Pravata, V. M. | bioRxiv Science

bioRxiv2023

An O-GlcNAc transferase pathogenic variant that affects pluripotent stem cell self-renewal

O-linked {beta}-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential enzyme that modifies proteins with O-GlcNAc. Inborn OGT genetic variants were recently shown to mediate a novel type of Congenital Disorder of Glycosylation (OGT-CDG) which is characterized by X-linked intellectual disability (XLID) and developmental delay. Here, we report an OGTC921Y variant which co-segregates with XLID and epileptic seizures, and results in loss of catalytic activity. Colonies formed by mouse embryonic stem cells carrying OGTC921Y show decreased levels of protein O-GlcNAcylation accompanied by decreased levels of Oct4, Sox2 and extracellular alkaline phosphatase (ALP), implying reduced self-renewal capacity. These data establish a link between OGT-CDG and embryonic stem cell self-renewal, providing a foundation for examining the developmental aetiology of this syndrome. Summary statementWe show that the C921Y O-GlcNAc transferase variant found in patients with intellectual disability leads to a defect in pluripotent stem cell self-renewal and decreased levels of stem cell markers.

molecular biology↗

bioRxiv2019

TIMP-3 facilitates binding of target metalloproteinases to the endocytic receptor LRP-1 and promotes scavenging of MMP-1

Matrix metalloproteinases (MMPs) and the related families of disintegrin metalloproteinases (ADAMs) and ADAMs with thrombospondin repeats (ADAMTSs) play a crucial role in the turnover of extracellular matrix (ECM) components and shedding of cell-surface molecules. The proteolytic activity of metalloproteinases is post-translationally regulated by their endogenous inhibitors, known as tissue inhibitors of metalloproteinases (TIMPs). Several MMPs, ADAMTSs and TIMPs have been reported to be endocytosed by the low-density lipoprotein receptor-related protein-1 (LRP-1). Different binding affinities of these proteins for the endocytic receptor correlate with different turnover rates which, together with differences in their mRNA expression, determines their nett extracellular levels. In this study, we used surface plasmon resonance to evaluate the affinity between LRP-1 and a number of MMPs, ADAMs, ADAMTSs, TIMPs and metalloproteinase/TIMP complexes. This identified MMP-1 as a new LRP-1 ligand. Among the proteins analysed, TIMP-3 bound to LRP-1 with highest affinity (KD = 1.68 nM). Additionally, we found that TIMP-3 can facilitate the clearance of its target metalloproteinases by bridging their binding to LRP-1. For example, the free form of MMP-1 was found to have a KD of 34.6 nM for LRP-1, while the MMP-1/TIMP-3 complex had a 7-fold higher affinity (KD = 4.96 nM) for the receptor. TIMP-3 similarly bridged binding of MMP-13 and MMP-14 to LRP-1. TIMP-1 and TIMP-2 were also found to increase the affinity of target metalloproteinases for LRP-1, albeit to a lesser extent. These findings suggest that LRP-1 scavenging of TIMP/metalloproteinase complexes may be a general mechanism by which inhibited metalloproteinases are removed from the extracellular environment.

biochemistry↗