Journal: Advanced Science
Article Title: Humanized Biomimetic Nanovesicles for Neuron Targeting
doi: 10.1002/advs.202101437
Figure Lengend Snippet: Physiochemical and biomimetic characterization of neural biomimetic NVs. A) iNeurons were directly generated from a genetically engineered human pluripotent stem cell (hPSC) line containing a doxycycline (dox)‐inducible neurogenin 2 (ngn2) transgene. A pure population was obtained within 7 days of differentiation. B) A stable membrane‐bound green fluorescent protein (memGFP) transgene was incorporated into the hPSC line to track protein carry‐over. Scale: 100 µm. C) A microfluidic approach was utilized for the synthesis of neural biomimetic NVs with cell‐specific membrane proteins and two different lipid formulations (i.e., A and B). Three NV groups were fabricated using each lipid formulation: “liposomes” (lipo‐, L ) containing no protein, “plurisomes” (pluri‐, P ) containing hPSC‐derived proteins, and “neurosomes” (neuro‐, N ) containing iNeuron‐derived proteins. D) Immunoblotting revealed the transfer of mem‐GFP in plurisomes and neurosomes (NVs originating from hPSCs and iNeurons, respectively) as well as the transfer of neuronal membrane protein (MP) marker NCAM1 in neurosomes of both formulations. (Bands are replicated from Figure S1E (Supporting Information), with dividing lines indicating splicing from original image.) E) Cryo‐TEM images illustrated that all NV formulations had similar lipid bilayer morphologies containing a spherical bilayer structure. Scale: 50 nm. F) Physiochemical properties including NV size, PDI, and zeta potential were assessed. Though neither NV size nor PDI were significantly altered between NVs of different lipid formulations, NVs from lipid formulation B displayed a less negative zeta potential ( n = 3–7 independent NV batches per group; see Figure S1F in the Supporting Information). For Figure , results are shown as mean ± SEM. One‐way ANOVA followed by Tukey's multiple comparison test was used to determine statistical probabilities between NVs of different protein sources within the same formulation (A or B), with * p < 0.05.
Article Snippet: The aqueous phase for formulation A consisted of 1× PBS alone (for liposomes) or 1× PBS with extracted membrane proteins (for plurisome and neurosomes) or with recombinant human NCAM1 protein (R&D Systems).
Techniques: Generated, Membrane, Formulation, Liposomes, Derivative Assay, Western Blot, Marker, Zeta Potential Analyzer, Comparison
![NCAM2 and NCAM1 are BACE1 substrates in the mouse olfactory bulb. A–B , schematic diagram of the two mouse NCAM2 (transmembrane [TM] and GPI-anchored) isoforms ( A ) and three major mouse NCAM1 (transmembrane [NCAM1-180 and NCAM1-140] and GPI-anchored [NCAM1-120]) isoforms ( B ) resulting from alternative splicing. NCAM2 and NCAM1 have five immunoglobulin (Ig)-like domains represented by ovals and two fibronectin type III repeats (FN) represented by rectangles. C , schematic presentation of NCAM2-TM and polysialylated NCAM1-140 illustrating antibody-binding sites. NCAM2 contains eight putative Asn-linked glycosylation sites (N-glycosylation) indicated by blue triangles, while NCAM1 has six N-glycosylation sites. In the fifth Ig (Ig5) of NCAM1, two of three N-glycosylation sites are polysialylated, represented by a chain of green circles (sialic acid). D , representative immunoblot of PBS soluble fraction (Soluble) and membrane fraction (Membrane) of olfactory bulb samples from 4-month-old BACE1+/+ and BACE1−/− mice using anti-NCAM2 (sc-136328), anti-NCAM1 (AF-2408), BACE1 (D10E5), GAPDH (MAB374), and anti-calnexin (610523) antibodies. BACE1-specific soluble NCAM2 (sNCAM2β) and NCAM1 (sNCAM1β) are observed in BACE1+/+ mice, but not in BACE1−/− mice. Full-length NCAM1 (NCAM1-180 and NCAM1-140, but not NCAM1-120) levels were slightly increased in BACE1−/−, compared with BACE1+/+ mice. However, levels of full-length NCAM2 (NCAM2-TM and NCAM2-GPI) were similar in BACE1+/+ and BACE1−/− mice. E–G , representative immunoblot of membrane fractions of olfactory bulb samples from 4-month-old BACE1+/+ and BACE1−/− mice using two different anti-C-terminal NCAM1 (rabbit pAb; AB5032 [ E ] and mouse mAb; 0B11 [ F ]) and anti-C-terminal NCAM2 (goat pAb; GTX89311 [ G ]) antibodies. After longer exposure, a ∼75 kDa NCAM1-CTF was detected in BACE1+/+ mice, but it was greatly decreased in BACE1−/− mice, and thus termed NCAM1-βCTF (arrow in D and E ). However, a BACE1-specific NCAM2-βCTF was not observed ( G ). H , sagittal section from 12-month-old BACE1+/+ olfactory bulb was stained with anti-BACE1 (3D5, magenta ), anti-NCAM2 (AF778, green ), and anti-NCAM1 (AB5032, red ) antibodies. NCAM1 and NCAM2 immunostaining with BACE1 are significantly overlapped in glomeruli. Scale bar represents 100 μm. D (BACE1+/+; n = 5–6, BACE1−/−; n = 5); E–G (BACE1+/+; n = 3 and BACE1−/−; n = 3); H (BACE1+/+; n = 4, two sagittal ( H ) and two coronal [ <xref ref-type=](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9136/pmc07949136/pmc07949136__gr1.jpg)
Fig. S3 ] sections). " width="100%" height="100%">
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: NCAM2 and NCAM1 are BACE1 substrates in the mouse olfactory bulb. A–B , schematic diagram of the two mouse NCAM2 (transmembrane [TM] and GPI-anchored) isoforms ( A ) and three major mouse NCAM1 (transmembrane [NCAM1-180 and NCAM1-140] and GPI-anchored [NCAM1-120]) isoforms ( B ) resulting from alternative splicing. NCAM2 and NCAM1 have five immunoglobulin (Ig)-like domains represented by ovals and two fibronectin type III repeats (FN) represented by rectangles. C , schematic presentation of NCAM2-TM and polysialylated NCAM1-140 illustrating antibody-binding sites. NCAM2 contains eight putative Asn-linked glycosylation sites (N-glycosylation) indicated by blue triangles, while NCAM1 has six N-glycosylation sites. In the fifth Ig (Ig5) of NCAM1, two of three N-glycosylation sites are polysialylated, represented by a chain of green circles (sialic acid). D , representative immunoblot of PBS soluble fraction (Soluble) and membrane fraction (Membrane) of olfactory bulb samples from 4-month-old BACE1+/+ and BACE1−/− mice using anti-NCAM2 (sc-136328), anti-NCAM1 (AF-2408), BACE1 (D10E5), GAPDH (MAB374), and anti-calnexin (610523) antibodies. BACE1-specific soluble NCAM2 (sNCAM2β) and NCAM1 (sNCAM1β) are observed in BACE1+/+ mice, but not in BACE1−/− mice. Full-length NCAM1 (NCAM1-180 and NCAM1-140, but not NCAM1-120) levels were slightly increased in BACE1−/−, compared with BACE1+/+ mice. However, levels of full-length NCAM2 (NCAM2-TM and NCAM2-GPI) were similar in BACE1+/+ and BACE1−/− mice. E–G , representative immunoblot of membrane fractions of olfactory bulb samples from 4-month-old BACE1+/+ and BACE1−/− mice using two different anti-C-terminal NCAM1 (rabbit pAb; AB5032 [ E ] and mouse mAb; 0B11 [ F ]) and anti-C-terminal NCAM2 (goat pAb; GTX89311 [ G ]) antibodies. After longer exposure, a ∼75 kDa NCAM1-CTF was detected in BACE1+/+ mice, but it was greatly decreased in BACE1−/− mice, and thus termed NCAM1-βCTF (arrow in D and E ). However, a BACE1-specific NCAM2-βCTF was not observed ( G ). H , sagittal section from 12-month-old BACE1+/+ olfactory bulb was stained with anti-BACE1 (3D5, magenta ), anti-NCAM2 (AF778, green ), and anti-NCAM1 (AB5032, red ) antibodies. NCAM1 and NCAM2 immunostaining with BACE1 are significantly overlapped in glomeruli. Scale bar represents 100 μm. D (BACE1+/+; n = 5–6, BACE1−/−; n = 5); E–G (BACE1+/+; n = 3 and BACE1−/−; n = 3); H (BACE1+/+; n = 4, two sagittal ( H ) and two coronal [ Fig. S3 ] sections).
Article Snippet: After washing the beads with PBS, PSA-NCAM1 or non-PSA-NCAM1 proteins were sequentially incubated in a reaction buffer (100 mM sodium acetate, pH 4.5) including recombinant human BACE1 (931-AS-050; R&D systems) at 37 °C overnight.
Techniques: Alternative Splicing, Binding Assay, Western Blot, Membrane, Staining, Immunostaining
Figure 1 . One-way ANOVA with Tukey's multiple comparison test was applied. ∗ p < 0.05, ∗∗ p <0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, A–D , n = 3. Full-length versions of the western blots in C are shown in
Figure S4B . ns, not significant. " width="100%" height="100%">
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: NCAM1 is cleaved by metalloproteinases or BACE1, but not sequentially cleaved by γ-secretase in HEK cells. HEK cells were transfected with an NCAM1-Myc-DDK expression vector (NCAM1-140 isoform) or an empty vector (EV) as a control and then treated with DMSO only or indicated inhibitors dissolved in DMSO; GI254023X (GI; selective ADAM10 inhibitor, 5 μM), GM6001 (GM; broad spectrum of MMPs inhibitor, 2.5 μM), C3 (inhibitor IV, 10 μM), and DAPT (γ-secretase inhibitor, 10 μM) for 24 h as indicated. Total DNA concentration was kept constant at 1.5 μg for the empty vector. A , representative immunoblot of cell lysates (Lysate) and conditioned media (CM) using anti-Myc (2272), anti-NCAM1 (AF2408), anti-BACE1 (D10E5), and anti-GAPDH (MAB374) antibodies. Ectopically expressed NCAM1 undergoes proteolysis with the production of three C-terminal NCAM1 fragments (NCAM1-CTFs): a major fragment of ∼34 kDa, and two additional fragments of ∼37 kDa, and ∼47 kDa (see arrowheads ) and secreted soluble fragments (sNCAM1) in conditioned media. The asterisk denotes nonspecific bands. GI and GM treatments significantly decreased the levels of NCAM1-CTF (∼34 kDa) and sNCAM1, indicating that NCAM1 is cleaved by ADAM10 and MMPs. C , HEK cells were cotransfected with NCAM1 and BACE1 or empty vector as a control and then treated with DMSO, C3, or DAPT for 24 h. Note that the ectopic expression of BACE1 results in cleavage of NCAM1 and the production of BACE1-specific NCAM1-CTF (NCAM1-βCTFs; ∼38 kDa and ∼45 kDa) and soluble NCAM1 (sNCAM1β). However, NCAM1-βCTFs were not accumulated by DAPT treatment, suggesting that NCAM1-βCTFs were not sequentially cleaved by γ-secretase. The asterisk denotes nonspecific bands. B and D , graphs represent densitometric quantification of 34 kDa NCAM1-CTF/NCAM1-FL, NCAM1-FL/GAPDH or NCAM1-CTF/GAPDH ratio in lysates and secreted soluble NCAM1 (sNCAM1) in CM. Note that a different protein ladder was used in this figure compared with Figure 1 . One-way ANOVA with Tukey's multiple comparison test was applied. ∗ p < 0.05, ∗∗ p <0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, A–D , n = 3. Full-length versions of the western blots in C are shown in Figure S4B . ns, not significant.
Article Snippet: After washing the beads with PBS, PSA-NCAM1 or non-PSA-NCAM1 proteins were sequentially incubated in a reaction buffer (100 mM sodium acetate, pH 4.5) including recombinant human BACE1 (931-AS-050; R&D systems) at 37 °C overnight.
Techniques: Transfection, Expressing, Plasmid Preparation, Concentration Assay, Western Blot, Comparison
Fig. 2 C ) and NCAM1-βCTF (∼38 kDa, in
Fig. 3 C ) were cut from the gel and sequenced by microcapillary LC/MS/MS to identify the BACE1 cleavage site. NCAM2-FL and NCAM1-FL were also sequenced as a control. A , comparison of BACE1 cleavage sites on mouse and human APP, NCAM2, and NCAM1. BACE1 cleaves APP at Asp1 within Aβ peptides ( yellow box ) and also cleaves APP at Glu11 (G 680 Y 681 ↓ E 682 V 683 ) within Aβ peptides, which are located 28 and 18 residues distant from the transmembrane region ( gray box ), respectively. APP numbering is based on the 770 amino acid isoform. NCAM2 and NCAM1 numbering is that of the mouse NCAM2-TM and mouse NCAM1-180 amino acid isoform, respectively. The ↓ symbol denotes the scissile (cleavage) bond. BACE1 cleavage sites of NCAM2 (G 661 Y 662 ↓ E 663 V 664 ) and NCAM1 (E 669 Y 670 ↓ E 671 V 672 ) in the second fibronectin type-III repeat domain ( green box , see FN2 in
Fig. 1 C ) are located 35 and 41 residues distant from the transmembrane region ( gray box ), respectively. Notably, these cleavage sites are identical or similar to the secondary BACE1 cleavage site (β′) of human APP site (GY↓EV). The cleavage sites of NCAM2 at Asp 693 are indicated by the arrowhead. B and C , ion chromatogram of the peptide peak (EVQITAANR; m/z = 501.27–501.29) from the stained bands (32-kDa NCAM2-βCTF and NCAM2-FL as control). D and E , ion chromatogram of the peptide peak (EVQITAANR; m/z = 682.34–682.35) from the stained bands (38-kDa NCAM1-βCTF and NCAM1-FL as control). F and G , BACE1 cleavage site is denoted by a down arrow depicting the scissile bond (P1-P1′). All of the site-directed mutageneses in P1-P1′ amino acids are denoted. HEK cells were cotransfected with BACE1 and wild-type (WT) NCAM or mutant NCAM and then conditioned for 24 h with solvent only (DMSO) or BACE inhibitor (C3, 10 μM) dissolved in DMSO. Total DNA concentration was kept constant at 1.5 μg with empty vector. Cell lysates were analyzed by immunoblot to assess the BACE1 processing of mutant NCAM2 (E663H, YE662/663AH, E663K, YE662/663VK) ( F ) or NCAM1 (E671H, YE670/671AH, E671K, YE670/671VK) ( G ) compared with WT. When BACE1 is overexpressed, only NCAM2-YE662/663AH prohibits the generation of BACE1-specific 32-kDa NCAM2-βCTF ( arrowhead ). Instead, additional bands are produced, but these bands are abolished by BACE inhibition (C3). These data indicate that BACE1 cleaves NCAM2 between Y 662 and E 663 . NCAM1-E671H mutation did not prevent the generation of NCAM1-βCTFs at ∼38 kDa and ∼45 kDa ( arrowhead ). The ectopic expression of NCAM1-YE670/671AH, NCAM1-E671K, and NCAM1-YE670/671VK generates additional C-terminal fragments of NCAM1 at ∼41 kDa (41-kDa NCAM1-CTF) compared with NCAM1-WT even in the absence of BACE1 expression ( open arrowhead ). The coexpression of NCAM1-E671K with BACE1 resulted in increased 38-kDa NCAM1-βCTF levels, concurrently with reduced 45-kDa NCAM1-βCTF levels, most likely by favoring the proteolysis of NCAM1 at Glu 671 by BACE1. In contrast, only double mutations at the P1-P1′ site of NCAM1 (YE670/671AH and YE670/671VK) abolished the generation of BACE1-specfic 38-kDa NCAM1-βCTF and 45-kDa NCAM1-βCTF, but generated 41-kDa NCAM1-CTF, which was reduced by BACE inhibition (C3). These data support that BACE1 cleaves NCAM1 between Y 670 and E 671 . F and G , n = 3. " width="100%" height="100%">
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: Identification and validation of the BACE1 cleavage site in NCAM2 and NCAM1. HEK cells were cotransfected with BACE1 and transmembrane NCAM2 (NCAM2-TM) or NCAM1-140. NCAM2-FL and NCAM2-βCTFs; or NCAM1-FL and NCAM1-βCTF were immunoprecipitated in cell lysates using anti-Myc (911B) antibody with agarose beads. After electrophoresis of immunoprecipitated samples, Coomassie stained bands of NCAM2-βCTF (∼32 kDa, in Fig. 2 C ) and NCAM1-βCTF (∼38 kDa, in Fig. 3 C ) were cut from the gel and sequenced by microcapillary LC/MS/MS to identify the BACE1 cleavage site. NCAM2-FL and NCAM1-FL were also sequenced as a control. A , comparison of BACE1 cleavage sites on mouse and human APP, NCAM2, and NCAM1. BACE1 cleaves APP at Asp1 within Aβ peptides ( yellow box ) and also cleaves APP at Glu11 (G 680 Y 681 ↓ E 682 V 683 ) within Aβ peptides, which are located 28 and 18 residues distant from the transmembrane region ( gray box ), respectively. APP numbering is based on the 770 amino acid isoform. NCAM2 and NCAM1 numbering is that of the mouse NCAM2-TM and mouse NCAM1-180 amino acid isoform, respectively. The ↓ symbol denotes the scissile (cleavage) bond. BACE1 cleavage sites of NCAM2 (G 661 Y 662 ↓ E 663 V 664 ) and NCAM1 (E 669 Y 670 ↓ E 671 V 672 ) in the second fibronectin type-III repeat domain ( green box , see FN2 in Fig. 1 C ) are located 35 and 41 residues distant from the transmembrane region ( gray box ), respectively. Notably, these cleavage sites are identical or similar to the secondary BACE1 cleavage site (β′) of human APP site (GY↓EV). The cleavage sites of NCAM2 at Asp 693 are indicated by the arrowhead. B and C , ion chromatogram of the peptide peak (EVQITAANR; m/z = 501.27–501.29) from the stained bands (32-kDa NCAM2-βCTF and NCAM2-FL as control). D and E , ion chromatogram of the peptide peak (EVQITAANR; m/z = 682.34–682.35) from the stained bands (38-kDa NCAM1-βCTF and NCAM1-FL as control). F and G , BACE1 cleavage site is denoted by a down arrow depicting the scissile bond (P1-P1′). All of the site-directed mutageneses in P1-P1′ amino acids are denoted. HEK cells were cotransfected with BACE1 and wild-type (WT) NCAM or mutant NCAM and then conditioned for 24 h with solvent only (DMSO) or BACE inhibitor (C3, 10 μM) dissolved in DMSO. Total DNA concentration was kept constant at 1.5 μg with empty vector. Cell lysates were analyzed by immunoblot to assess the BACE1 processing of mutant NCAM2 (E663H, YE662/663AH, E663K, YE662/663VK) ( F ) or NCAM1 (E671H, YE670/671AH, E671K, YE670/671VK) ( G ) compared with WT. When BACE1 is overexpressed, only NCAM2-YE662/663AH prohibits the generation of BACE1-specific 32-kDa NCAM2-βCTF ( arrowhead ). Instead, additional bands are produced, but these bands are abolished by BACE inhibition (C3). These data indicate that BACE1 cleaves NCAM2 between Y 662 and E 663 . NCAM1-E671H mutation did not prevent the generation of NCAM1-βCTFs at ∼38 kDa and ∼45 kDa ( arrowhead ). The ectopic expression of NCAM1-YE670/671AH, NCAM1-E671K, and NCAM1-YE670/671VK generates additional C-terminal fragments of NCAM1 at ∼41 kDa (41-kDa NCAM1-CTF) compared with NCAM1-WT even in the absence of BACE1 expression ( open arrowhead ). The coexpression of NCAM1-E671K with BACE1 resulted in increased 38-kDa NCAM1-βCTF levels, concurrently with reduced 45-kDa NCAM1-βCTF levels, most likely by favoring the proteolysis of NCAM1 at Glu 671 by BACE1. In contrast, only double mutations at the P1-P1′ site of NCAM1 (YE670/671AH and YE670/671VK) abolished the generation of BACE1-specfic 38-kDa NCAM1-βCTF and 45-kDa NCAM1-βCTF, but generated 41-kDa NCAM1-CTF, which was reduced by BACE inhibition (C3). These data support that BACE1 cleaves NCAM1 between Y 670 and E 671 . F and G , n = 3.
Article Snippet: After washing the beads with PBS, PSA-NCAM1 or non-PSA-NCAM1 proteins were sequentially incubated in a reaction buffer (100 mM sodium acetate, pH 4.5) including recombinant human BACE1 (931-AS-050; R&D systems) at 37 °C overnight.
Techniques: Immunoprecipitation, Electrophoresis, Staining, Liquid Chromatography with Mass Spectroscopy, Comparison, Mutagenesis, Solvent, Concentration Assay, Plasmid Preparation, Western Blot, Produced, Inhibition, Expressing, Generated
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Figure S1, A and C . " width="100%" height="100%">
