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

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 ( pmm2 sa10150 ) 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 pmm2 sa10150 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 pmm2 sa10150 mutant embryos. Importantly, pharmacologic and genetic manipulation of proconvertase activity restored matrix metalloproteinase activity, N-cadherin processing, and cartilage pathology in pmm2 sa10150 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.

Tracer metabolomics reveals the role of aldose reductase in glycosylation

“…The pmm2 mutant zebrafish harbors a mutation in an essential splice site ( pmm2 sa1050 ), which results in a hypomorphic pmm2 allele. 23 By 6 days post fertilization (dpf), PMM activity is significantly decreased in pmm2 mutant zebrafish embryos ( Figure 1 D), 23 with levels comparable with the enzymatic activity measured in PMM2-CDG patient fibroblasts. 19 Like the in vitro PMM2-CDG fibroblast model, GDP-mannose levels were reduced in pmm2 mutant embryos compared with wild type ( Figure 1 D).…”

“…Then, using an automated behavioral tracking system ( Figures 3 A and 3B), we asked if epalrestat improved the previously characterized defects in pmm2 zebrafish swim behavior. 23 We chose 6-dpf embryos, as the pmm2 mutant zebrafish first exhibit differences in swim behavior at 5–6 dpf. These differences become more pronounced as the animals age, resulting in death between 10 and 13 dpf.…”

“…These differences become more pronounced as the animals age, resulting in death between 10 and 13 dpf. 23 Therefore, we analyzed epalrestat’s effect at 6–10 dpf. This time window provides the best chance to evaluate epalrestat’s ability to improve swim behavior without the complicating effects of global system death.…”

“…This time window provides the best chance to evaluate epalrestat’s ability to improve swim behavior without the complicating effects of global system death. Choosing this time window is further supported by the fact that the pmm2 mutant zebrafish have other tissue defects (beyond altered swim behavior) 23 that may also contribute to their early mortality. Specifically, development of craniofacial cartilage is disrupted in pmm2 mutants, which raises the possibility that feeding problems also contribute to system failure in these animals.…”

“…Animals were maintained according to standard protocols. The zebrafish strains were originally obtained from the Zebrafish International Resource Center (ZIRC, Eugene, OR) 23 . Staging was done according to established criteria.…”