Placental Protein Citrullination Signatures Are Modified in Early- and Late-Onset Fetal Growth Restriction

Fetal growth restriction (FGR) is an obstetric condition most frequently caused by placental dysfunction. It is a major cause of perinatal morbidity with limited treatment options, so identifying the underpinning mechanisms is important. Peptidylarginine deiminases (PADs) are calcium-activated enzymes that mediate post-translational citrullination (deimination) of proteins, through conversion of arginine to citrulline. Protein citrullination leads to irreversible changes in protein structure and function and is implicated in many pathobiological processes. Whether placental protein citrullination occurs in FGR is poorly understood. We assessed protein citrullination and PAD isozyme abundance (PAD1, 2, 3, 4 and 6) in human placental samples from pregnancies complicated by early- and late-onset FGR, compared to appropriate-for-gestational-age (AGA) controls. Proteomic mass spectrometry demonstrated that the placental citrullinome profile changed in both early- and late-onset FGR, with 112 and 345 uniquely citrullinated proteins identified in early- and late-onset samples, respectively. Forty-four proteins were citrullinated only in control AGA placentas. The proteins that were uniquely citrullinated in FGR placentas were enriched for gene ontology (GO) terms related to neurological, developmental, immune and metabolic pathways. A greater number of GO and human phenotype pathways were functionally enriched for citrullinated proteins in late- compared with early-onset FGR. Correspondingly, late-onset but not early-onset FGR was associated with significantly increased placental abundance of PAD2 and citrullinated histone H3, determined by Western blotting. PAD3 was downregulated in early-onset FGR while abundance of PAD 1, 4 and 6 was less altered in FGR. Our findings show that placental protein citrullination is altered in FGR placentas, potentially contributing to the pathobiology of placental dysfunction.