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Protease-dependent defects in N-cadherin processing drive PMM2-CDG pathogenesis
Elsenoor J. Klaver, … , Richard Steet, Heather Flanagan-Steet
Elsenoor J. Klaver, … , Richard Steet, Heather Flanagan-Steet
Published November 16, 2021
Citation Information: JCI Insight. 2021;6(24):e153474. https://doi.org/10.1172/jci.insight.153474.
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Research Article Development

Protease-dependent defects in N-cadherin processing drive PMM2-CDG pathogenesis

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Abstract

The genetic bases for the congenital disorders of glycosylation (CDG) continue to expand, but how glycosylation defects cause patient phenotypes remains largely unknown. Here, we combined developmental phenotyping and biochemical studies in a potentially new zebrafish model (pmm2sa10150) of PMM2-CDG to uncover a protease-mediated pathogenic mechanism relevant to craniofacial and motility phenotypes in mutant embryos. Mutant embryos had reduced phosphomannomutase activity and modest decreases in N-glycan occupancy as detected by matrix-assisted laser desorption ionization mass spectrometry imaging. Cellular analyses of cartilage defects in pmm2sa10150 embryos revealed a block in chondrogenesis that was associated with defective proteolytic processing, but seemingly normal N-glycosylation, of the cell adhesion molecule N-cadherin. The activities of the proconvertases and matrix metalloproteinases responsible for N-cadherin maturation were significantly altered in pmm2sa10150 mutant embryos. Importantly, pharmacologic and genetic manipulation of proconvertase activity restored matrix metalloproteinase activity, N-cadherin processing, and cartilage pathology in pmm2sa10150 embryos. Collectively, these studies demonstrate in CDG that targeted alterations in protease activity create a pathogenic cascade that affects the maturation of cell adhesion proteins critical for tissue development.

Authors

Elsenoor J. Klaver, Lynn Dukes-Rimsky, Brijesh Kumar, Zhi-Jie Xia, Tammie Dang, Mark A. Lehrman, Peggi Angel, Richard R. Drake, Hudson H. Freeze, Richard Steet, Heather Flanagan-Steet

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Figure 1

mRNA splicing is disrupted in pmm2m/m transcripts generating a hypomorphic allele.

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mRNA splicing is disrupted in pmm2m/m transcripts generating a hypomorph...
(A) Schematic illustrates zebrafish pmm2. (B) High resolution melting curve (HRM) analysis shows melting curves for pmm2+/+, pmm2+/m (heterozygous for the sa10150 allele), and pmm2 [G/A] mutants homozygous for the sa10150 allele (pmm2m/m). (C) Schematic shows use of embryo fin for HRM genotyping. Reverse transcriptase PCR (RT-PCR) analyses reveal 3 unique pmm2 gene products in pmm2m/m embryos (forms 1–3). (D) Sequencing of individual RT-PCR products shows a frameshift in forms 1 and 3 with early stop codons. Form 2 contains an in-frame truncation of exon 5, explaining the hypomorphic allele. (E) Pmm activity measured in embryonic lysates shows a progressive decrease in activity in pmm2m/m embryos. n = 3 experiments of 25 embryos per sample. Error bars show SEM, Dunnett’s test, **P < 0.01, ***P < 0.001. (F) Bright-field images of embryos 4 and 7 dpf show no obvious differences between pmm2+/+ and pmm2m/m embryos. Scale bar: 100 μm. (G) Schematic illustrates several key structures of embryonic jaw, including Meckel’s cartilage (M) and the ceratohyal (CH), with arrowed lines demonstrating parameters measured. Alcian blue staining of ventral structures of 6 dpf embryos reveals differences in the shape of M and CH cartilages. Flatmount preparations show morphological alterations are associated with immature chondrocytes that are round and disorganized. n = 25–30 embryos per condition over 3 experiments. Scale bars: 10 μm. (H) Quantitation of CH cartilage angle, CH length, and M cartilage length show multiple pmm2+/+ and pmm2m/m embryos. n = 25–30 embryos per condition over 3 experiments. Error bars show SEM, Student’s t test, **P < 0.01, ***P < 0.001.

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