Figure 1. NeuropsinC208S forms a high-molecular-weight complex on the surface of hippocampal neurons. A, Neuropsin structure (Protein Data Bank code 1NPM). The catalytic triad (marked in yellow) consists of His73, Asp120, and Ser212. The disulfide bond-forming cysteines are marked SS1–SS6 (blue lines). Asp206 (green) in the S1-specific pocket and Cys208 (red) are indicated. Neuropsin forms eight loops and has one N-glycosylated site (GlcNac). Loops C and G are designated in the structure. B, Neuropsin domains and mutation sites used in this study. The signal sequence, activity-masking amino acid sequence (Q29GSK), Myc-tag, and His-tag are indicated. For modifications, Ser was substituted for Cys208 in loop G, or Ser87-Leu-Gln-Ser-Arg-Asp-Gln-Pro in loop C was deleted (ΔS87LQSRDQP). To modify the protease activity pocket, Val was substituted for Asp206 in the S1-specific pocket and Ala for Ser212 in the catalytic triad. C, Quantitative analysis of neuropsin-immunoreactive signal under the conditions specified in D. Bar diagram represents the average values (AU) obtained for different cultures (n = 5 different cultures, 2 neurons per cultures in each condition). *p < 0.05; **p < 0.01. Error bars indicate the SEM. D, Extracellular protein complex bound to mutant neuropsin. Immunostained images are merged with those showing intracellular EGFP fluorescence (green). Neuropsin was detected using an anti-neuropsin antibody (red) under nonpermeabilized conditions. Hippocampal neurons were transfected with preneuropsin, preneuropsinC208S, preproneuropsinC208S, preneuropsinD206V, or preneuropsinS212A. Scale bar, 10 μm. Bottom, High-magnification images of the rectangles in the top. Scale bar, 2 μm. E, Double immunostaining of preneuropsinC208S-transfected hippocampal neurons with an anti-neuropsin antibody under nonpermeabilized conditions (red; top) and an anti-PSD-95 antibody under permeabilized conditions (blue; middle), and their superimposed images (bottom). Neuropsin immunoreactivity was localized to the surface of dendrites in close apposition with intracellular PSD-95. Scale bar, 1 μm. F, BlueNative-PAGE of preproneuropsin-, preneuropsin-, preproneuropsinC208S-, or preneuropsinC208S-transfected cell lysate, immunoblotted and probed using an anti-neuropsin antibody. An ∼1-MDa high-molecular-weight protein complex was detected in the preneuropsinC208S cell lysate (arrow) (n = 3). G, SDS-PAGE of preneuropsinC208S-transfected cell lysates under nonreducing (preneuropsinC208S), boiling (+boil) or reducing conditions (+DTT). Although the high-molecular-weight band (arrow) was weaker or disappeared after boiling or DTT pretreatment, a monomeric neuropsin protein band was apparent in both cases (arrowhead) (n = 3). H, Top, Cell lysates transfected with preneuropsinC208S immunoprecipitated with an anti-neuropsin antibody. Immunoprecipitates were treated with heparitinase to degrade glycosaminoglycans and immunoblotted with an anti-Myc antibody. Note that the high-molecular-weight complex (black arrow) was weaker after heparitinase pretreatment, and an ∼150 kDa band (red arrow) appeared (n = 3). IP, Immunoprecipitation; IB, immunoblotting. Bottom, The high-molecular-weight band (arrow) was weaker after neuropsin pretreatment (n = 3).