Abstract

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) β-binding protein 1 (LTBP1), microfibrils regulate TGF-β 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-β 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.

Highlights

  • Fibrillins are extracellular matrix (ECM) glycoproteins that form microfibrils, which act as a scaffold for elastin deposition [1]

  • To confirm whether human FBN1 is modified with Oglucose, we first analyzed endogenous human FBN1 secreted from human dermal fibroblasts

  • We confirmed FBN1, FBN2, and LTBP1 are heavily modified with O-glucose by Protein O-glucosyltransferase 2 (POGLUT2) and/or protein O-glucosyltransferase 3 (POGLUT3)

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Summary

RESEARCH ARTICLE

POGLUT2 and POGLUT3 O-glucosylate multiple EGF repeats in fibrillin-1, -2, and LTBP1 and promote secretion of fibrillin-1. Haltiwanger1,2,* From the 1Complex Carbohydrate Research Center, 2Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA

Edited by Chris Whitfield
Results
Discussion
Experimental procedures
Protein expression and purification
Mass spectral analysis
Western blotting
Full Text
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