Journal: The Journal of Biological Chemistry

Article Title: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo

doi: 10.1016/j.jbc.2021.100372

Figure Lengend Snippet: PSA-NCAM1 is cleaved by BACE1. A , HEK cells were transiently transfected with the indicated expression vectors (NCAM1, ST8SIA2, and BACE1) for 48 h. Representative immunoblot of cell lysates (Lysate) and conditioned media (CM) using anti-PSA-NCAM1 (2-2B), anti-Myc (2272), anti-turboGFP (OTI2H8), anti-NCAM1 (AF2408), anti-BACE1 (D10E5), and anti-GAPDH (MAB374) antibodies. ST8SIA2 ectopic expression induces an increase in PSA-NCAM1 levels. Note that the ectopic expression of BACE1 produces BACE1-specific NCAM1-βCTFs (∼38 and ∼45 kDa) in the cell lysates and released sNCAM1β in the CM. Also, secreted soluble PSA-NCAM1 levels were increased. This result indicates BACE1 cleaves PSA-NCAM1 as well as non-PSA-NCAM1 in HEK cells. n = 3. B–D , NCAM1 proteins (PSA-NCAM1 and non-PSA-NCAM1) from P10 hemibrain lysates were immunoprecipitated using anti-C-terminal NCAM1 antibody (5B8). Immunoprecipitants were incubated with or without sialidase (P0722) to remove sialic acid modification and were then incubated with or without human recombinant BACE1 (h-BACE1) ( B ). After treatment, samples were directly analyzed by western blot (WB) using anti-PSA-NCAM1 (2-2B) ( C ) or anti-N-terminal-NCAM1 (AF2408) ( D ) antibodies. As expected, PSA-NCAM1 was almost absent in samples incubated with sialidase (lanes 1 and 3). Immunoblot with anti-N-terminal-NCAM1 (AF2408) antibodies revealed the presence of an N-terminal NCAM1 fragment in samples incubated with h-BACE1 independently of sialylation status of NCAM1 ( arrowheads ). E and F , immunoprecipitated endogenous APP using anti-C-terminal APP antibody (C1/6.1) was incubated with human BACE1 (h-BACE1) as a positive control for the BACE1 enzymatic cleavage experiment. While C99 and C89 represent BACE1-mediated C-terminal APP fragments (APP-βCTFs), C83 is an α-secretase-mediated C-terminal APP fragment (APP-αCTFs). APP-CTFs are present as phosphorylated (pC99, pC89, and pC83) and nonphosphorylated (C99, C89, and C83) forms. h-BACE1 cleaves APP-FL and increased C99 and pC99 levels, concurrently decreased C89/pC89 and C83/pC83 levels. Bands around at 50 kDa ( double open arrowheads ) and 25 kDa ( open arrowheads ) correspond to the heavy and light chain of precipitated primary antibody (C1/6.1), respectively. While a Bis-Tris gel was used to see the full-length APP ( E ), Tris-Tricine gels were used to separate APP-CTFs ( F ).

Article Snippet: For polysialic acid modification of NCAM1, expression plasmid containing C-terminally turboGFP tagged mouse ST8SIA2 (MG205823) was purchased from ORIGENE.

Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation, Incubation, Modification, Recombinant, Positive Control