Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Sez6, Sez6L, and Sez6L2 are expressed by principal (excitatory, pyramidal) neurons of the mouse hippocampus at much higher levels than other known complement regulators (namely Crry, C4BP, CFH, C1-INH, DAF, and MCP). Expression data was obtained from Hipposeq: a comprehensive RNA-Seq database of gene expression in hippocampal principal neurons ( http://hipposeq.janelia.org ; ). The RNA samples used in this database were isolated from mouse hippocampal principal neurons micro-dissected from the CA1, CA3, or Dentate Gyrus (DG) cell layers of the hippocampus at Postnatal Day 25-32. Differential gene expression is shown in the heatmap with the relative units of FPKM (Fragments Per Kilobase of Exon Per Million Reads Mapped.) B) Brain sections from adult WT mice or Sez6 triple knockout mice (TKO) were immuno-stained for Sez6L2 (green) and DAPI and imaged in the CA1 region of the hippocampus. Scale Bar= 27μm. High density Sez6L2 staining occurs around cell bodies in the pyramidal layer, but significant Sez6L2 is also found in the stratum radiatum and stratum oriens. C) Higher magnification images of sections immuno-stained for Sez6L2 (green) and the postsynaptic protein, Homer1 (red), shows Sez6L2 is found near or co-localized with synapses in the stratum radiatum. Scale bar = 1.8μm.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Expressing, RNA Sequencing Assay, Isolation, Triple Knockout, Staining

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Purified Sez6L2-MH is shown by a coomassie stained gel and by western blot with anti-Sez6L2 and anti-Myc antibodies. Lanes with the coomassie stain are from the same gel. B) Schematic of Sez6L2 and Sez6L2-MH domain structures. CCP=Domain abundant in c omplement c ontrol p roteins. CCP domains are also known as SUSHI repeats or short complement-like repeat (SCR) CUB= Domains named after complement C 1r/C1s, u EGF, and B MP1 TM=Transmembrane region. Sez6L2-MH was made by replacing the transmembrane and cytoplasmic tail domains with a tandem Myc, 6xHis tag. C) and D) Classical pathway hemolysis assay. Antibody-coated sheep erythrocytes were exposed to human serum pre-incubated with purified Sez6L2-MH, C1-INH, FH, C4BP, H-DAF, or BSA. After 30 mins, the percent of cell lysis was measured by spectrophotometry (A415). C1-INH, FH, C4BP, and H-DAF are known complement inhibitors and were used as positive controls. BSA was used as a negative control for comparison. H-DAF is His-tagged DAF. 1-way ANOVA (P <0.0001; F(6,14)=314.4). N=3 (1 experiment with 3 replicates; Representative of 4 independent experiments with Sez6L2 and buffer and 2-3 experiments with each control protein). D) Supernatants from classical pathway hemolysis assays were further analyzed for C3 cleavage products by western blot. Supernatants in D are from same experiment shown in C. Sez6L2-MH increases the amount of the C3dg cleavage product similar to other complement regulators that work at the level of the C3 convertase. Sez6L2-MH runs just above the C3α band of C3. When large amounts of Sez6L2 are present it can cause C3α to run lower on the gel and sometimes mildly interferes with antibody binding to C3α. The membrane blotted for C3 was also stained with ponceau S to reveal total protein and shows the presence of the purified proteins in each sample. E) Schematic of C3 cleavage products. The C3d region recognized by our C3 antibody is highlighted in dark grey. F) Alternative pathway hemolysis assay. Rabbit erythrocytes were exposed to human serum pre-incubated with Sez6L2-MH, complement regulators, or BSA in presence of 12.5mM MgEGTA to block the classical pathway. Then the percent of cell lysis was measured by spectrophotometry (A415). 1-way ANOVA (P =0.0122; F(4,7)=7.298). Results are the mean of three independent experiments which tested 4 independent Sez6L2-MH preps multiple times. N=3 for buffer and Sez6L2-MH; N=2 for FH, H-DAF, and BSA. For all graphs * = p <0.05; ** = p<0.01

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Purification, Staining, Western Blot, Hemolysis Assay, Incubation, Lysis, Spectrophotometry, Negative Control, Binding Assay, Blocking Assay

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Schematic of Factor I and cofactor cleavage of C3b and iC3b. B) Factor I cleavage assay of C3b. C3b and Factor I (FI) were incubated alone, with Factor H (FH), or C4BP, or with concentrations of Sez6L2-MH ranging from 1 to 10 μg/mL for 2 hours at 37°C. Then samples were analyzed by western blot using antibodies that recognize C3d, a region within the C3α chain (and highlighted by the black rectangle in the schematics in (A)). Coomassie stained gels are also shown. FH and C4BP are known co-factors of FI towards C3b and served as positive controls. Incubation of C3b and FI with Sez6L2-MH also generated the C3 cleavage products α’1 and α’2 showing Sez6L2-MH is a cofactor for Factor I cleavage of C3b. C) Schematic of Factor I + cofactor cleavage of C4b. D) C4b and Factor I (FI) were incubated alone, with FH, C4BP, or with concentrations of Sez6L2-MH ranging from 1 to 10 μg/mL for 2 hours at 37°C. Then samples were analyzed by western blot using a C4 polyclonal antibody or coomassie stained gels. C4 components recognized by the C4 antibody are colored black in the schematic in C. C4BP is a known cofactor of FI for C4b cleavage and served as a positive control. Incubation of C4b and FI with Sez6L2-MH did not result in the appearance of C4b cleavage products.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Cleavage Assay, Incubation, Western Blot, Staining, Generated, Positive Control

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: Factor I cleavage assay of C3b (A) or C4b (B). C3b or C4b and Factor I (FI) were incubated alone, with Factor H (FH, 1ug), C4BP (1ug), or Sez6L2-MH (1ug or 5ug) for 4 or 8 hours at 37°C. Then samples were analyzed by coomassie stained gels. Sez6L2-MH aids significant factor I cleavage of C3b but not C4b at 4 and 8 hours resulting in appearance of the C3b cleavage products, C3α’1 and C3α’2, but not the C4b cleavage products, C4d and C4α3.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Cleavage Assay, Incubation, Staining

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A and B) Alternative C3 convertase assay: A 96 well plate coated with C3b was incubated with Factor B and Factor D to form the C3 convertase C3bBb, then incubated with Sez6L2-MH or FH at concentrations ranging from 0 to 500 μg/mL to assess their decay accelerating activity. Factor B remaining bound to the plate (as C3bBb) was detected using an anti-Factor B antibody ELISA in (A) and Bb released into the supernatant is shown via western blot (B). C) Classical C3 convertase assay: A plate coated with C4b was incubated with C2 and C1s-enzyme to form the classical/lectin pathway C3 convertase C4b2a, then incubated with Sez6L2-MH or H-DAF at concentrations ranging from 0 to 500 μg/mL to assess their decay accelerating activity. C2 remaining bound to the plate (presumably as C4b2a) was detected using an anti-C2 antibody ELISA. For A and C ELISAs: N=3 (1 experiment with 3 replicates; representative of 2-3 independent experiments). Statistics: 1-way ANOVAS with Holms-Sidak multiple comparison’s tests were performed for each complement inhibitor with comparisons to 0 ug/mL controls. * p<0.05.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Convertase Assay, Incubation, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A-B) Sez6L2 inhibits C3 deposition at a range of serum concentrations. CHO cells were transfected with plasmids for GFP alone or with Myc-tagged Sez6L2 (M-Sez6L2) or His-tagged DAF (H-DAF). CHO cells were coated with antibodies and exposed to 0-20% C5 depleted human serum for 1 hour and then labeled with anti-C3b/C3c antibodies and analyzed by flow cytometry. One experiment is shown that is representative of two independent experiments. B) C3 deposition on GFP transfected cells with or without M-Sez6L2 or H-DAF at 15% serum. ANOVA (P=0.0016; F(2,6)=22.51). N=3; 1 experiment with 3 replicates (representative of 3+ independent experiments). C) Schematic of Sez6L2, Sez6, and Sez6L protein domain structures. D-I) CHO cells were transfected with the indicated Myc-tagged cDNAs and processed as outlined in A with 15% C5 depleted serum, except that an anti-Myc antibody was used in place of GFP to identify transfected and expressing CHO cells. D) 5% Contour plots of C3 versus Myc fluorescence (top layer) and C3 fluorescence histograms (bottom layer) of the same samples normalized to mode and compared to baseline cells not exposed to serum. For Contour plots, boxed regions highlight cells designated as myc positive (top box) and myc negative (lower box) populations. For C3 histograms, dark grey, solid line population = Myc positive cells; Light grey, dotted line population= Myc negative cells; White, dashed grey line population = baseline. Representative of 4+ independent experiments. E) Quantification of the average median C3 fluorescence intensity from myc positive and myc negative cells within each sample. Statistics = t-tests. N=3 (1 experiment with 3 replicates; Representative of 4+ independent experiments). F) Average median C3 fluorescence intensities after normalization to the myc negative cells from each experimental group. ANOVA between Myc+ cell populations (p<0.001; F(4, 15)=64.53). Sez6L2 inhibits C3 deposition at a level comparable to positive control MCP. Sez6 is a stronger complement inhibitor than Sez6L2 and Sez6L is a weaker inhibitor. F) Average median Myc fluorescence intensity from myc positive cells ANOVA (p<0.001; F(4, 15)=36.79). G) Average % of Myc positive cells in each experimental group (ANOVA, p=0.115; F(4, 15)=2.224). For sections F-H, N=4 (4 independent experiments). I) Sez6 blocks complement deposition more efficiently than Sez6L2 and Sez6L even when comparing similar levels of myc surface expression. Average C3 median fluorescence intensity normalized to internal myc negative populations for M-Sez6, M-Sez6L2, and M-Sez6L samples shown relative to the myc median fluorescence intensity. N=3 (1 experiment with 3 replicates, Representative of 3 independent experiments). For all graphs * = p <0.05; ** = p<0.01; # = p<0.001 for all Myc+ groups compared to M-CR2.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Transfection, Labeling, Flow Cytometry, Expressing, Fluorescence, Positive Control

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: Sez6 family FASTAs sequences were uploaded into the web-based program provided by Ojha et al ( http://coredo.nccs.res.in/meme-5.0.3/CoReDo/home.html ). Classical model complement regulatory proteins (CRPs) have motifs in the order of M5, M3, M1, M2, and either M4 or M1 spaced across 3 consecutive CCP domains. Sez6L2 has this same pattern, but Sez6 and Sez6L do not.

Article Snippet: CHO (Freestyle) cells were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc tags obtained from Sinobiological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567; similar to NM 001114100.2; Uniprot ACC: Q6UXD5-6;), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM 178860.4; Uniprot ACC: Q53EL9-1;), M-SEZ6L (HG20982-NM; modified from NM 001184773.1 and contains the point mutation 1428A/C that does not alter the amino acid sequence; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594 ), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM 001877.3 ) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques:

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Sez6 family expression in the hippocampus. (A) Sez6, Sez6L, and Sez6L2 are expressed by principal (excitatory, pyramidal) neurons of the mouse hippocampus at much higher levels than other known complement regulators (namely Crry, C4BP, CFH, C1-INH, DAF, and MCP). Expression data was obtained from Hipposeq: a comprehensive RNA-Seq database of gene expression in hippocampal principal neurons [ http://hipposeq.janelia.org ]. The RNA samples used in this database were isolated from mouse hippocampal principal neurons micro-dissected from the CA1, CA3, or Dentate Gyrus (DG) cell layers of the hippocampus at Postnatal Day 25-32. Differential gene expression is shown in the heatmap with the relative units of FPKM (Fragments per Kilobase of Exon per Million Reads Mapped.) (B) Brain sections from adult WT mice or Sez6 triple knockout mice (TKO) were immuno-stained for Sez6L2 (green) and DAPI and imaged in the CA1 region of the hippocampus. Scale Bar= 27 µm. High density Sez6L2 staining occurs around cell bodies in the pyramidal layer, but significant Sez6L2 is also found in the stratum radiatum and stratum oriens. (C) Higher magnification images of sections immuno-stained for Sez6L2 (green) and the postsynaptic protein, Homer1 (red), shows a subset of Sez6L2 is found near or co-localized with synapses in the stratum radiatum. Scale bar = 1.8 µm.

Article Snippet: CHO cells (a Chinese hamster ovary cell line) were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc-tags obtained from Sino Biological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567, similar to NM_001114100.2; Uniprot ACC: Q6UXD5-6), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM_178860.4; Uniprot ACC: Q53EL9-1), M-SEZ6L (HG20982-NM; modified from NM_001184773.1; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM_001877.3) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Expressing, RNA Sequencing Assay, Isolation, Triple Knockout, Staining

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Full Length Sez6L2, Sez6, and Sez6L inhibit C3b/iC3b opsonization of CHO cells by the classical pathway. (A, B) Sez6L2 inhibits C3b/iC3b opsonization at a range of serum concentrations. CHO cells were transfected with plasmids for GFP alone or with Myc-tagged Sez6L2 (M-Sez6L2) or His-tagged DAF (H-DAF). CHO cells were coated with antibodies and exposed to 0-20% C5-depleted human serum for one hour and then immuno-stained with anti-C3b/iC3b antibodies and analyzed by flow cytometry. One experiment is shown that is representative of two independent experiments. (B) C3b/iC3b on GFP transfected cells with or without M-Sez6L2 or H-DAF at 15% serum. ANOVA (P=0.0016; F(2,6)=22.51). N=3; one experiment with three replicates (representative of 3+ independent experiments). (C) Schematic of Sez6L2, Sez6, and Sez6L protein domain structures. (D–I) CHO cells were transfected with the indicated Myc-tagged cDNAs and processed as outlined in A with 15% C5 depleted serum, except that an anti-Myc antibody was used in place of GFP to identify transfected and expressing CHO cells. (D) 5% Contour plots of C3b/iC3b versus Myc fluorescence (top layer) and C3b/iC3b fluorescence histograms (bottom layer) of the same samples normalized to mode and compared to baseline cells not exposed to serum. For Contour plots, boxed regions highlight cells designated as Myc-positive (top box) and Myc-negative (lower box) populations. For C3b/iC3b histograms, dark grey, solid line population = Myc-positive cells; Light grey, dotted line population= Myc-negative cells; White, dashed grey line population = baseline. Representative of 4+ independent experiments. (E) Quantification of the average median C3b/iC3b fluorescence intensity from Myc-positive and Myc-negative cells within each sample. Statistics = t-tests. N=3 (one experiment with three replicates; Representative of 4+ independent experiments). (F) Average median C3b/iC3b fluorescence intensities after normalization to the Myc-negative cells from each experimental group. ANOVA between Myc-positive cell populations (p<0.001; F(4, 15)=64.53). Sez6L2 inhibits C3b/iC3b opsonization at a level comparable to positive control MCP. Sez6 is a stronger complement inhibitor than Sez6L2 and Sez6L is a weaker inhibitor. (F) Average median Myc fluorescence intensity from Myc-positive cells. ANOVA (p<0.001; F(4, 15)=36.79). (G) Average % of Myc-positive cells in each experimental group (ANOVA, p=0.115; F(4, 15)=2.224). For sections (F–H) , N=4 (four independent experiments). (I) Sez6 blocks complement opsonization more efficiently than Sez6L2 and Sez6L even when comparing similar levels of Myc surface expression. Average C3b/iC3b median fluorescence intensity normalized to internal Myc-negative populations for M-Sez6, M-Sez6L2, and M-Sez6L samples shown relative to the Myc median fluorescence intensity. N=3 (one experiment with three replicates, Representative of three independent experiments). For all graphs *p < 0.05; **p < 0.01; # p < 0.001 for all Myc-positive groups compared to M-CR2.

Article Snippet: CHO cells (a Chinese hamster ovary cell line) were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc-tags obtained from Sino Biological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567, similar to NM_001114100.2; Uniprot ACC: Q6UXD5-6), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM_178860.4; Uniprot ACC: Q53EL9-1), M-SEZ6L (HG20982-NM; modified from NM_001184773.1; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM_001877.3) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Transfection, Staining, Flow Cytometry, Expressing, Fluorescence, Positive Control

Journal: Frontiers in Immunology

Article Title: The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

doi: 10.3389/fimmu.2021.607641

Figure Lengend Snippet: Full Length Sez6L2, Sez6, and Sez6L inhibit C3b/iC3b opsonization of CHO cells by the alternative pathway. CHO cells were transfected with the indicated Myc-tagged cDNAs and then coated with a low level of antibodies and exposed to 20% C5-depleted human serum for one hour in the presence of 10 mM EGTA and 10 mM MgCl 2 to block the classical pathway. Cells were then labeled with anti-C3b/iC3b and anti-Myc antibodies and analyzed by flow cytometry. (A) 5% Contour plots of C3b/iC3b versus Myc fluorescence (top layer) and C3b/iC3b fluorescence histograms (bottom layer) of the same samples normalized to mode and compared to baseline cells not exposed to serum. For Contour plots, boxed regions highlight cells designated as Myc-positive (top box) and Myc-negative (lower box) populations. For C3b/iC3b histograms, dark grey, solid line population = Myc-positive cells; Light grey, dotted line population= Myc-negative cells; White, dashed grey line population = baseline. Representative of 3+ independent experiments with technical replicates. (B) Quantification of the average median C3b/iC3b fluorescence intensity from Myc-positive and Myc-negative cells within each sample. N=3 (one experiment with three replicates; Representative of 3+ independent experiments) Statistics = t-tests. E) Average median C3b/iC3b fluorescence intensities after normalization to the Myc-negative cells from each experimental group. ANOVA (p<0.001; F(4, 10)=74.47. N=3 (3 independent experiments). For all graphs *p < 0.05; **p < 0.01 # p < 0.001 for all Myc-positive groups compared to M-CR2.

Article Snippet: CHO cells (a Chinese hamster ovary cell line) were grown in serum-free Freestyle media (Gibco, 12651-014) and transfected with human cDNA expression plasmids with N-terminal Myc-tags obtained from Sino Biological: M-SEZ6L2 (HG13969-NM; modified from NCBI RefSeq BC000567, similar to NM_001114100.2; Uniprot ACC: Q6UXD5-6), M-SEZ6 (HG13436-NM; modified from NCBI RefSeq NM_178860.4; Uniprot ACC: Q53EL9-1), M-SEZ6L (HG20982-NM; modified from NM_001184773.1; Uniprot ACC: Q9BYH1-6), M-MCP (HG12239-NM; modified from NCBI RefSeq BC030594), or M-CR2 (HG10811-NM; modified from NCBI RefSeq NM_001877.3) using FectoPro transfection reagent (Polyplus, 116-001).

Techniques: Transfection, Blocking Assay, Labeling, Flow Cytometry, Fluorescence

Journal: Nature Communications

Article Title: Structural basis for xenobiotic extrusion by eukaryotic MATE transporter

doi: 10.1038/s41467-017-01541-0

Figure Lengend Snippet: Overall structure of AtDTX14. a Cartoon representations of the crystal structure of AtDTX14, viewed parallel to the membrane. The structures are colored from blue to red from the N terminus to the C terminus. b Surface representations and cross-sections of AtDTX14 and NorM-VC (PDB ID: 3MKT). The cross-sections are viewed perpendicular to the membrane, for comparison of their cavities. c Comparison of the helix axis angles of TM7 between AtDTX14 (blue) and NorM-VC (orange). All molecular graphics were created with the program CueMol ( http://www.cuemol.org/ )

Article Snippet: The AtDTX14 gene (UniProt ID: Q9C994, NCBI Reference Sequence: NP_177270.1, Supplementary Table ) was artificially synthesized (GenScript).

Techniques: Membrane, Comparison

Journal: Nature Communications

Article Title: Structural basis for xenobiotic extrusion by eukaryotic MATE transporter

doi: 10.1038/s41467-017-01541-0

Figure Lengend Snippet: Internal cavity, intracellular gate, and predicted extracellular gate. Crystal structure of AtDTX14 ( a – c ) and the modeled structure of the inward-open state ( d – f ), viewed parallel to the membrane. The model structure was created by, respectively, superimposing the N-lobe and C-lobe of AtDTX14 onto the N-lobe and C-lobe of the crystal structure of MurJ (PDB ID: 5T77), using the SSM superpose algorithm . The resulting RMSD values are 2.90 Å and 2.06 Å for the N-lobe and C-lobe, respectively, indicating their structural similarities despite the low-sequence identity (~4%). b Close-up view of the bottom of the internal cavity. The bottom is closed by the salt-bridge network, which is represented by dashed lines. c Close-up view of the intracellular gate. The intracellular Gly pair (G97 and G319) enables the side-by-side interaction between TM2 and TM8. e , f Close-up views of the putative extracellular gate, viewed parallel to the membrane ( e ) and perpendicular to the membrane ( f ). The blue spheres and lines indicate the residue pairs with high EC scores, calculated by the program EVfold_membrane ,

Article Snippet: The AtDTX14 gene (UniProt ID: Q9C994, NCBI Reference Sequence: NP_177270.1, Supplementary Table ) was artificially synthesized (GenScript).

Techniques: Membrane, Sequencing, Residue

Journal: Nature Communications

Article Title: Structural basis for xenobiotic extrusion by eukaryotic MATE transporter

doi: 10.1038/s41467-017-01541-0

Figure Lengend Snippet: In vivo complementation analysis of AtDTX14. Norfloxacin-resistance growth assay of AtDTX14 and its mutants was performed, using the 6-knock-out E. coli strain. AtDTX14 expression was induced by 0 mM (top panel, non induction control, 12 h) and 0.25 mM IPTG, with 0 μg ml −1 (middle panel, 18 h) or 0.02 μg ml −1 (bottom panel, 12 h) of norfloxacin. The mutants that decreased the complementary activity are labeled with (+) or (++), and those that showed the wild-type AtDTX14 level of complementary activity are labeled with (+++). The transformant of the single knock-out strain ( BW25113 acrAB::Δ macAB::Δ yojHI::Δ ) harboring the isogenic wild-type acrB inducible expression vector was included as an induction control (CTRL)

Article Snippet: The AtDTX14 gene (UniProt ID: Q9C994, NCBI Reference Sequence: NP_177270.1, Supplementary Table ) was artificially synthesized (GenScript).

Techniques: In Vivo, Growth Assay, Knock-Out, Expressing, Control, Activity Assay, Labeling, Plasmid Preparation

Journal: Nature Communications

Article Title: Structural basis for xenobiotic extrusion by eukaryotic MATE transporter

doi: 10.1038/s41467-017-01541-0

Figure Lengend Snippet: Homology model of hMATE1 and transport assay. a Homology model of hMATE1 and close-up view of the hydrogen-bonding network in the C-lobe. The model structure was generated by the program MODELLER , using the AtDTX14 structure as the template. The residues involved in the hydrogen-bonding network were manually adjusted, using the program COOT . In the close-up view, Trp274 is represented by a CPK model (gray). b , c Uptake of [1- 14 C]-labeled TEA ( b ), and [ N -methyl- 3 H]-labeled cimetidine ( c ) into HEK-293 cells expressing hMATE1 mutants. Activities relative to that of the wild type (WT) are shown. The control activity (100%) of TEA and cimetidine correspond to 6 ± 0.17 nmol mg −1 protein and 47 ± 2.7 pmol mg −1 protein, respectively. The labels on the horizontal axis indicate the mutated amino-acid residues of each hMATE1-mutant (AtDTX14 numbering is indicated in parentheses), n = 8–33. Data are means ± SEM, ** P < 0.01 (two-tailed paired Student’s t test)

Article Snippet: The AtDTX14 gene (UniProt ID: Q9C994, NCBI Reference Sequence: NP_177270.1, Supplementary Table ) was artificially synthesized (GenScript).

Techniques: Transport Assay, Generated, Labeling, Expressing, Control, Activity Assay, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Structural basis for xenobiotic extrusion by eukaryotic MATE transporter

doi: 10.1038/s41467-017-01541-0

Figure Lengend Snippet: Structural comparison between the N-lobes of AtDTX14 and PfMATE. a , b Close-up views of the N-lobe of AtDTX14 ( a ) and that of PfMATE ( b )

Article Snippet: The AtDTX14 gene (UniProt ID: Q9C994, NCBI Reference Sequence: NP_177270.1, Supplementary Table ) was artificially synthesized (GenScript).

Techniques: Comparison

Journal: Nature Communications

Article Title: Structural basis for xenobiotic extrusion by eukaryotic MATE transporter

doi: 10.1038/s41467-017-01541-0

Figure Lengend Snippet: Structural comparison of the C-lobes of AtDTX14 and NorM-VC. a , b Close-up views of the putative substrate pocket of AtDTX14 ( a ) and that of NorM-VC ( b ) located at the C-lobe. The gray sphere represents a rubidium ion, and the dashed circle indicates the putative substrate pocket of NorM-VC. The double-headed arrows indicate the distances between the glutamate and aspartate residues, which are essential for the transport activity and the substrate recognition

Article Snippet: The AtDTX14 gene (UniProt ID: Q9C994, NCBI Reference Sequence: NP_177270.1, Supplementary Table ) was artificially synthesized (GenScript).

Techniques: Comparison, Activity Assay