Fig 1.
HNK-1 epitope is expressed on the N-glycan of tenascin-C.
(A) Soluble fractions prepared from embryonic day 16 (E16), postnatal day 0 and 7 (P0 and P7, respectively), 2- and 10-week-old (2W and 10W, respectively) mouse brains were immunoblotted with HNK-1 mAb, anti-TNC pAb, and anti-Tubulin mAb (for loading control). Each blot was representative of 12 images of western blots. 50, 80, and 20 μg proteins were loaded to each lane for HNK-1, TNC, and Tubulin, respectively. (B) Soluble fractions of E16, P0, P7, and 2W mouse brains were immunoprecipitated with anti-TNC pAb and antibody-bound (Bound) and unbound (Unbound) fractions were obtained. Each blot was representative of 8 images of western blots. 200 μg proteins were used for each immunoprecipitation. (C) Soluble fractions of E16, P0, P7, and 2W mouse brains were treated with or without peptide N-glycosidase F (PNGase F or No treatment, respectively). The blot was representative of 5 images of western blots. 20 μg proteins were loaded to each lane.
Fig 2.
HNK-1 epitope on alternatively spliced FNIII repeats promotes neurite outgrowth.
(A) Purified seven fibronectin type-III repeats (FNx7) with or without HNK-1 synthesis enzymes (HNK-1(+) or HNK-1(-), respectively) were subjected to SDS-PAGE and stained with CBB or immunoblotted with anti-myc pAb and HNK-1 mAb. Each blot was representative of 2 images of western blots. 1 and 0.2 μg proteins were loaded to each lane for CBB and western blots, respectively. (B) Primary hippocampal neurons were cultured on poly-D-lysine (PDL), PDL coated with FNx7 without HNK-1 expression (FNx7 HNK-1(-)), or PDL coated with FNx7 with HNK-1 expression (FNx7 HNK-1(+)). At day 3 in vitro (DIV3), neurons were immunostained with anti-Tau-1 mAb. (C) Estimation of neurite length on each coating. Green, PDL; yellow, FNx7 HNK-1(-); and orange, FNx7 HNK-1(+). Numbers of neurons measured on PDL, FNx7 HNK-1(-), and FNx7 HNK-1(+) were 63, 138, 43, respectively. This experiment was performed across seven independent cultures, and the representative result obtained from a single culture was shown. Error bars represent standard error of the mean (SEM). ***p < 0.001.
Fig 3.
HNK-1 epitope on the C domain is effective for promoting neurite outgrowth.
(A–C) Primary hippocampal neurons were cultured on PDL, PDL coated with FNx6 (A), FNx5 (B), or FNx3 (C) without HNK-1 expression (HNK-1(-)), or PDL coated with FNx6 (A), FNx5 (B), or FNx3 (C) with HNK-1 expression (HNK-1(+)). The cultured neurons were immunostained and analyzed at DIV3. Green, PDL (n = 130, 131, and 86 for A, B, and C, respectively); yellow, FNx6, FNx5, or FNx3 HNK-1(-) (n = 161, 241, and 124 for FNx6, FNx5, and FNx3 HNK-1(-), respectively); and orange, FNx6, FNx5, or FNx3 HNK-1(+) (n = 152, 143, and 92 for FNx6, FNx5, and FNx3 HNK-1(+), respectively). Numbers of measured neurons were indicated in parentheses. These experiments were performed with three independent cultures and the representative results obtained from a single culture were shown. Error bars represent SEM. ***p < 0.001; n.s. (not significant), p > 0.05.
Fig 4.
HNK-1 epitope on N1534 of the C domain affects neurite outgrowth.
(A) Scheme of FNIIIx7N1534Q-myc-His (N1534Q). N-glycosylation site (Asn-X-Ser/Thr; highlighted in yellow) at N1534 was mutated to glutamine. (B) N1534Q was purified with or without HNK-1 synthesis enzymes (HNK-1(+) or HNK-1(-), respectively), subjected to SDS-PAGE and stained with CBB or immunoblotted with anti-myc pAb and HNK-1 mAb. Each blot was representative of 2 images of western blots. 1 and 0.2 μg proteins were loaded to each lane for CBB and western blots, respectively. (C) Primary hippocampal neurons were cultured on PDL, PDL coated with N1534Q without HNK-1 expression (HNK-1(-)), or PDL coated with N1534Q with HNK-1 expression (HNK-1(+)). Neurons were immunostained and analyzed at DIV3. Green, PDL; yellow, N1534Q HNK-1(-); and orange, N1534Q HNK-1(+). Numbers of neurons measured on PDL, N1534Q HNK-1(-), and N1534Q HNK-1(+) were 184, 197, 195, respectively. This experiment was performed across two independent cultures, and the representative result obtained from a single culture was shown. Error bars represent SEM. ***p < 0.001; n.s., p > 0.05.
Fig 5.
Contactin-1 mediates neurite outgrowth derived from the HNK-1 epitope on tenascin-C.
Primary hippocampal neurons were cultured on PDL, PDL coated with FNx7 without HNK-1 expression (HNK-1(-)), or PDL coated with FNx7 with HNK-1 expression (HNK-1(+)). Normal goat IgG (+ normal IgG) or anti-CNTN pAb (+ CNTN Ab) was added to the culture medium at a final concentration of 30 μg/mL after 18 h of culture. Neurons were immunostained and analyzed at DIV3. Green, PDL with normal goat IgG (n = 219); light green, PDL with anti-CNTN pAb (n = 130); yellow, FNx7 HNK-1(-) with normal goat IgG (n = 139); light yellow, FNx7 HNK-1(-) with anti-CNTN pAb (n = 163); orange, FNx7 HNK-1(+) with normal goat IgG (n = 154); and light orange, FNx7 HNK-1(+) with anti-CNTN pAb (n = 166). Numbers of measured neurons were indicated in parentheses. This experiment was performed across two independent cultures, respectively, and the representative result obtained from a single culture was shown. Error bars represent SEM. ***p < 0.001; n.s., p > 0.05.
Fig 6.
Model of neurite regulation by the HNK-1 epitope on tenascin-C and contactin-1.
In E16 mouse brains, the HNK-1 epitope is expressed on both TNC and CNTN, inducing maximal neurite outgrowth, which could promote brain development and/or neuronal circuit formation (left panel). As development progresses, the HNK-1 epitope on TNC is nearly lost completely in the P7 mouse brain, whereas the epitope is expressed by CNTN. However, TNC is still expressed in the brain, and exhibits neurite-promoting activity, which might induce efficient neurite outgrowth via HNK-1-expressing CNTN in early development (middle panel). However, in the P14 mouse brain, TNC expression is almost abolished, resulting in weak neurite promotion (right panel).