Altered protein O-GlcNAcylation in placentas from mothers with diabetes causes aberrant endocytosis in placental trophoblast cells

Victoria Palin,Melissa Westwood,Matthew Russell,Robert Graham,John D Aplin

doi:10.1038/s41598-021-00045-8

Abstract

Women with pre-existing diabetes have an increased risk of poor pregnancy outcomes, including disordered fetal growth, caused by changes to placental function. Here we investigate the possibility that the hexosamine biosynthetic pathway, which utilises cellular nutrients to regulate protein function via post-translationally modification with O-linked N-acetylglucosamine (GlcNAc), mediates the placental response to the maternal metabolic milieu. Mass spectrometry analysis revealed that the placental O-GlcNAcome is altered in women with type 1 (n = 6) or type 2 (n = 6) diabetes T2D (≥ twofold change in abundance in 162 and 165 GlcNAcylated proteins respectively compared to BMI-matched controls n = 11). Ingenuity pathway analysis indicated changes to clathrin-mediated endocytosis (CME) and CME-associated proteins, clathrin, Transferrin (TF), TF receptor and multiple Rabs, were identified as O-GlcNAcylation targets. Stimulating protein O-GlcNAcylation using glucosamine (2.5 mM) increased the rate of TF endocytosis by human placental cells (p = 0.02) and explants (p = 0.04). Differential GlcNAcylation of CME proteins suggests altered transfer of cargo by placentas of women with pre-gestational diabetes, which may contribute to alterations in fetal growth. The human placental O-GlcNAcome provides a resource to aid further investigation of molecular mechanisms governing placental nutrient sensing.

Highlights

Women with pre-existing diabetes have an increased risk of poor pregnancy outcomes, including disordered fetal growth, caused by changes to placental function
In order to understand how protein O-GlcNAcylation status is altered in women who have type 1 or type 2 diabetes (Table 1), and whether such changes affect placental function, proteins isolated from placental lysates using succinylated wheat germ agglutinin (sWGA) pull-down were analysed by mass spectrometry. 961 proteins were identified in total; 668 and 695 in type 1 diabetes (T1D) and T2D placenta, respectively (Supplementary Table S1); some of which were unique to particular sample groups (Supplementary Table S2)
We quantified alterations to global protein O-GlcNAcylation in placentas exposed to an altered metabolic environment in vivo—pre-existing maternal diabetes—to investigate whether this molecular switch might contribute to an aberrant phenotype

Summary

Introduction

Women with pre-existing diabetes have an increased risk of poor pregnancy outcomes, including disordered fetal growth, caused by changes to placental function. We investigate the possibility that the hexosamine biosynthetic pathway, which utilises cellular nutrients to regulate protein function via post-translationally modification with O-linked N-acetylglucosamine (GlcNAc), mediates the placental response to the maternal metabolic milieu. Fetal overgrowth is often perceived to be the consequence of the increased availability, and supply, of maternal nutrients[7,8] This hypothesis assumes that the placenta, which forms the interface between mother and fetus, allows passive transfer of nutrients. Both animal and clinical studies show that the placenta actively regulates transfer to the fetus[9], modifying its structure and function to achieve nutrient transfer that is optimal for fetal development and growth. Rodent studies have shown high abundance of these two enzymes in p lacenta[12,13]

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