Cell lines and cell culture
Human embryonic kidney (HEK293) cells (ATCC, Manassas, VA) and LoVo cells, which have a point mutation in the endopeptidase furin (a gift from Dr. Wilson, CBER, FDA), were used in all transfection experiments. A panel of liver cells was tested for the localization and expression of ADAMTS13: Hep3B (ATCC), Huh7, Alexander (a gift from Sara Ladu, National Cancer Institute (NCI), NIH), 7404 cells (a gift from Michael M. Gottesman, NCI, NIH), and hepatic stellate cells (LX2)29
. All cells were grown in Dulbecco’s Modified Eagle Medium with 1% glutamine, 1% penicillin- streptomycin and 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37 °C under humid conditions in 5% CO2
pcDNA4-ADAMTS13 (a gift from Evan Sadler, St. Louis, MO), which carries the full ADAMTS13 cDNA (including the prodomain) conjugated to V5 and poly (His) tags was used for transfection as the WT construct. A plasmid expressing enhanced green fluorescence protein (Clonetech peGFP-C1, referred here as eGFP) was used to create a C-terminal eGFP fusion construct that contains 70 amino acids (Pro30-Asp99) that follow the secretory signal peptide in ADAMTS13, thus including the putative NLS in ADAMTS13 and its flanking residues. The region of ADAMTS to be fused to eGFP was amplified with the following primers (5’ EcoR1 and BamH1 sites underlined): F- 5’ CCGGAATTCGCCCTCCCATTTCCAGCAGA R- 5’ CGCGGATCCTGTGTCCTCCTGGTGAGCCT. Following amplification, products and plasmid were digested with EcoR1 and BamH1 and ligated to create a C-terminal fusion construct. We also used an rADAMTS13 construct that lacks its prodomain (a gift from Elaine M. Majerus) alongside pcDNA4-ADAMTS13 to assess changes in cellular localization with and without the influence of the putative NLS found within the prodomain.
In preparation for confocal microscopy or for immunoblot, cells were plated in MatTeK dishes (MatTeK, Ashland, MA), or 6-well plates or T-75 flasks 24 hours before transfection at a confluency of 80–90%. Cells were transfected with 2–20 µg plasmid DNA (respective to the container size) using Lipofectamine Plus or Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After transfection, the medium was replaced with OPTI-MEM medium (Invitrogen).
Confocal microscopy and immunostaining
Transfected cells were washed twice with phosphate-buffered saline (PBS) with 0.1% bovine serum albumin (BSA), and then permeabilized and fixed with 4% paraformaldehyde for 30 minutes and an optional step of 70% ethanol for 15 minutes. Antibody labeling was performed for one hour at room temperature (). Immediately before imaging the dish, 5 µl of DAPI (4',6-diamidino-2-phenylindole) (Invitrogen, Molecular Probes) were added to stain the nucleus from a stock concentration of 1 µg/ml, which was dissolved in water.
Confocal images were sequentially acquired with Zeiss AIM software on a Zeiss LSM 510 Confocal system (Carl Zeiss Inc, Thornwood, NY) with a Zeiss Axiovert 100M inverted microscope and 50 mW argon UV laser tuned to 364 nm, a 25 mW Argon visible laser tuned to 488 nm and a 1 mW HeNe laser tuned to 543 nm. A 63× Plan-Neofluar 1.4 NA oil immersion objective was used at various digital zoom settings. Emission signals after sequential excitation of DAPI, Alexa Fluor 488 and Texas Red by the 364 nm, 488 nm or 543 nm laser lines were collected with a BP 435–485, BP 505–550 or LP 560 filter respectively, using individual photomultipliers. Fluorescence intensities were measured using regions of interest (ROI) for the entire cell and nucleus on 8-bit images. The intensity value for cytoplasm was calculated for each cell by subtracting the ROI value for the nucleus from the entire cell ROI value. Imaging transfected cells with secondary antibody only set the background conditions for time-course experiments.
Preparation of nuclear and cytoplasmic lysates
LoVo cells were scraped and washed twice with PBS 24 hours post-transfection. The cells were trypsinized, pelleted then resuspended in 200 µL of buffer 1 (25 mM HEPES (pH 7.9), 5 mM KCl, 0.5 mM MgCl2, 1 mM DTT, 1 mM PMSF) and 200 µL of buffer 2 (1% IGEPAL) was added. The mixture was mixed gently at 4°C for 15 minutes and centrifuged at 2000 × g for one minute. The resulting supernatant contained the cytoplasmic fraction. The pellet was then washed with buffer 3 (1:1 mixture of buffer 1 and buffer 2) and pelleted as before with the supernatant being discarded afterwards. Finally, the pellet was mixed gently at 4°C for 1 hour in 500 µL of buffer 4 (25 mM HEPES, 350 mM NaCl, 10% Sucrose, 0.05% NP-40, 1 mM DTT, 1 mM PMSF). The contents were centrifuged at 13,000 × g for 10 minutes. The supernatant contained the nuclear fraction. The cytoplasmic and nuclear fractions were stored at −80 °C.
Protein concentrations for the nuclear and whole cell lysates were determined using a BCA Protein Assay Reagent kit (Pierce, Rockford, IL) as per the manufacture’s protocol. For electrophoresis, subcellular fractions were loaded at 20 µg of total protein. The samples were mixed with SDS Sample Buffer (Invitrogen) and boiled for 5 minutes at 95 °C. The samples were electrophoresed on 7% Tris-Acetate SDS-PAGE gel, 12% Bis-Tris or 10% Tris-Glycine with corresponding running buffer (Invitrogen). Immunoblotting was performed using antibodies as detailed in and secondary anti-mouse HRP antibody (Invitrogen).
Structural and sequence analysis of the CUB domain
Structure similarity searches were conducted using the standalone version of the DALI program called DaliLite with the query structures scanned against a local current version of Protein Data Bank (PDB) which has all chains as separate entries (construction detailed below). These hits were also confirmed by querying the DALI database. The structural hits for each query were collected and parsed for congruence of strand orientation with nucleoplasmin structures (e.g. 1×e0). This was further confirmed by visual examination of each structure. Protein structures were visualized using the Swiss-PDB viewer30
and cartoon representations (figures) were constructed with the PyMOL program (http://www.pymol.org
). Protein secondary structure predictions were made with the JPRED program (http://www.compbio.dundee.ac.uk/~www-jpred
, using multiple alignments as queries. The residues of the CUB-2 domain that potentially interact with other proteins were deduced using alignment, which was generated by PCMA (Profile Consistency Multiple sequence Alignment)32
for the structural hits with the ADAMTS13 CUB-2 domain. Further alignment was adjusted manually based on secondary structure prediction by the JPRED program.
Construction of the DaliLite searchable local database
Over 30,000 atomic structures were downloaded from the PDB database, split into over 50,000 PDB chains and the corresponding amino acid sequences for each PDB chain were generated. The FASTA-formatted primary sequences were collated in into a single file, and they were then clustered into unique, non-overlapping groups using BLASTCLUST with a sequence identity cut-off of 40% and a length threshold of 70%. A representative PDB chain from each group was identified, considering the following in order of preference: (1) resolution (2) R-factor (3) X-Ray > NMR (4) largest sequence length. The identified representative PDB chain from each group was used to build the local PDB database. Over 8,000 PDB chains were ultimately used to construct the local DaliLite searchable database.
The PSORT/PSORT II suite of programs was used to automatically search for an NLS via its web portal (http://psort.nibb.ac.jp/form2.html
). The non-redundant (NR) database of protein sequences (National Center for Biotechnology Information, NIH, Bethesda, MD) was searched for sequence homologs of human ADAMTS13 protein with the BLASTP program33
. Profile searches were conducted using the PSI-BLAST program34
with a profile inclusion expectation (e) value threshold of 0.01. Searches were iterated until convergence. Multiple alignments were constructed using the PCMA program32