Journal: Journal of Biological Chemistry

Article Title: Physiological IgM Class Catalytic Antibodies Selective for Transthyretin Amyloid

doi: 10.1074/jbc.m114.557231

Figure Lengend Snippet: FIGURE 3. Characterization of polyclonal IgM hydrolytic activity. A, comparison of hydrolytic activity of individual pIgM preparations (90 g/ml) from non-aged (35 years, n 12) and aged humans (70 years, n 20) determined by electrophoresis. Shown are the hydrolysis levels estimated using the preaggregated 125I-TTR substrate (160 nM; misTTR content, 60%; 18-h incubation) and non-boiled reaction mixtures. B, rates of preaggregated 125I-TTR hydrolysis by MMP9 and pIgM (left axis). MMP9 and pIgM concentrations were 133 nM. Other conditions were as in A. T50 (right axis) is the time required to hydrolyze 50% of 1 nM misTTR at the MMP9 or pIgM concentrations in blood (3.2 nM and 2.2 M, respectively, computed from the equation, S S0(1 e[Eo]kt), where S0 is substrate concentration at time 0, E0 is the enzyme concentration at time 0, k is the second order rate constant derived from the equilibrium binding and catalytic constants, and t is reaction time. Inset, SDS gel showing reduced 1-mer misTTR band following MMP9 treatment (lane 2) compared with diluent treatment (lane 1). C, pIgM immunoadsorption. misTTR hydrolysis by unfractionated human serum and Ab-free serum was measured as in A. The protein contentofunfractionatedserum(diluted1:100)andtheantibody-freeserumwasadjustedtobeidentical(1.14mg/ml).IgMconcentrationsinhydrolysisassays using unfractionated serum and antibody-free serum were 20 and 0.01 g/ml, respectively. Data are expressed as percentage depletion of the 1-mer misTTR band compared with the control substrate reaction mixture treated with diluent (100% value 22.5 5.6 nM). Inset, electrophoresis showed identical product fragments (13, 10, and 7 kDa bands) generated by digesting preaggregated 125I-TTR with unfractionated serum (lane 3, 1:100 dilution) and purified IgM (lane 4, 120 g/ml). Lane 1, substrate treated with diluent. Lane 2, substrate treated with antibody-free serum. D, distribution of hydrolytic activity of unfractionated serum. Only the IgM-containing 900-kDa fraction (retention volume 8–11 ml) obtained by FPLC gel filtration of human serum (blue trace) displayed detectable misTTR hydrolytic activity assayed as in A (red bars). IgM concentration in the assay, 20 g/ml (equivalent to IgM concentration in 1% unfractionated serum). Elution of reference purified IgM (black trace) is coincident with elution of the serum hydrolytic activity. Non-IgM serum fractions were tested as two pools at a concentration corresponding to 1% unfractionated serum equivalents (retention volume 0–8.0 and 11.0–22.0 ml; concentrated using a 3-kDa ultrafilter). Inset, reducing SDS gels of 900-kDa serum IgM fraction showing anti- antibody-stained 70-kDa heavy chain band (lane 1) and anti-/ antibody-stained 25-kDa light chain band (lane 2). Lanes 3 and 4, respectively, are similarly stained heavy and light chain bands of the reference IgM, respectively. H, heavy chain; L, light chain. E, protease inhibitor effects. misTTR hydrolysis was measured using IgM pretreated (1 h) with diluent or the indicated inhibitors of metallopro- teases, cysteine proteases, acid proteases, and serine proteases. The structure of phosphonate 1 is shown. SAP is an amyloid-binding protein. Residual hydrolyticactivityofinhibitor-treatedIgMswascomputedasapercentageofIgMactivityaftertreatmentwithdiluent(100%value244nMTTR).Hydrolysis was measured using boiled reaction mixtures of 100 nM preaggregated 125I-TTR incubated with 60 g IgM/ml for 18 h. F, unimpeded hydrolysis of preaggre- gated 125I-TTRbypIgMinthepresenceofAb-freeserum.Plottedare1-mermisTTRhydrolyticactivitiesexpressedasapercentageoftheactivityofpurified(130 g/ml) alone (90 3 nM). Reaction conditions were as in A. Error bars, S.D.

Article Snippet: Aggregated TTR—Wild type, purified TTR from human plasma (Cell Sciences, Canton, MA) was labeled with 125I (125I-TTR) using 1,3,4,6-tetrachloro-3 ,6 -diphenylglycouril (Thermo Fisher Scientific), followed by removal of free 125I by gel filtration (BioSpin-6 column; Bio-Rad).

Techniques: Activity Assay, Comparison, Electrophoresis, Incubation, Concentration Assay, Derivative Assay, Binding Assay, SDS-Gel, Control, Generated, Purification, Filtration, Staining, Protease Inhibitor

Journal: Journal of Biological Chemistry

Article Title: Physiological IgM Class Catalytic Antibodies Selective for Transthyretin Amyloid

doi: 10.1074/jbc.m114.557231

Figure Lengend Snippet: FIGURE 6. Monoreactive and oligoreactive misTTR hydrolyzing monoclonal IgMs. A, no hydrolysis of non-amyloid and non-superantigen proteins. Shown are SDS electrophoresis gels containing reaction mixtures of the following biotinylated proteins treated for 22 h with pIgM (pooled from 12 humans) or mIgM 1802 (130 g/ml): extracellular domain of epidermal growth factor receptor (exEGFR), bovine serum albumin (BSA), ovalbumin (OVA), transferrin (Trans), and two S. aureus virulence factors, LukS and collagen adhesion protein CNA. mIgM-P1, control reaction mixture containing IgM 1802 inhibited by electrophilic phosphonate 1. B–D, no fibrillar A hydrolysis by mIgM 1802. FPLC gel filtration profiles of formic acid-solubilized reaction mixtures containing fibrillar 125I-A42 (30,000 cpm) treated for 16 h with diluent (B), monoclonal mIgM 1802 (120 g/ml) (C), or the A-hydrolyzing immunoglobulin V domain fragment (IgV 2E6, 10 g/ml) (D). No depletion of intact 125I-A42 (computed mass 4,630 Da) or product appearance was evident by mIgM treatment, whereas IgV 2E6 generated a radioactive fragment with a mass of 1654 Da corresponding to hydrolysis at the His14–Gln15 bond (29). E, discordant misTTR and A hydrolytic activities of mIgMs (n 9). A40 hydrolysis data are from Ref. 7. Connecting lines identify individual mIgMs. In parentheses are the number of mIgMs without detectable activity. misTTR-monoreactive mIgM 1814 and oligoreactive mIgM Yvo are identified. F, discordant misTTR and Protein A hydrolytic activities of mIgMs(n16).HydrolysisofbiotinylatedProteinA(72nM)followingIgMtreatment(45g/ml,72h)wasdeterminedelectrophoretically.Solidanddottedlines, least-square regression fit and 95% confidence bands, respectively (p 0.05, r2 0.001; two-tailed Pearson analysis). misTTR-monoreactive mIgMs 1802 and 1814 and oligoreactive mIgM Yvo are identified. Inset, SDS-gel electrophoresis lanes of Protein A treated with diluent (lane 1), non-hydrolytic mIgM 1801 (lane 2), and hydrolytic mIgM Yvo showing depletion of intact Protein A and the appearance of smaller mass products (lane 3). G, hydrolysis of biotinylated gp120 (100 nM) following IgM treatment (45 g/ml, 18 h) was determined electrophoretically. Solid and dotted lines are the least-square regression fit and 95% confidence bands, respectively (p 0.05, Pearson r2 0.007). misTTR-monoreactive mIgMs 1802 and 1814 and oligoreactive mIgM Yvo are identified. Inset, SDS-gel electrophoresis of gp120 treated with diluent (lane 1), non-hydrolytic mIgM 1801 (lane 2), and hydrolytic mIgM Yvo showing depletion of intact gp120 and appearance of smaller mass product bands (lane 3). H, inhibition of mIgM Yvo catalyzed misTTR hydrolysis by alternate substrates. Only A inhibited the misTTR hydrolytic activity. Like the irrelevant protein ovalbumin (OVA), the superantigens Protein A and gp120 were non-inhibitory. Concentration of alternate substrates was 1 M, and that of mIgM Yvo was 130 g/ml. Other reaction conditions were as in Fig. 4A. Error bars, S.D.

Article Snippet: Aggregated TTR—Wild type, purified TTR from human plasma (Cell Sciences, Canton, MA) was labeled with 125I (125I-TTR) using 1,3,4,6-tetrachloro-3 ,6 -diphenylglycouril (Thermo Fisher Scientific), followed by removal of free 125I by gel filtration (BioSpin-6 column; Bio-Rad).

Techniques: Electrophoresis, Control, Filtration, Generated, Activity Assay, Two Tailed Test, SDS-Gel, Inhibition, Concentration Assay

Journal:

Article Title: GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

doi:

Figure Lengend Snippet: Ypt1p-Q67L is defective in intrinsic and GAP-stimulated GTP hydrolysis. GTP hydrolysis was monitored by the charcoal binding assay. Wild-type (squares) and Ypt1p-Q67L (triangles) proteins were preloaded with [γ-32P]GTP for 15 min at 30°C. Unbound nucleotide was removed with two successive acrylamide spin columns. GTP hydrolysis assays were performed by incubating 2 nM preloaded Ypt1p with a P12 subcellular fraction (5 mg/ml) prepared from GPY60 cells (GAP-stimulated hydrolysis; open symbols) or without the P12 fraction (intrinsic hydrolysis; closed symbols) at 30°C. Aliquots were removed at the indicated time points and added to ice-cold activated charcoal to stop the reaction. The charcoal was pelleted, and an aliquot of the supernatant was removed and quantified by scintillation counting. The counts measured at time zero were subtracted as background. GTP binding was slightly less efficient for Ypt1p-Q67L, but hydrolysis rates were normalized for the amount of Ypt1p bound to GTP. Data shown are typical of three independent experiments.

Article Snippet: Preload reactions were diluted with 50 μl of reaction buffer (20 mM HEPES [pH 7.2], 5 mM magnesium acetate, 300 mM sorbitol, 1 mM DTT) plus 0.5 mg of BSA per ml, and unbound nucleotide was removed at 4°C with two successive acrylamide spin columns (BioSpin6; Bio-Rad Laboratories, Hercules, Calif.) equilibrated with reaction buffer plus BSA.

Techniques: Binding Assay

Journal:

Article Title: GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

doi:

Figure Lengend Snippet: Mutant Ypt1p-Q67L is partially defective in prenylation. (A) A smaller fraction of the mutant Ypt1p-Q67L than of wild-type Ypt1p is prenylated, as determined by Triton X-114 phase partitioning and urea-acrylamide gradient gel electrophoresis. Wild-type (NSY125) and ypt1-Q67L mutant (NSY406) total cell lysates were subjected to phase partitioning with 1% Triton X-114. Total (T), aqueous (A), and detergent (D) phases were electrophoresed on 4 to 8 M urea–10 to 15% acrylamide gels and processed for Western blot analysis with anti-Ypt1p antibodies. Note that the aqueous phase contains all of the unprenylated form and the detergent phase contains all of the prenylated Ypt1p-Q67L. (B) Unprenylated and some prenylated mutant Ypt1p-Q67L is mislocalized to the cytoplasm (S100 fraction). Wild-type and ypt1-Q67L mutant cells were lysed with glass beads and centrifuged at 100,000 × g to generate supernatant (S) and pellet (P) fractions (T, total cell lysate). Proteins were resolved by SDS-PAGE on 4 to 8 M urea–10 to 15% acrylamide gradient gels, transferred to nylon membranes, and processed for Western blot analysis with affinity-purified anti-Ypt1p antibodies. The upper form of Ypt1p is unprenylated; the lower form is prenylated. Quantification indicates that there is the same amount of prenylated Ypt1p in wild-type and mutant strains. Data are typical of three independent experiments.

Article Snippet: Preload reactions were diluted with 50 μl of reaction buffer (20 mM HEPES [pH 7.2], 5 mM magnesium acetate, 300 mM sorbitol, 1 mM DTT) plus 0.5 mg of BSA per ml, and unbound nucleotide was removed at 4°C with two successive acrylamide spin columns (BioSpin6; Bio-Rad Laboratories, Hercules, Calif.) equilibrated with reaction buffer plus BSA.

Techniques: Mutagenesis, Nucleic Acid Electrophoresis, Western Blot, SDS Page, Affinity Purification

Journal:

Article Title: GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

doi:

Figure Lengend Snippet: ypt1-Q67L is not dominant for growth or secretion when overexpressed. (A) Growth phenotypes. Wild-type YPT1 (WT; pNS326), ypt1-Q67L (pNS330), and YPT1-N121I (pNS327) were expressed from the galactose-inducible GAL10 promoter on a CEN URA-marked plasmid in the strain NSY125. Tenfold serial dilutions of cells were spotted onto SRaf-Ura or SRaf-Ura-plus-2% galactose plates and grown at 30°C. (B) CPY transport. The strains were grown overnight in SRaf-Ura minus methionine at 30°C to mid-logarithmic phase and then switched to inducing media (SRaf-Ura minus methionine plus 2% galactose) for 3 h at 30°C. Cells were harvested and pulse-labeled with Tran35S-label for 6 min at 30°C and chased for the indicated times (minutes). Cells were then lysed, and CPY was immunoprecipitated with anti-CPY antibodies and separated on SDS–8% polyacrylamide gels. p1, ER form; p2, Golgi form; m, mature vacuolar form. (C) Western blot analysis of Ypt1p expression. Cells used for the CPY assay were tested for Ypt1p expression. Ypt1p expression was induced with 2% galactose for 3 h at 30°C. Cells were lysed, and equivalent quantities of extract were run on 4 to 8 M urea–10 to 15% acrylamide SDS-containing gels. Proteins were transferred to nylon membranes and processed for Western blotting with anti-Ypt1p antibodies. The unprenylated and prenylated forms of Ypt1p are indicated.

Article Snippet: Preload reactions were diluted with 50 μl of reaction buffer (20 mM HEPES [pH 7.2], 5 mM magnesium acetate, 300 mM sorbitol, 1 mM DTT) plus 0.5 mg of BSA per ml, and unbound nucleotide was removed at 4°C with two successive acrylamide spin columns (BioSpin6; Bio-Rad Laboratories, Hercules, Calif.) equilibrated with reaction buffer plus BSA.

Techniques: Plasmid Preparation, Labeling, Immunoprecipitation, Western Blot, Expressing