POGLUT2 and POGLUT3 O-glucosylate multiple EGF repeats in fibrillin-1, -2, and LTBP1 and promote secretion of fibrillin-1.

Fibrillin-1 (FBN1) is the major component of extracellular matrix microfibrils, which are required for proper development of elastic tissues, including the heart and lungs. Through protein-protein interactions with latent transforming growth factor (TGF) ß-binding protein 1 (LTBP1), microfibrils regulate TGF-ß signaling. Mutations within the 47 epidermal growth factor-like (EGF) repeats of FBN1 cause autosomal dominant disorders including Marfan Syndrome, which is characterized by disrupted TGF-ß signaling. We recently identified two novel protein O-glucosyltransferases, Protein O-glucosyltransferase 2 (POGLUT2) and 3 (POGLUT3), that modify a small fraction of EGF repeats on Notch. Here, using mass spectral analysis, we show that POGLUT2 and POGLUT3 also modify over half of the EGF repeats on FBN1, fibrillin-2 (FBN2), and LTBP1. While most sites are modified by both enzymes, some sites show a preference for either POGLUT2 or POGLUT3. POGLUT2 and POGLUT3 are homologs of POGLUT1, which stabilizes Notch proteins by addition of O-glucose to Notch EGF repeats. Like POGLUT1, POGLUT2 and 3 can discern a folded versus unfolded EGF repeat, suggesting POGLUT2 and 3 are involved in a protein folding pathway. In vitro secretion assays using the N-terminal portion of recombinant FBN1 revealed reduced FBN1 secretion in POGLUT2 knockout, POGLUT3 knockout, and POGLUT2 and 3 double-knockout HEK293T cells compared with wild type. These results illustrate that POGLUT2 and 3 function together to O-glucosylate protein substrates and that these modifications play a role in the secretion of substrate proteins. It will be interesting to see how disease variants in these proteins affect their O-glucosylation.

Global computational mutagenesis of domain structures associated with inherited eye disease.

Multidomain proteins account for 70% of the eukaryotic proteome. In genetic disease, multidomain proteins are often affected by numerous mutations, but the effects of these mutations on protein stability and their roles in genetic disease are not well understood. Here, we analyzed protein globular domains to understand how genetic mutations affect the stability of multidomain proteins in inherited disease. In total, 291 domain atomic structures from nine multidomain proteins were modeled by homology, equilibrated using molecular dynamics in water, and subjected to global computational mutagenesis. The domains were separated into 7 groups based on protein fold homology. Mutation propensities within each group of domains were then averaged to select residues critical for domain fold stability. The consensus derived from the sequence alignment shows that the critical residues determined by global mutagenesis are conserved within each group. From this analysis, we concluded that 80% of known disease-related genetic variants are associated with critical residues and are expected to have significant destabilizing effects on domain structure. Our work provides an in silico quantification of protein stability and could help to analyze the complex relationship among missense mutations, multidomain protein stability, and disease phenotypes in inherited eye disease.

Fibrillin-2 and Tenascin-C bridge the age gap in lung epithelial regeneration.

Organ engineering based on native matrix scaffolds involves combining regenerative cell populations with corresponding biological matrices to form functional grafts on-demand. The extracellular matrix (ECM) that is retained following lung decellularization provides essential structure and biophysical cues for whole organ regeneration after recellularization. The unique ECM composition in the early post-natal lung, during active alveologenesis, may possess distinct signals that aid in driving cell adhesion, survival, and proliferation. We evaluated the behavior of basal epithelial stem cells (BESCs) isolated from adult human lung tissue, when cultured on acellular ECM derived from neonatal (aged < 1 week) or adult lung donors (n = 3 donors per group). A significant difference in cell proliferation and survival was found. We next performed in-depth proteomic analysis of the lung scaffolds to quantify proteins significantly enriched in the neonatal ECM, and identified the glycoproteins Fibrillin-2 (FBN-2) and Tenascin-C (TN-C) as potential mediators of the observed effect. BESCs cultured on Collagen Type IV coated plates, supplemented with FBN-2 and TN-C demonstrated significantly increased proliferation and decreased cellular senescence. No significant increase in epithelial-to-mesenchymal transition was observed. In vitro migration was also increased by FBN-2 and TN-C treatment. Decellularized lung scaffolds treated with FBN-2 and TN-C prior to re-epithelialization supported greater epithelial proliferation and tissue remodeling. BESC distribution, matrix alignment, and overall tissue morphology was improved on treated lung scaffolds, after 3 and 7 days of ex vivo lung culture. These results demonstrate that scaffold re-epithelialization is enhanced on neonatal lung ECM, and that supplementation of FBN-2 and TN-C to the native scaffold may be a valuable tool in lung tissue regeneration.

Exome Sequencing Identified a Novel FBN2 Mutation in a Chinese Family with Congenital Contractural Arachnodactyly.

Congenital contractural arachnodactyly (CCA) is an autosomal dominant disorder of connective tissue. CCA is characterized by arachnodactyly, camptodactyly, contrature of major joints, scoliosis, pectus deformities, and crumpled ears. The present study aimed to identify the genetic cause of a three-generation Chinese family with CCA. We successfully identified a novel missense mutation p.G1145D in the fibrillin-2 (FBN2) gene as the pathogenic mutation by whole exome sequencing (WES). The p.G1145D mutation occurs in the 12th calcium-binding epidermal growth factor-like (cbEGF) domain. The p.G1145D mutation caused a hydrophobic to hydrophilic substitution, altering the amino acid property from neutral to acidic. Three-dimensional structural analysis showed that this mutation could alter the conformation of the residue side chain, thereby producing steric clashes with spatially adjacent residues, disrupting the formation of H bonds and causing folding destabilization. Therefore, this amino acid appears to play an important role in the structure and function of FBN2. Our results may also provide new insights into the cause and diagnosis of CCA and may have implications for genetic counseling and clinical management.