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ERO1 alpha deficiency impairs angiogenesis by increasing N-glycosylation of a proangiogenic VEGFA.

ABSTRACT: N-glycosylation and disulfide bond formation are two essential steps in protein folding that occur in the endoplasmic reticulum (ER) and reciprocally influence each other. Here, to analyze crosstalk between N-glycosylation and oxidation, we investigated how the protein disulfide oxidase ERO1-alpha affects glycosylation of the angiogenic VEGF¹²¹, a key regulator of vascular homeostasis. ERO1 deficiency, while retarding disulfide bond formation in VEGF¹²¹, increased utilization of its single N-glycosylation sequon, which lies close to an intra-polypeptide disulfide bridge, and concomitantly slowed its secretion. Unbiased mass-spectrometric analysis revealed interactions between VEGF¹²¹ and N-glycosylation pathway proteins in ERO1-knockout (KO), but not wild-type cells. Notably, MAGT1, a thioredoxin-containing component of the post-translational oligosaccharyltransferase complex, was a major hit exclusive to ERO1-deficient cells. Thus, both a reduced rate of formation of disulfide bridges, and the increased trapping potential of MAGT1 may increase N-glycosylation of VEGF¹²¹. Extending our investigation to tissues, we observed altered lectin staining of ERO1 KO breast tumor xenografts, implicating ERO1 as a physiologic regulator of protein N-glycosylation. Our study, highlighting the effect of ERO1 loss on N-glycosylation of proteins, is particularly relevant not only to angiogenesis but also to other cancer patho-mechanisms in light of recent findings suggesting a close causal link between alterations in protein glycosylation and cancer development.

SUBMITTER: Varone E

PROVIDER: S-EPMC9463388 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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ERO1 alpha deficiency impairs angiogenesis by increasing N-glycosylation of a proangiogenic VEGFA.

Varone Ersilia E Chernorudskiy Alexander A Cherubini Alessandro A Cattaneo Angela A Bachi Angela A Fumagalli Stefano S Erol Gizem G Gobbi Marco M Lenardo Michael J MJ Borgese Nica N Zito Ester E

Redox biology 20220827

N-glycosylation and disulfide bond formation are two essential steps in protein folding that occur in the endoplasmic reticulum (ER) and reciprocally influence each other. Here, to analyze crosstalk between N-glycosylation and oxidation, we investigated how the protein disulfide oxidase ERO1-alpha affects glycosylation of the angiogenic VEGF<sup>121</sup>, a key regulator of vascular homeostasis. ERO1 deficiency, while retarding disulfide bond formation in VEGF<sup>121</sup>, increased utilizati ...[more]

PMID: 36063727

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Project description:Mice lacking the EF-hand Ca2+ sensor S100A1 display endothelial dysfunction because of distorted Ca2+ -activated nitric oxide (NO) generation.To determine the pathophysiological role of S100A1 in endothelial cell (EC) function in experimental ischemic revascularization.Patients with chronic critical limb ischemia showed almost complete loss of S100A1 expression in hypoxic tissue. Ensuing studies in S100A1 knockout (SKO) mice subjected to femoral artery resection unveiled insufficient perfusion recovery and high rates of autoamputation. Defective in vivo angiogenesis prompted cellular studies in SKO ECs and human ECs, with small interfering RNA-mediated S100A1 knockdown demonstrating impaired in vitro and in vivo proangiogenic properties (proliferation, migration, tube formation) and attenuated vascular endothelial growth factor (VEGF)-stimulated and hypoxia-stimulated endothelial NO synthase (eNOS) activity. Mechanistically, S100A1 deficiency compromised eNOS activity in ECs by interrupted stimulatory S100A1/eNOS interaction and protein kinase C hyperactivation that resulted in inhibitory eNOS phosphorylation and enhanced VEGF receptor-2 degradation with attenuated VEGF signaling. Ischemic SKO tissue recapitulated the same molecular abnormalities with insufficient in vivo NO generation. Unresolved ischemia entailed excessive VEGF accumulation in SKO mice with aggravated VEGF receptor-2 degradation and blunted in vivo signaling through the proangiogenic phosphoinositide-3-kinase/Akt/eNOS cascade. The NO supplementation strategies rescued defective angiogenesis and salvaged limbs in SKO mice after femoral artery resection.Our study shows for the first time downregulation of S100A1 expression in patients with critical limb ischemia and identifies S100A1 as critical for EC function in postnatal ischemic angiogenesis. These findings link its pathological plasticity in critical limb ischemia to impaired neovascularization, prompting further studies to probe the microvascular therapeutic potential of S100A1.

Dataset Information

ERO1 alpha deficiency impairs angiogenesis by increasing N-glycosylation of a proangiogenic VEGFA.

Publications

ERO1 alpha deficiency impairs angiogenesis by increasing N-glycosylation of a proangiogenic VEGFA.

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