Transcript peptide assay

Anti-OPN Monoclonal Antibodies as Probes of OPN Structure and Function
Christian C. Kazanecki, Josephine Cassella, Yao Li, Cassandra Louis, Tanya Gordonov, Esben S. Sørensen and David T. Denhardt
ABSTRACT: OPN, a primarily secreted protein found in
all body fluids, is post-translationally modified; there is
also an intracellular form. It is functionally important in
bone remodeling, the progression of autoimmune disease,
cancer metastasis, inflammatory processes and stress
responses. Our research has focused on how OPN interacts
with cells and the consequences of these interactions. We
have used MAbs to known epitopes in the protein and
peptides representative of functional sequences in OPN to
shed light on how OPN signals cells and the consequences
of those signaling events. In this work we present the
properties of some novel anti-OPN MAbs and we describe
studies that suggest a possible interaction of the Cterminus of the protein with the central RGDSVVYGL
sequence important in signal transduction pathways.
OVERVIEW: Our characterization of the newer MAbs has
used uncloned hybridomas because of our desire to identify
MAbs that might not be effectively detected by Western
Blotting or ELISAs (because the relevant epitope was bound to
the plastic or PVDF membrane). Thus we use two additional
screening methodologies to detect antigen-antibody
associations free in solution (a peptide assay and a magnetic
bead assay). We use both native (modified) and recombinant
(unmodified) OPN to identify MAbs that specifically recognize
either the native or the recombinant form. Antibodies specific
only for the native form would be candidates for MAbs
recognizing an epitope that is post-translationally modified;
antibodies specific only for the recombinant form would
presumably recognize peptide sequences that are posttranslationally modified.
The peptide assay makes use of peptides that collectively
cover the entire OPN molecule with 2-4 amino acid overlaps;
in some cases both a phosphorylated peptide and its nonphosphorylated counterpart were used. These peptides possess
a biotin at the C terminus coupled to the peptide by two
glycine residues; via the biotin, the peptides bind to the surface
of neutravidin-coated wells in a 96-well format. The magnetic
bead assay employs two different forms of OPN, the highly
post-translationally modified native OPN purified from human
milk and the unmodified form synthesized in E. coli. In this
novel assay the two forms (differentially labeled) are mixed
and allowed to react in solution with the a specific MAb. The
antigen-antibody complexes are then captured on magnetic
beads coupled to protein G. After washing the beads, the
adsorbed IgG is eluted at low pH, and the fluorescence at the
two wavelengths determined. MAbs that selectively bind the
native protein are presumed to be antibodies that recognize
post-translational modifications. We believe this novel method
is important in that it detects the antibody-antigen reaction in
solution. This is in contrast to the methods currently in use
which entail the immobilization of the antigen on a plastic
surface (ELISA) or a membrane (Western blotting).
Methods to characterize the MAbs
Results
ELISA:
Performed to see how well an
antibody binds to modified
(native) and non-modified
(recombinant) forms of OPN
adherent to a plastic surface
WESTERN BLOTTING:
Used to test whether an MAb binds
to native or recombinant OPN
bound to a PVDF membrane.
Monoclonal antibody recognition of OPN.
Western blotting results showing antibody
recognition of murine OPN. Conditioned media
from various cell lines or 50 ng of recombinant
murine OPN (GST-mOPN) were separated on
12% SDS-PAGE gels and transferred to PVDF
membranes. The membranes were then cut into
strips which were blotted with MAbs at 1 g/ml
or polyclonal control at a 1:3000 dilution. 275-32: ras-tranformed murine fibroblast cell line.
275: non-transformed murine fibroblast 3T3 cell
line. MC3T3E1: pre-osteoblast cell line induced
to differentiate for 12 days.
Antibody inhibition of cell adhesion to
recombinant human OPN. 96-well plates
were coated with 150M human recombinant
his-tagged OPN, then blocked with 1% BSA.
Antibodies were then added at 125 M and
allowed to bind OPN for 2 hr. The wells were
then washed and 5x104 275-3-2 cells were
added and allowed to adhere for 3.5 h. Nonadherent cells were removed by washing and
adherent cells were quantitated by staining with
crystal violet. Data are representative of two
independent experiments for the 275-3-2 cell
line (n=4). *, p<0.001 Student’s t test.
FROM: Characterization of Anti-Osteopontin Monoclonal Antibodies: Binding
Sensitivity to Post-Translational Modifications. Christian C. Kazanecki, Aaron J.
Kowalski, Tony Ding, Susan R. Rittling, and David T. Denhardt (JCB, in press)
The post-translational modifications of OPN include phosphorylation, glycosylation, and
cross-linking by transglutaminase. Here we describe the generation of unique monoclonal
antibodies raised against recombinant OPN utilizing the OPN knock-out mouse. The
antibodies exhibit differential binding to OPN produced by different cell lines. Most of the
antibodies generated recognize OPN produced by ras-transformed mouse fibroblasts,
however only one antibody recognizes the more phosphorylated protein produced by the
differentiating pre-osteoblast murine cell line MC3T3E1. Using a novel biopanning
procedure combining T7 phage gene fragment display and protein G precipitation, we have
epitope-mapped these antibodies. Several of the antibodies bind to regions of the OPN
molecule that are phosphorylated, and one binds the region of OPN that is glycosylated.
Using phosphorylated and non-phosphorylated peptides, we show that the binding of two
antibodies to the C-terminal end of OPN is inhibited by phosphorylation of this region. In
addition, these two antibodies are able to inhibit cell adhesion to recombinant and weakly
modified OPN. The sensitivity of these antibodies to PTMs suggests that caution must be
taken when choosing anti-OPN monoclonal antibodies to detect this highly modified protein.
PEPTIDE ASSAY:
Tests which specific 18-mer OPN
peptide can bind an MAb
•Similar to an ELISA, only wells
are coated with a peptide via an Nterminal biotin-GG linker
•Peptides cover the entire OPN
protein in overlapping pieces.
•This reveals the approximate
location of the epitope to which the
MAb binds.
MAGNETIC BEAD ASSAY:
Recombinant and native OPN are
differentially labeled and allowed
to react with an MAb. ProteinG
coupled to magnetic beads is then
used to extract the MAb from the
reaction. The ratio of the
fluorescent signals indicates
whether the MAb binds to rec or
nat OPN (or both or neither).
SHOW EXAMPLES WITHOUT THE TEXT
Representative data
MODEL OF OPN FOLDING
From Kazanecki, Uzwiak and Denhardt, in press
Acknowledgements
Putative binding sites for some of the MAbs.
This research was supported by funds from a Busch Biomedical Research Award,
the Rutgers Technology Commercialization fund, and by a grant from the National
Multiple Sclerosis Society. The support of Aresty Summer Fellowships to Tanya
Gordonov and Cassandra Louis are gratefully acknowledged. During portions of this
study Yao Li received support from a SURF Fellowship and Christian Kazanecki from
an IGERT Fellowship. We thank Dr. Larry Steinman for many generous contributions
including the peptides used in this research.