Cosmc-dependent mucin-type<i>O</i>-linked glycosylation is essential for podocyte function

Stotter, Brian R.; Talbot, Brianna E.; Capen, Diane E.; Artelt, Nadine; Zeng, Junwei; Matsumoto, Yasuyuki; Endlich, Nicole; Cummings, Richard D.; Schlöndorff, Johannes

doi:10.1152/ajprenal.00399.2019

Cited by 19 publications

(20 citation statements)

References 64 publications

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“… 43 In addition, PODO447 showed significant binding to the Tn antigen (GalNAcα1-OSer), which has previously been reported to be abundantly expressed on Podxl in the kidney. 44 However, the Tn antigen is most likely not involved in PODO447’s recognition of cancer cells, as we observe a very low number of Tn-positive cells on cell lines expressing high levels of the PODO447 epitope ( online supplemental figure 3 ). In addition, the anti-Tn Ab, clone 5F4, did not interfere with the ability of PODO447 to bind its epitope ( online supplemental figure 4 ).…”

Section: Resultsmentioning

confidence: 72%

PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin

Hernaez

Hughes

Dean

et al. 2020

J Immunother Cancer

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BackgroundThe success of new targeted cancer therapies has been dependent on the identification of tumor-specific antigens. Podocalyxin (Podxl) is upregulated on tumors with high metastatic index and its presence is associated with poor outcome, thus emerging as an important prognostic and theragnostic marker in several human cancers. Moreover, in human tumor xenograft models, Podxl expression promotes tumor growth and metastasis. Although a promising target for immunotherapy, the expression of Podxl on normal vascular endothelia and kidney podocytes could hamper efforts to therapeutically target this molecule. Since pathways regulating post-translational modifications are frequently perturbed in cancer cells, we sought to produce novel anti-Podxl antibodies (Abs) that selectively recognize tumor-restricted glycoepitopes on the extracellular mucin domain of Podxl.MethodsSplenic B cells were isolated from rabbits immunized with a Podxl-expressing human tumor cell line. Abs from these B cells were screened for potent reactivity to Podxl+ neoplastic cell lines but not Podxl+ primary endothelial cells. Transcripts encoding heavy and light chain variable regions from promising B cells were cloned and expressed as recombinant proteins. Tumor specificity was assessed using primary normal tissue and an ovarian cancer tissue microarray (TMA). Mapping of the tumor-restricted epitope was performed using enzyme-treated human tumor cell lines and a glycan array.ResultsOne mAb (PODO447) showed strong reactivity with a variety of Podxl+ tumor cell lines but not with normal primary human tissue including Podxl+ kidney podocytes and most vascular endothelia. Screening of an ovarian carcinoma TMA (219 cases) revealed PODO447 reactivity with the majority of tumors, including 65% of the high-grade serous histotype. Subsequent biochemical analyses determined that PODO447 reacts with a highly unusual terminal N-acetylgalactosamine beta-1 (GalNAcβ1) motif predominantly found on the Podxl protein core. Finally, Ab–drug conjugates showed specific efficacy in killing tumor cells in vitro.ConclusionsWe have generated a novel and exquisitely tumor-restricted mAb, PODO447, that recognizes a glycoepitope on Podxl expressed at high levels by a variety of tumors including the majority of life-threatening high-grade serous ovarian tumors. Thus, tumor-restricted PODO447 exhibits the appropriate specificity for further development as a targeted immunotherapy.

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Section: Resultsmentioning

confidence: 72%

PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin

Hernaez

Hughes

Dean

et al. 2020

J Immunother Cancer

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“…To calculate podocyte density, kidney sections were stained for immunofluorescence microscopy as described previously [ 48 ], using rabbit monoclonal anti-WT1 (Abcam, ab89901) and appropriate fluorophore-labeled secondary antibody (Jackson ImmunoResearch Laboratories), and counterstained with fluorescently labeled wheat germ agglutinin (Invitrogen, W7024) and Hoechst 33342. Podocyte number and glomerular cross-section were obtained for 20 glomeruli per animal.…”

Section: Methodsmentioning

confidence: 99%

“…Washed platelets were resuspended directly in NP-40 lysis buffer containing Complete protease inhibitors (Roche). Glomeruli were isolated using magnetic bead perfusion as previously described [ 48 , 51 ], utilizing high iron content magnetic particles (AMS-40-10H, Spherotech). For western blot, glomeruli were resuspended in NP-40 lysis buffer with protease inhibitors.…”

Section: Methodsmentioning

confidence: 99%

Gain-of-function, focal segmental glomerulosclerosis Trpc6 mutation minimally affects susceptibility to renal injury in several mouse models

et al. 2022

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Mutations in TRPC6 are a cause of autosomal dominant focal segmental glomerulosclerosis in humans. Many of these mutations are known to have a gain-of-function effect on the non-specific cation channel function of TRPC6. In vitro studies have suggested these mutations affect several signaling pathways, but in vivo studies have largely compared wild-type and Trpc6-deficient rodents. We developed mice carrying a gain-of-function Trpc6 mutation encoding an E896K amino acid change, corresponding to a known FSGS mutation in TRPC6. Homozygous mutant Trpc6 animals have no appreciable renal pathology, and do not develop albuminuria until very advanced age. The Trpc6E896K mutation does not impart susceptibility to PAN nephrosis. The animals show a slight delay in recovery from the albumin overload model. In response to chronic angiotensin II infusion, Trpc6E896K/E896K mice have slightly greater albuminuria initially compared to wild-type animals, an effect that is lost at later time points, and a statistically non-significant trend toward more glomerular injury. This phenotype is nearly opposite to that of Trpc6-deficient animals previously described. The Trpc6 mutation does not appreciably impact renal interstitial fibrosis in response to either angiotensin II infusion, or folate-induced kidney injury. TRPC6 protein and TRPC6-agonist induced calcium influx could not be detected in glomeruli. In sum, these findings suggest that a gain-of-function Trpc6 mutation confers only a mild susceptibility to glomerular injury in the mouse.

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“…[44] Washed platelets were resuspended directly in NP-40 lysis buffer containing Complete protease inhibitors (Roche). Glomeruli were isolated using magnetic bead perfusion as previously described, [48,51] utilizing high iron content magnetic particles (AMS-40-10H, Spherotech). For western blot, glomeruli were resuspended in NP-40 lysis buffer with protease inhibitors.…”

Section: Platelet and Glomerular Isolationmentioning

confidence: 99%

Gain-of-function, focal segmental glomerulosclerosis Trpc6 mutation minimally affects susceptibility to renal injury in several mouse models

Brown

Boekell

Stotter

et al. 2022

Preprint

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Mutations in TRPC6 are a cause of autosomal dominant focal segmental glomerulosclerosis in humans. Many of these mutations are known to have a gain-of-function effect on the non-specific cation channel function of TRPC6. In vitro studies have suggested these mutations affect several signaling pathways, but in vivo studies have largely compared wild-type and Trpc6-deficient rodents. We developed mice carrying a gain-of-function Trpc6 mutation encoding an E896K amino acid change, corresponding to a known FSGS mutation in TRPC6. Homozygous mutant Trpc6 animals have no appreciable renal pathology, and do not develop albuminuria until very advanced age. The animals show a slight delay in recovery from the albumin overload model. In response to chronic angiotensin II infusion, the mice have slightly greater albuminuria initially compared to wild-type animals, an effect that is lost at later time points, and a statistically non-significant trend toward more glomerular injury. This phenotype is nearly opposite to that of Trpc6-deficient animals previously described. The Trpc6 mutation does not appreciably impact renal interstitial fibrosis in response to either angiotensin II infusion, or folate-induced kidney injury. TRPC6 protein and TRPC6-agonist induced calcium influx could not be detected in glomeruli. In sum, these findings suggest that gain-of-function Trpc6 mutations confer only a mild susceptibility to glomerular injury in the mouse.

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Cosmc-dependent mucin-typeO-linked glycosylation is essential for podocyte function

Cited by 19 publications

References 64 publications

PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin

PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin

Gain-of-function, focal segmental glomerulosclerosis Trpc6 mutation minimally affects susceptibility to renal injury in several mouse models

Gain-of-function, focal segmental glomerulosclerosis Trpc6 mutation minimally affects susceptibility to renal injury in several mouse models

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