its standard deviation (SDNeg). The Q value of each compound (as well as the untreated ∆glycan positive controls) was calculated by using the following equation: Q=(R-RNeg)/SDNeg. Background fell within the range −2.5 ≤ Q ≤ 3 and the average Q∆glycan was 135 and 38 for the T2 and T3 sensors, respectively.

Secondary screen

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Glycosylation assays using recombinant ppGalNAc-T2 and ppGalNAc-T3 were carried out using the UDP-Glo Glycosyltransferase assay kit (Promega, Madison, WI, Cat#V6962), according to the manufacturer’s recommendation. The reaction (25 µl) included 2.5 ng/ µl purified enzyme, 25 µM UDP-GalNAc (Sigma-Aldrich, Cat#U5252), 12.5 µM EA2 peptide (AnaSpec, Mucin 10, AA153-165, PTTDSTTPAPTTK, Cat#AS-63841), 25 mM Tris-HCl (pH7.5) (Fisher Scientific, Cat#77-86-1), 5 mM MnCl2 (Fisher Scientific, Cat#M87-500), 2.5 mM CaCl2 (Fisher Scientific, Cat#C70-500) and 50 µM compound. The negative control was vehicle only (same reaction mixture with a matched percentage of DMSO instead of compound), whereas background was from the reaction carried out without enzyme and DMSO instead of compound. All reactions were incubated at 37°C in a water bath for 30 min and then cooled to room temperature. Aliquots (5 µl) were then added to a 384-well plate (Thermo Scientific, Waltham, MA, Cat#164610) to which 5 µl of UDP Detection Reagent was also added. Duplicate measures were made for all reactions. After 1 hr at room temperature the luminescent signals were determined using a Tecan Infinite M1000 (Tecan Group Ltd., Männedorf, Switzerland) with integration time set to 1000 ms. The background-subtracted average for each compound was expressed as a percentage of the negative control (taken as 100%). Compounds with ≥50% effect were considered direct modulators.

Titration assays

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For the sensor assay, about 200,000 cells expressing either sensor were seeded into Greiner Bio-One 24-well plates (Sigma-Aldrich, Cat#662160). After 24 hr, the cells were incubated for another 6 hr in the presence of 0–50 µM compound. To release the cells the medium was replaced with 200 µl 5 mM EDTA/PBS containing 110 nM MG dye. After 5 min at 37°C, fluorescence measurements (20,000 cells/well) were carried out as described above. For the biochemical assay, the assay conditions were identical except for variations in the compound or substrate concentrations as indicated in the figure legends. Data analysis was using Prism (Graph Pad Prism Inc., SCR_002798).

Tryptophan fluorescence quenching assay

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The purified lumenal domain of ppGalNAc-T3 (30 ng/ µl) was incubated with 0–500 µM compound at room temperature for 10 min and 200 µl aliquots were transferred to a Greiner Bio-One 96-well glass-bottom plate (Sigma-Aldrich, Cat#655892) and the fluorescent emission was scanned (300–450 nm) using a Tecan Infinite M1000 with excitation at 290 nm, gain set to 150, number of flashes at 50 and flash frequency at 400 Hz. The value at the peak of emission at 324 nm was used for the binding curve analysis by Prism.

Microscopy

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For determination of sensor activation, spinning-disk confocal microscopy was used exactly as described (Song et al., 2014). To assess possible effects of compounds on Golgi markers, including ppGalNAc-T2 and ppGalNAc-T3, immunofluorescence was carried on HeLa cells treated with 10 µM compound for 6 hr. Briefly, the cells were grown on 12 mm diameter coverslips (Fisher Scientific, Cat#12-545-81) for 48 hr, treated with the compounds, washed with PBS and fixed with 3% paraformaldehyde (Sigma-Aldrich, Cat#P6148) for 15 min. Blocking, Triton X-100 permeabilization, antibody incubations and image capture by spinning-disk confocal were as described (Mukhopadhyay et al., 2010). Monoclonal antibodies against ppGalNAc-T2 and T3 were used undiluted and the polyclonal against GPP130 was used at 1:2000. All corresponding images were acquired and adjusted using identical parameters.

Lectin staining

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HeLa cells (treated with T3Inh-1 for 24 hr at the indicated concentrations) were washed twice in PBS containing 0.5% FBS then stained for 30 min with fluorescent lectins ConA (Cat#FL-1001), WGA (Cat#FL-1021), SNA (Cat#FL-1301) or VVA (Cat#FL-1231) (Vector Laboratories, Burlingame, CA) at 1:100 dilutions (except WGA used at 1:1000) in the wash buffer. After staining, the cells were washed twice, lysed with 0.2% Triton X-100 (Fisher Scientific, Cat#BP151-100) for 15 min at 4°C, centrifuged at 14,000 x g for 15 min at 4°C, and the supernatants were read in a Greiner Bio-One 96-well glass-bottom plate using a Tecan Infinite M1000 with excitation at 495 nm and emission from 510–550 nm. Three independent trials were carried out and the peak value (520 nm) was used for quantification.

Proliferation assay

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Equal numbers of HEK or MDA-MB231 cells were plated in Greiner Bio-One 24-well dishes in growth medium containing 0–50 µM T3Inh-1. After 24, 48 or 72 hr, the cells were released using trypsin and counted twice using a hemocytometer for three independent trials.

Cell invasion assay

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Breast cancer MDA-MB231 cells were grown in DMEM medium (Corning, NY, Cat#10–013-CV) with 10% FBS and 100 IU/ml penicillin-streptomycin at 37°C and 5% CO2 and then plated at a density of 1.32 × 104 in 0.3 ml of DMEM medium without FBS into the upper chamber of a Bioboat insert fitted with a 8.0 µm PET membrane (Corning, NY, Cat#354578). For migration assays, the filter was uncoated. For invasion assays, it was pre-coated with 100 µl Matrigel (BD Biosciences, Cat#356234) at concentration of 272 µg /ml for 1–2 hr at room temperature. Medium containing 10% FBS (0.6 ml) was placed into the lower chamber as a chemo attractant. The compound (final concentration of 5 µM) or a matching amount of DMSO was added to both chambers. After 24 hr or 48 hr the cells were fixed with −20°C methanol for 15 min and then stained with Trypan blue for 5 min. Cells on the upper surface were removed using cotton swabs. Cells present on the underside of the membrane were photographed using an EVOS FL Cell Imaging System (Invitrogen, CA) and the images were thresholded for presentation and counting using Image J (National Institutes of Health, Bethesda, MD, SCR_003070). The assays involving MCF7 cells were identical except that they were performed 24 hr post transfection.

Immunoblotting

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The FLAG-tagged FGF23, Myc-tagged ANGPTL3 with the T225G modification (Song et al., 2014), and untagged ppGalNAc-T3 (from [Kato et al., 2006] and cloned into PCDNA 3.0 using BamH1 sites) were transfected into HEK cells using the JetPEI transfection reagent (VWR International, Radnor, PA, Cat#101–40N) according to the manufacturer’s instructions. After 24 hr, the medium was replaced with serum-free MEM containing the compound for 6 hr. The medium and cells were then collected and, after trichloroacetic acid precipitation of the medium, analyzed by immunoblot using anti-FLAG antibody at 1:1000 or anti-Myc antibody at 1:2000 and then the peroxidase-coupled secondary antibodies. Emission was captured and quantified using a ChemiDoc Touch Imaging System with Image Lab Software (BioRad, SCR_014210). For ppGalNAc-T3 determinations in different cell lines, cells were collected and lysed with 100 µl buffer (10 mM Tris-HCl (pH8.0), 1 mM EDTA (ACROS ORGANICS, Cat#446085000), 1% Triton X-100, 0.1% sodium deoxycholate (Fisher Scientific, Cat#BP349-100), 0.1% SDS (Fisher Scientific, Cat#BP166-500), 140 mM NaCl (Fisher Scientific, Cat#S271-3), 1 mM PMSF (Sigma-Aldrich, Cat#PMSF-RO). Then 15 µl of each lysate was analyzed by immunoblotting using the purchased anti-ppGalNAc-T3 and anti-α-tubulin antibodies.

Animal analysis

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Wild-type C57BL/6 six to eight week old mice were purchased from Charles River Laboratories international Inc. (Wilmington, MA). Protocols, handling, and care of the mice conformed to protocols approved by the Institutional Animal Care and Use Committee of Carnegie Mellon University (CMU IACUC protocol AS16-005). The compound was dissolved in DMSO at 25 and 50 mg/ml then further diluted with PEG400 (Hampton Research, CA, USA, HR2-603) to create 5 and 10 mg/ml stocks for injection. Control (vehicle only: 20% DMSO, 80% PEG400) and experimental (25 or 50 mg/kg compound) animals received either single or double (separated by 24 hr) intraperitoneal injections and, 24 hr after the last injection, a cardiac blood draw was carried out. The cleaved/intact FGF23 ratio was determined using ELISA kits from Immunotopics (Carlsbad, VA, USA, Cat#60–6800, Cat#60–6300) with cleaved equaling total minus intact.

References

    1. Kuan SF
    2. Byrd JC
    3. Basbaum C
    4. Kim YS
    (1989)
    Inhibition of mucin glycosylation by aryl-N-acetyl-alpha-galactosaminides in human Colon cancer cells
    The Journal of Biological Chemistry 264:19271–19277.
    1. Röttger S
    2. White J
    3. Wandall HH
    4. Olivo JC
    5. Stark A
    6. Bennett EP
    7. Whitehouse C
    8. Berger EG
    9. Clausen H
    10. Nilsson T
    (1998)
    Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the golgi apparatus
    Journal of Cell Science 111:45–60.

Decision letter

  1. Christopher G Burd
    Reviewing Editor; Yale School of Medicine, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Inhibitor of GalNAc-T3-mediated O-glycosylation blocks cancer cell invasiveness and lowers FGF23 levels" for consideration by eLife. Your article has been reviewed by two peer reviewers, one of whom, Christopher G Burd (Reviewer #1), is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Vivek Malhotra as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Song and Linstedt report the results of a small molecule screen designed to identify modifiers of the GalNAc-T2 and GalNAc-T3 GalNAc-transferases. The screen implemented clever, previously published and validated, cell-based assays that report GalNAc-T2 and GalNAc-T3 dependent modifications that generate fluorescent signals. A compound that inhibits GalNAc-T3, termed "T3Inh-1", was identified and characterized. Evidence is presented that T3Inh-1 inhibits GalNAc-T3 activity by binding to the lumenal portion of the enzyme. Assays of migration and invasion using cultured MDA-MB231 cells indicate that T3Inh-1 inhibits these properties of the cells.

Essential revisions:

1) The conclusion regarding the specificity of T3Inh-1 for Galnt3 is not adequately supported. The effect of T3Inh-1 on the in vitro activity of purified Galnt6, which is more closely related to Galnt3 than to Galnt2 (see: Revoredo et al., 2016), should be included in the manuscript in order to more thoroughly validate the specificity of T3Inh-1.

2) The cell-based fluorescence lectin binding data does not adequately support the authors' conclusion that T3Inh-1 has no significant effects on synthesis of the major classes of glycans. For example, GSII is not appropriate since few complex N-glycans terminate with GlcNAc. Lectins that detect terminal Gal or sialic acid and differential N-glycan branching, as well as missing O-GalNAc glycans should be investigated. A broader range of lectins needs to be implemented and the cells should be exposed to increasing concentrations of T3Inh-1 for at least 24 and 48 hours. It is recommended that changes in glycosylation should be determined by a more quantitative approach than that presented in the manuscript, such as quantitative flow cytometry.

3) Regarding the effects of T3Inh-1 of FGF23 cleavage and signaling, a concern lies with the presentation and interpretation of the FGF23 processing assays shown in Figure 4. As the authors point out that loss of modification of FGF23 by GalNAc-T3 should result in a T3Inh-1 dose-dependent increase in the amount of cleaved product. The data that tests this, however, show a dose-dependent decrease in the amounts of the intact protein, but the amounts of the cleaved product remain essentially constant (or perhaps it decreases a bit?). The authors need to explain how these data support their conclusion that T3Inh-1 increases cleavage of FGF23, rather than elicits some other outcome, such as failure to secrete the intact protein and/or degradation of the intact protein. The authors state, but do not show, that FGF23 levels in cell lysates were unaffected by T3Inh-1; the reviewers feel these data should be shown because it might be insightful for determining how T3Inh-1 causes the observed change in the ratio of cleaved/uncleaved protein. Perhaps the unglycosylated, intact form is retained and/or degraded within the cell, but the cleaved form is not. Better yet would be a pulse-chase assay of radiolabeled FGF23, which would directly test the authors' hypothesis that the intact form is unstable/degraded in the culture medium. Related, the authors interpret change in the ratio of cleaved/uncleaved protein to reflect a net change in "signaling activity" (Results section), but no assays of signaling activity are reported. The text should be modified to accurately reflect this.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Inhibitor of GalNAc-T3-mediated O-glycosylation blocks cancer cell invasiveness and lowers FGF23 levels" for further consideration at eLife. Your revised article has been favorably evaluated by Vivek Malhotra (Senior editor), a Reviewing editor and one reviewer.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below. All of these points can be addressed by revisions to the text.

1) The authors must use the established nomenclature throughout the text and in all figures. The enzymes that add GalNAc to protein are called polypeptide GalNAc-transferases. The shorthand for each of the 20 members of the family is ppGalNAcT-1 etc, and the genes are named GALNT1 etc. The leaving out of polypeptide by the authors confuses the enzymes under study with GalNAcTs which transfer GalNAc to other sugars, a totally distinct class of enzyme.

2) Regarding the new lectin binding assay, the authors should qualify their conclusions by stating that no major change in the binding of lectins that detect N- or O-glycans was induced by inhibitor treatment. The lectin binding assay, though acceptable, is not standard practice for the field (flow cytometry is). Additionally, the sensitivity of the fluorescence assay is not clear since there are no positive controls with altered N- or O-glycans. There are many other types of glycans that would not be detected by the lectins used in their assay. The amount of time that cells were incubated with inhibitor before lectin binding was assayed must be indicated.

3) The authors should explain the origin of the band at ~25 kD in Figure 4A. This gel does not show a visible increase in the ~15 kD cleavage product which is very abundant at time zero. More explanation of these data is warranted.

4) The Materials and methods should include precise details regarding origin, clone name, catalogue number and dilution used for each antibody or reagent and precise conditions of use in the relevant experiment. For example the source of GPP130 is given as the reference for Puri, Bachert, Fimmel and Linstedt, 2002, which is not sufficient. Enough information should be provided so that precise replication of each experiment is possible.

https://doi.org/10.7554/eLife.24051.029

Author response

Essential revisions:

1) The conclusion regarding the specificity of T3Inh-1 for Galnt3 is not adequately supported. The effect of T3Inh-1 on the in vitro activity of purified Galnt6, which is more closely related to Galnt3 than to Galnt2 (see: Revoredo et al., 2016), should be included in the manuscript in order to more thoroughly validate the specificity of T3Inh-1.

As requested, we have performed a test of the inhibitor (T3Inh-1) against GalNAc transferase-6, which according to a phylogenetic analysis is most closely related to GalNAc-T3. These data are now added to Figure 2B. Gratifyingly, there was no detectable activity despite the full and expected potency against GalNAc-T3 in parallel assays.

2) The cell-based fluorescence lectin binding data does not adequately support the authors' conclusion that T3Inh-1 has no significant effects on synthesis of the major classes of glycans. For example, GSII is not appropriate since few complex N-glycans terminate with GlcNAc. Lectins that detect terminal Gal or sialic acid and differential N-glycan branching, as well as missing O-GalNAc glycans should be investigated. A broader range of lectins needs to be implemented and the cells should be exposed to increasing concentrations of T3Inh-1 for at least 24 and 48 hours. It is recommended that changes in glycosylation should be determined by a more quantitative approach than that presented in the manuscript, such as quantitative flow cytometry.

As requested, we expanded our survey of lectin staining to include Wheat germ agglutinin (WGA) and Sambucus Nigra (SNA), which recognize most major surface glycans. Additionally, the assay was made more rigorously quantifiable by using a plate reader allowing determinations from thousands of cells (n=3 independent trials). These data are present as a new figure: Figure 1—figure supplement 2. There was no significant diminishment of staining for any lectin tested supporting our conclusion that T3Inh-1 is selective against GalNAc-T3 rather than acting as a general inhibitor of glycosylation enzymes.

3) Regarding the effects of T3Inh-1 of FGF23 cleavage and signaling, a concern lies with the presentation and interpretation of the FGF23 processing assays shown in Figure 4. As the authors point out that loss of modification of FGF23 by GalNAc-T3 should result in a T3Inh-1 dose-dependent increase in the amount of cleaved product. The data that tests this, however, show a dose-dependent decrease in the amounts of the intact protein, but the amounts of the cleaved product remain essentially constant (or perhaps it decreases a bit?). The authors need to explain how these data support their conclusion that T3Inh-1 increases cleavage of FGF23, rather than elicits some other outcome, such as failure to secrete the intact protein and/or degradation of the intact protein. The authors state, but do not show, that FGF23 levels in cell lysates were unaffected by T3Inh-1; the reviewers feel these data should be shown because it might be insightful for determining how T3Inh-1 causes the observed change in the ratio of cleaved/uncleaved protein. Perhaps the unglycosylated, intact form is retained and/or degraded within the cell, but the cleaved form is not. Better yet would be a pulse-chase assay of radiolabeled FGF23, which would directly test the authors' hypothesis that the intact form is unstable/degraded in the culture medium. Related, the authors interpret change in the ratio of cleaved/uncleaved protein to reflect a net change in "signaling activity" (Results section), but no assays of signaling activity are reported. The text should be modified to accurately reflect this.

Figure 4—figure supplement 1A-D has been added to address issues concerning the intact and cleaved bands of FGF23. The main question is why the loss of intact FGF23 does not appear to be accompanied by a quantitatively comparable increase in the cleaved fragment. There is previous evidence that the cleaved FGF23 fragment is difficult to recover in cultured cell media samples (Kato et al., 2006). Also, note that even in ∆T3 cells the cleaved fragment is relatively under- represented in the media. It also bears mentioning that we do detect a strong increase in this cleaved FGF23 fragment in the serum of injected mice (Figure 4C). Finally, the new figure presents the data more thoroughly and shows that for the quantified data the discrepancy is minor. First we present an additional representative blot from the 3 identical trials (panel A of the figure) to illustrate that there was some variation in our recovery of the cleaved fragment. This blot better represents the slight increase that the quantified data show. This quantified data, showing the increase on average in cleaved FGF23 and the loss of intact, is presented in panel B. Taken together with the data in the main body (Figure 4A-B) this provides a comprehensive representation of the result we obtained. Indeed, in data not shown, the experiment was carried out for an additional cell type and with different parameters and, in each case, yielded the same result: a significant loss of intact and a slight increase of cleaved FGF23. In part C-E, we have now added our data for the cell extracts. Neither the intact nor cleaved FGF23 showed a significant change (C-D). Also, panel E compares cells extracts from untreated and chloroquine-treated cells (to block lysosomal degradation). Chloroquine did not cause any accumulation. All together the data in cells and mice argue against changes in expression, sorting, or degradation and support the conclusion that T3Inh- 1 increases FGF23 cleavage.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below. All of these points can be addressed by revisions to the text.

1) The authors must use the established nomenclature throughout the text and in all figures. The enzymes that add GalNAc to protein are called polypeptide GalNAc-transferases. The shorthand for each of the 20 members of the family is ppGalNAcT-1 etc, and the genes are named GALNT1 etc. The leaving out of polypeptide by the authors confuses the enzymes under study with GalNAcTs which transfer GalNAc to other sugars, a totally distinct class of enzyme.

The text has been changed accordingly using ppGalNAcT to refer to the enzymes.

2) Regarding the new lectin binding assay, the authors should qualify their conclusions by stating that no major change in the binding of lectins that detect N- or O-glycans was induced by inhibitor treatment. The lectin binding assay, though acceptable, is not standard practice for the field (flow cytometry is). Additionally, the sensitivity of the fluorescence assay is not clear since there are no positive controls with altered N- or O-glycans. There are many other types of glycans that would not be detected by the lectins used in their assay. The amount of time that cells were incubated with inhibitor before lectin binding was assayed must be indicated.

The text and methods were revised to state that it was a 24 h treatment (already in figure legend). The text was also revised to make clear that our results speak only to the abundant glycans detected by the lectins we used.

3) The authors should explain the origin of the band at ~25 kD in Figure 4A. This gel does not show a visible increase in the ~15 kD cleavage product which is very abundant at time zero. More explanation of these data is warranted.

The legend to Figure 4A was revised to indicate that the origin and identity of the 25kD band is unknown and its presence in the independent trials was variable. The identity of intact and cleaved FGF23 (marked on the figure and explained in the legend) is clear in these experiments because of their migration on the gel, their presence only in transfected cells, and their expected behavior in cells lacking expression of ppGalNAc-T3.

4) The Materials and methods should include precise details regarding origin, clone name, catalogue number and dilution used for each antibody or reagent and precise conditions of use in the relevant experiment. For example the source of GPP130 is given as the reference for Puri, Bachert, Fimmel and Linstedt, 2002, which is not sufficient. Enough information should be provided so that precise replication of each experiment is possible.

The Materials and methods section was extensively revised by addition of information regarding the origin of the reagents used. To our knowledge all dilutions are specified. The reference for anti-GPP130 is the paper describing the origin and first use of the antibody.

https://doi.org/10.7554/eLife.24051.030

Article and author information

Author details

  1. Lina Song

    Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, United States
    Contribution
    LS, Conceptualization, Data curation, Formal analysis, Writing—original draft
    Competing interests
    The authors declare that no competing interests exist.
  2. Adam D Linstedt

    Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, United States
    Contribution
    ADL, Conceptualization, Supervision, Funding acquisition, Methodology, Project administration, Writing—review and editing
    For correspondence
    linstedt@andrew.cmu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0754-1638

Funding

National Institutes of Health (1R21DE026714)

  • Adam Linstedt

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr. Henrik Clausen (University of Copenhagen) for contribution of essential reagents and advice with the manuscript, HaiBing Teng for help with the imaging, Yehuda Creeger for help of flow cytometry, Collin Bachert and Dr. Lingtao Jin for technical and editorial help and Dr. Tina Lee, Dr. Somshuvra Mukhopdhyay and Emily Simon for helpful manuscript suggestions. Funding was from NIH grant 1R21DE026714 to ADL.

Ethics

Animal experimentation: Protocols, handling, and care of the mice conformed to protocols approved by the Institutional Animal Care and Use Committee of Carnegie Mellon University. (CMU IACUC protocol AS16-005).

Reviewing Editor

  1. Christopher G Burd, Yale School of Medicine, United States

Publication history

  1. Received: December 8, 2016
  2. Accepted: March 30, 2017
  3. Accepted Manuscript published: March 31, 2017 (version 1)
  4. Version of Record published: April 27, 2017 (version 2)
  5. Version of Record updated: May 12, 2017 (version 3)

Copyright

© 2017, Song et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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