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. 2020 Jun 22;39(15):e103457. doi: 10.15252/embj.2019103457

Figure 4. Loss of SEZ6 impairs glycosylation and maturation of GluK2/3.

Figure 4

  • A
    In WT neurons, GluK2/3 was seen as two closely comigrating bands, whereas the upper one of lower intensity was missing or running even more closely to the lower band of main intensity in SEZ6KO neurons (schematic representation on the right). When the total lysates of SEZ6KO and WT neurons were digested with endoglycosidase H (EndoH)—which removes immature, but not mature sugars—the two closely comigrating bands were converted to three bands of a lower apparent molecular weight, consistent with full deglycosylation of the lowest band and a partial deglycosylation of the upper two bands (marked in light blue, red, and green in the right panel). In SEZ6KO neurons, the uppermost (light blue) band was missing and a new band of lower apparent molecular weight was seen that was overlapping with the red labeled band and was indicated with the purple asterisk in the SEZ6KO. No difference in the glycosylation of GluA2 was detectable, pointing to a specific effect of SEZ6 on GluK2/3.
  • B
    The running pattern of GluK2/3 in SEZ6KO and WT brains was compared upon EndoH digestion. Similar to primary neurons, GluK2/3 displayed an immature glycosylation pattern in SEZ6KO brains.
  • C
    Total GluK2/3 levels were quantified and showed a significant but moderate reduction in SEZ6KO compared to WT neuronal lysates (plot shows mean ± SEM, 9 replicates in 3 independent biological experiments, Mann–Whitney **P‐value = 0.0012).
  • D
    No significant change in GluK2/3 total levels was detectable in SEZ6KO and WT adult brains (plot shows mean ± SEM, 6 replicates, Mann–Whitney test was used).
  • E
    Synaptosomes from knock‐out (KO) brains of single SEZ6 family members (SEZ6, SEZ6L, SEZ6L2), triple knock‐out (TKO), and respective controls (WT and TWT—triple WT) were digested with EndoH. GluK2/3 displayed the immature glycosylation in SEZ6KO and TKO brains, but not in SEZ6L and SEZ6L2 brains, demonstrating non‐redundant functions for the SEZ6 family members (n = 4).
  • F
    Brain homogenates from SEZ6KO and WT mice at different ages were digested with EndoH. GluK2/3 displayed the immature glycosylation at all developmental stages of SEZ6KO mice, suggesting that no compensation effect is occurring with aging.

Source data are available online for this figure.