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. Author manuscript; available in PMC: 2019 Dec 1.
Published in final edited form as: Dev Biol. 2018 Jun 27;444(Suppl 1):S237–S251. doi: 10.1016/j.ydbio.2018.06.018

Figure 2. Multiple Cad6B NTFs of various lengths are shed from cranial neural crest cells during EMT.

Figure 2.

(A) Cad6B proteolysis in cranial neural crest cells generates multiple shed ectodomains or NTFs during pre-EMT (5ss) and EMT (6ss) stages. Neural crest domains were excised, pooled, and converted to lysates for Concanavalin A enrichment. Glycoprotein eluates were equally divided with one half serving as a control to the other half that was treated with Peptide-N-Glycosidase F (PNGase F) to perform N-linked deglycosylation. In untreated samples (lane 1, 5ss, and lane 3, 6ss), a 122 kDa full-length Cad6B protein (#), 95 kDa NTF (L-NTF, +), and smaller 83 kDa NTF (S-NTF, *) were observed. Full removal of N-linked glycosylations through treatment with PNGase F in both the 5ss sample (lane 2) and the 6ss sample (lane 4) decreased molecular weights of full-length Cad6B (#) by 16 kDa to 106 kDa, L-NTF (+) by 15 kDa to 80 kDa, and S-NTF (*) by 13 kDa to 70 kDa. These comparable shifts (13–16 kDa) indicate that the NTFs are not post-translational modification variants of a single NTF but two NTFs of different length maintaining the original N-linked moieties existing on full-length Cad6B. (B) ADAM10 and an endogenous CHO cell protease differentially cleave Cad6B in vitro to generate two distinct NTFs. CHO cells transiently transfected with a Cad6B expression construct endogenously shed 88 kDa Cad6B L-NTFs into the supernatant (+, lanes 1 and 3). Treatment with PNGase F reduces its molecular weight by 17 kDa to produce a 71 kDa fragment (+, lane 2). In CHO cells transfected with both Cad6B and ADAM10 expression constructs, an additional ADAM10-specific 70 kDa S-NTF is shed (*, lane 1) that is reduced by 14kDa to 56 kDa following N-linked deglycosylation. Similar molecular weight shifts between L-NTFs (+) and S-NTFs (*) indicate that both NTFs exhibit the original N-linked glycosylation status as the full-length Cad6B. (C) Broad-spectrum protease inhibitor (GM6001) and MMP2/MMP9-specific Inhibitor V both significantly abrogate CHO cell protease shedding of Cad6B in vitro. 18 hours treatment of a Flp-In CHO cell line stably expressing Cad6B-HA with 25 μM GM6001 inhibits NTF shedding by 58.4% (black bars: GM6001 vs. CTRL, p < 0.05, n = 5), which subsequently increases Cad6B retention on CHO cell membranes by 72.8% (gray bars: GM6001 vs. CTRL, p < 0.05, n = 3). 18 hours treatment with 25 μM MMP2/MMP9 Inhibitor V also significantly inhibited NTF shedding by 52.1% (black bars: INH V vs. CTRL, p < 0.05, n = 5), which led to a 30.3% increase in Cad6B membrane retention (gray bars: INH V vs. CTRL, p = 0.065, n = 3). As a positive control, MMP2 was transiently transfected into Flp-In CHO Cad6B-HA cells to increase Cad6B shedding. Increasing supernatant MMP2 levels resulted in a 30.5% increase in 88 kDa L-NTF levels (gray bars: MMP2 vs. CTRL, p < 0.05, n = 5) and a significant reduction in CHO cell membrane Cad6B pools (45.9%, black bars: MMP2 vs. CTRL, p < 0.05, n = 3). No other bands were observed upon MMP2 overexpression, indicating that recombinant MMP2 and CHO cell MMP2/MMP9 cleave Cad6B at the same site in vitro. Treatment levels were analyzed by ANOVA, and asterisks denote treatments that are statistically different (p < 0.05). Immunoblots are representatives of independent experiments, and GAPDH served as a loading control for cell lysates. (D) HA-tagged versions of L-NTF and S-NTF constructs in Fig. S2 express secreted, N-linked glycosylated NTFS at similar molecular weights between neural crest cells in vivo and CHO cells in vitro. The HA-tagged long NTF or L-NTF-HA is comprised of the full Cad6B ectodomain including all 5 EC domains, and the fully N-linked deglycosylated forms are comparable in molecular weight when expressed in vivo (74 kDa, bottom left blot) or in vitro (72 kDa, top blot). The HA-tagged short NTF or S-NTF-HA construct, consisting of EC1-EC4.5 domains, expresses a 61 kDa deglycosylated fragment in vivo (bottom right blot), which is comparable in molecular weight to the deglycosylated 62 kDa NTF (top blot) expressed in CHO cells. The differences observed in glycosylated versions of L-NTF-HA expressed in vivo and in vitro (84 kDa versus 93 kDa, respectively) and S-NTF-HA expressed in vivo and in vitro (70 kDa versus 72 kDa, respectively) may be due to minor differences in N-linked glycosylation moieties between chicken neural crest cells and mammalian CHO cells.